UNIVERSITY OF OULU P .O. Box 8000 F I -90014 UNIVERSITY OF OULU FINLAND

A C T A U N I V E R S I T A T I S O U L U E N S I S

University Lecturer Tuomo Glumoff

University Lecturer Santeri Palviainen

Postdoctoral research fellow Sanna Taskila

Professor Olli Vuolteenaho

University Lecturer Veli-Matti Ulvinen

Planning Director Pertti Tikkanen

Professor Jari Juga

University Lecturer Anu Soikkeli

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-952-62-2028-4 (Paperback)ISBN 978-952-62-2029-1 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1481

AC

TAA

nnina Kelloniem

i

OULU 2018

D 1481

Annina Kelloniemi

NOVEL FACTORS REGULATING CARDIAC REMODELING IN EXPERIMENTAL MODELSOF CARDIAC HYPERTROPHY AND FAILURE

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;BIOCENTER OULU

ACTA UNIVERS ITAT I S OULUENS I SD M e d i c a 1 4 8 1

ANNINA KELLONIEMI

NOVEL FACTORS REGULATING CARDIAC REMODELING IN EXPERIMENTAL MODELS OF CARDIAC HYPERTROPHY AND FAILURE

Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defence inAuditorium F202 of the Faculty of Medicine (Aapistie 5 B),on 26 October 2018, at 12 noon

UNIVERSITY OF OULU, OULU 2018

Copyright © 2018Acta Univ. Oul. D 1481, 2018

Supervised byProfessor Heikki RuskoahoAssociate Professor Jaana Rysä

Reviewed byDocent Päivi LakkistoDocent Minna Kaikkonen-Määttä

ISBN 978-952-62-2028-4 (Paperback)ISBN 978-952-62-2029-1 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2018

Kelloniemi, Annina, Novel factors regulating cardiac remodeling in experimentalmodels of cardiac hypertrophy and failure. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; University ofOulu, Biocenter OuluActa Univ. Oul. D 1481, 2018University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Cardiac loading induces left ventricular hypertrophy and cardiac remodeling which whenprolonged, leads to heart failure, a complex syndrome affecting approximately 1-2% of the adultpopulation of the Western world with a prevalence increasing with age. Pathological remodelinginvolves functional and structural changes that are associated with fetal gene expression,sarcomeric re-organization, hypertrophy of cardiomyocytes, fibrosis, inflammation, oxidativestress and impairment of metabolism.

The aim of this study was to investigate the role of three novel factors during the cardiacremodeling process with different experimental models of cardiac overload. Phosphatase and actinregulator 1 (Phacr1) expression was rapidly downregulated due to myocardial infarction (MI).Adenovirus-mediated Phactr1 overexpression changed the skeletal α-actin to cardiac α-actin ratioin both healthy and infarcted rat hearts and cultured cardiomyocytes. Phactr1 could regulate theactin isoform switch via the serum response factor (SRF). The expression of transforming growthfactor (TGF)- β-stimulated clone 22 (TSC-22) was rapidly induced by multiple hypertrophicstimuli and was also evident post-MI. In addition, TSC-22 could regulate collagen 3a1 expressionin the heart. The expression of retinal degeneration 3-like (Rd3l) was downregulated in responseto pressure overload and also downregulated post-MI. Rd3l knockout mice expressed increasedmyocyte hypertrophy and cardiac dysfunction in response to a transverse aortic constriction(TAC) induced pressure overload.

This thesis provides novel information about Phactr1, TSC-22 and Rd3l in load-inducedcardiac hypertrophy and remodeling. Collectively these studies increase our understanding of theregulatory mechanisms underlying the progression of heart failure.

Keywords: cardiac hypertrophy, cardiac remodeling, gene expression, myocardialinfarction

Kelloniemi, Annina, Sydämen uudelleenmuovautumista säätelevät uudet tekijätsydämen hypertrofian ja vajaatoiminnan kokeellisissa malleissa. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Oulun yliopisto,Biocenter OuluActa Univ. Oul. D 1481, 2018Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Sydämen kuormitus saa aikaan vasemman kammion liikakasvun eli hypertrofian ja sydämenuudelleenmuovautumisen, mikä pitkittyessään johtaa sydämen vajaatoimintaan. Sydämen vajaa-toiminta on monimutkainen oireyhtymä, josta länsimaissa kärsii noin 1-2 % aikuisväestöstä, jaesiintyvyys nousee iän myötä. Patologisessa uudelleenmuovautumisessa tapahtuu toiminnallisiaja rakenteellisia muutoksia, joihin liittyy muutoksia geenien ilmentymisessä, sarkomeerin uudel-leen järjestäytymistä, sydänlihassolujen koon kasvua, fibroosia, tulehdusta, oksidatiivista stres-siä ja aineenvaihdunnan huonontumista.

Tämän työn tarkoituksena oli tutkia kolmen uuden tekijän roolia sydämen uudelleenmuovau-tumisessa erilaisissa kokeellisissa sydämen kuormituksen malleissa. Fosfataasin ja aktiinin sää-telijä 1:n (Phactr1) ilmentyminen väheni nopeasti infarktin seurauksena. AdenovirusvälitteinenPhactr1:n ylituotanto muutti luusto- ja sydänlihasaktiinien isomuotojen suhdetta sekä terveessäettä infarktisydämessä, samoin viljellyissä sydänlihassoluissa. Phactr1 saattaa säädellä isomuo-tojen suhdetta seerumiresponsiivisen tekijän (SRF) avulla. Transformoituvan kasvutekijä β1:nstimuloima proteiini 22:n (TSC-22) ilmentyminen nousi nopeasti usean hypertrofisen stimuluk-sen seurauksena sekä infarktin jälkeen. Lisäksi TSC-22 voisi säädellä kollageeni 3a1:n ilmenty-mistä sydämessä. Retinan degeneroituvan proteiinin 3 kaltaisen tekijän (Rd3l) ilmentyminenväheni sekä painekuormituksen että infarktin seurauksena. Rd3l-poistogeenisillä hiirillä aortanahtauman aiheuttama painekuormitus sai aikaan lisääntynyttä sydänlihassolujen hypertrofiaa jasydämen toimintahäiriöitä.

Tämä väitöskirjatutkimus tuo uutta tietoa Phactr1-, TSC-22- ja Rd3l-geeneistä kuormituksenaiheuttamassa sydämen hypertrofiassa ja uudelleenmuovautumisessa. Nämä tulokset auttavatosaltaan ymmärtämään monimutkaisia molekyylitason mekanismeja, jotka johtavat sydämenvajaatoiminnan kehittymiseen.

Asasanat: geenien ilmentyminen, hypertrofia, infarkti, sydämen uudelleen-muovautuminen

7

Acknowledgements

This work was carried out at the Department of Pharmacology and Toxicology,

Research Unit of Biomedicine, University of Oulu. I owe my deepest gratitude for

my supervisors Professor Heikki Ruskoaho and Associate Professor Jaana Rysä.

Heikki’s optimism, enthusiasm and vast knowledge of science is impressive. I am

very grateful to him for giving me the possibility to do research in his dynamic

research group in well-equipped environment. Jaana is acknowledged for her

excellent guidance and endless encouragement. Especially I am grateful for her

support in daily practical scientific matters and for always finding time to help.

Jaana’s contribution has been indispensable to this project.

I wish to thank Professor Emeritus Olavi Pelkonen and Professor Jukka

Hakkola, the former and current heads of the department for providing an inspiring

and relaxed atmosphere in which to work.

I thank Docents Päivi Lakkisto and Minna Kaikkonen-Määttä for their critical

review of the thesis and for their constructive comments. I also thank Ewen

MacDonald for excellent revision of my English and Anna Vuolteenaho for

revising the Finnish abstract.

I am grateful to all my co-authors Professor Olle Melander, Professor Thomas

Hedner, Professor Risto Kerkelä, Raija Soininen, Raisa Serpi, Zoltan Szabo, Juha

Näpänkangas, Jani Aro, Pauli Ohukainen, Erja Mustonen, Leena Kaikkonen, Elina

Koivisto, Olli Tenhunen, Margret Leosdottir and Zsolt Bagyura for their scientific

contribution.

I owe my gratitude to Sirpa Rutanen, Kati Lampinen, Marja Arbelius, Kaisa

Penttilä, Kirsi Salo and Pirjo Korpi for their skillful assistance and guidance in the

laboratory. Especially Kati’s contribution has been essential for completion of the

projects. Erja Tomperi and Mirja Vahera are also acknowledged for their technical

assistance with immunohistochemistry. I wish to thank the personnel at the

Laboratory Animal Centre and at the Biocenter Oulu Transgenic core facility. I am

also grateful for Raija Hanni, Esa Kerttula, Marja Räinä and Pirkko Viitala for

taking care of all the practical matters in the department.

All the past and present members of our research group are thanked for their

collaboration, company and help. Alicia Jurado Acosta, Leena Kaikkonen, Elina

Koivisto, Erja Mustonen, Virva Pohjolainen and Laura Vainio deserve special

thanks for their joyful company during unforgettable congress trips. Pauli

Ohukainen is greatly acknowledged for his invaluable friendship, support and

optimism that have helped me to overcome the final obstacles of this project. I

8

warmly thank Tarja Alakoski, Jani Aro, Sini Kinnunen, Anna-Maria Kubin, Hanne

Luosujärvi, Johanna Magga, Anne-Mari Moilanen, Ábel Perjés, Tuomas Peltonen,

Raisa Serpi, Zoltan Szabo, Hanna Säkkinen, Olli Tenhunen, Heikki Tokola, Anna-

Maria Tolonen and Maria Tölli for their excellent company in the lab, as well as

during the lunch breaks. Other colleagues in Farmis are warmly acknowledged as

well.

I cannot express enough my gratitude to all my friends. In particular, friendship

of Kristiina, Ari-Matti as well as Suvi, Sami and the other members of ”Kuukkelit”

has brought joy and meaning for my life. Thank you for persuading me out of lab

to enjoy outdoor activities and to hilarious social events.

I owe my deepest gratitude to my parents Marita and Pentti for their love and

care throughout my life as well as my dear sister Maaria and her family for all the

joy and support. Especially my nephew’s lovely and sunny smile makes me forget

all my worries. Finally, I am grateful to my ex-husband Antti for his friendship and

support for so many years. This project has been yours as much as mine.

This work was financially supported by the Academy of Finland (Center of

Excellence funding), Aarne Koskelo Foundation, Biocenter Oulu, the Finnish

Foundation for Cardiovascular Research, Ida Montin Foundation, Inkeri and Mauri

Vänskä Foundation, Orion Research Foundation and Sigrid Jusélius Foundation.

Oulu, August 2018 Annina Kelloniemi

9

Abbreviations

ACE angiotensin converting enzyme

Ang angiotensin

ANP atrial natriuretic peptide

AT1R angiotensin type 1 receptor

BNP B-type natriuretic peptide

BP blood pressure

CAD coronary artery disease

cGMP cyclic guanosine monophosphate

CMKII calcium-calmodulin-dependent kinase

COL collagen

DCM dilated cardiomyopathy

DN dominant negative

ECM extracellular matrix

ERK extracellular signal-regulated kinase

ESC European Society of Cardiology

EST expressed sequence tag

ET-1 endothelin-1

FS fractional shortening

GATA4 GATA binding protein 4

GPCR G protein-coupled receptor

gp130 glycoprotein 130

GWAS genome-wide association study

HCM hypertrophic cardiomyopathy

HDACs histone deacetylases

HF heart failure

HFpEF heart failure with preserved ejection fraction

HFrEF heart failure with reduced ejection fraction

HUVEC human umbilical vascular endothelial cell

IL interleukin

IP3 inositol triphosphate

JAK/STAT the janus kinase/signal transducers and activators of transcription

JNK c-jun NH2-terminal kinase

LV left ventricle

LVH left ventricular hypertrophy

LVEF left ventricular ejection fraction

10

MAPK mitogen-activated protein kinase

MAPKK mitogen-activated protein kinase kinase

MAPKKK MAPK kinase kinase

MEF-2 myocyte enhancer factor 2

MHC myosin heavy chain

MI myocardial infarction

MMP matrix metalloproteinase

MRTF myocardin-related transcription factor

NFAT calcineurin/nuclear factor of activated T cells

NRVMs neonatal rat ventricular myocytes

PAI-1 plasminogen activator inhibitor-1

PDE5A phosphodiesterase 5A

PHACTR1 phosphatase and actin regulator 1

PKC protein kinase C

PLC phospholipase C

RAA renin-angiotensin-aldosterone

Rd3l retinal degeneration 3-like

SERCA2 sarcoplasmic reticulum Ca2+ -ATPase

SHR spontaneously hypertensive rat

SNP single nucleotide polymorphism

SRE serum response elements

SRF serum response factor

STAT signal transducer and activator of transcription

TAC transverse aortic constriction

TGF transforming growth factor

TSC transforming growth factor β-stimulated clone

VEGF vascular endothelial growth factor

VHD valvular heart disease

11

List of original publications

This thesis is based on the following publications, which are referred throughout

the text by their Roman numerals:

I Kelloniemi A., Szabo Z., Serpi R., Näpänkangas J., Ohukainen P., Tenhunen O., Kaikkonen L., Koivisto E., Bagyura Z., Kerkelä R., Leosdottir M., Hedner T., Melander O., Ruskoaho H.*, Rysä J.* (2015). The early-onset myocardial infarction associated PHACTR1 gene regulates skeletal and cardiac alpha-actin gene expression. PLoS ONE 10(6). http://doi.org/10.1371/journal.pone.0130502.

II Kelloniemi A., Aro J., Näpänkangas J., Koivisto E., Mustonen E., Ruskoaho H., Rysä J. (2015). TSC-22 up-regulates collagen 3a1 gene expression in the rat heart. BMC Cardiovascular Disorders, 15(1). http://doi.org/10.1186/s12872-015-0121-2.

III Kelloniemi A., Szabo Z., Serpi R., Aro J., Soininen R., Ruskoaho H.*, Rysä J.* Genetic retinal degeneration 3-like (Rd3l) deficiency induces cardiac dysfunction in response to pressure overload. Manuscript.

* equal contribution.

12

13

Contents

Abstract

Tiivistelmä

Acknowledgements 7 Abbreviations 9 List of original publications 11 Contents 13 1 Introduction 15 2 Review of the literature 17

2.1 Heart failure ............................................................................................ 17 2.1.1 Coronary artery disease and myocardial infarction ...................... 18 2.1.2 Hypertension ................................................................................ 19 2.1.3 Other causes for heart failure ....................................................... 20

2.2 Cardiac remodelling ................................................................................ 21 2.2.1 Cardiac fibrosis ............................................................................. 24

2.3 Cardiac hypertrophy ................................................................................ 29 2.3.1 Cardiac hypertrophic stimuli ........................................................ 30 2.3.2 Intracellular signalling pathways .................................................. 31 2.3.3 Fetal gene program ....................................................................... 33 2.3.4 Actin proteins ............................................................................... 35 2.3.5 Phosphatase and actin regulatory proteins .................................... 37 2.3.6 Transcription factors in cardiac hypertrophy ................................ 39

2.4 Identifying genetic susceptibility and molecular markers for

heart failure ............................................................................................. 42 2.4.1 Genome-wide association studies ................................................. 42 2.4.2 Genome-wide gene expression profiling studies .......................... 45

3 Aims of the research 49 4 Materials and methods 51 5 Results and discussion 53

5.1 Phactr1 expression is rapidly reduced in experimental MI (I) ................ 53 5.2 Phactr1 regulates skeletal α-actin to cardiac α-actin switch (I) ............... 53 5.3 SRF DNA binding activity is enhanced due to Phactr1

overexpression in vitro (I) ....................................................................... 56 5.4 The effects of Phactr1 gene delivery on cardiac function and

structure in normal and infarcted hearts (I) ............................................. 57

14

5.5 MI associated PHACTR1 allele is not associated with cardiac

dysfunction in humans (I) ....................................................................... 57 5.6 TSC-22 is up-regulated by multiple hypertrophic stimuli and

post-MI (II) ............................................................................................. 59 5.7 Reduced TSC-22 expression levels after long-term treatment of

heart failure with metoprolol and hypertension with metoprolol

or losartan (II) ......................................................................................... 62 5.8 Hypertrophic stimuli inducible signalling pathways regulating

TSC-22 gene expression (II) ................................................................... 62 5.9 The effect of TSC-22 overexpression on cardiac gene expression

in vivo (II) ................................................................................................ 63 5.10 Rd3l gene expression and tissue distribution (III) ................................... 64 5.11 Rd3l expression levels in experimental models of hypertension

and MI in rats (III) ................................................................................... 65 5.12 Generation of the Rd3l knockout mouse line (III) .................................. 65 5.13 The effects of hemodynamic pressure overload on Rd3l-deficient

mice (III) ................................................................................................. 65 6 Summary and conclusions 69 References 71 Original articles 95

15

1 Introduction

The main function of the heart is to maintain a blood circulation to meet the body´s

needs. Cardiac remodeling is an adaptive process which occurs when the heart

responds to altered demands. Cardiac remodeling involves many structural and

functional changes ranging from those occurring at the cellular level to those

involving the entire left ventricle. Pathological stimuli, such as increased cardiac

load, can lead to a remodeling process which is characterized by the hypertrophic

growth of cardiomyocytes, initiation of fetal gene programs, alterations in the

extracellular matrix (ECM), energy metabolism and cardiac contractility. Initially,

cardiac remodeling is considered to be a compensatory mechanism to maintain

cardiac output but it becomes maladaptive when prolonged such as happens in

pathological processes and it can eventually lead to heart failure. The main causes

of heart failure are chronic hypertension and myocardial infarction (MI) (Mann,

Barger, & Burkhoff, 2012; Wu et al., 2017).

The main triggers of cardiac hypertrophy are stretch-sensitive mechanisms and

excessive neurohumoral activation, including the sympathetic nervous system, the

renin-angiotensin-aldosterone (RAA) system, vasoactive peptides and vasopressin

(van Berlo, Maillet, & Molkentin, 2013). These stimuli activate complex

intracellular signaling pathways leading to the triggering of several transcription

factors and altered gene expression. One early response to these hypertrophic

stimuli involves the activation of immediate-early genes such as c-fos, c-jun and

egr-1 (Rysä, Tokola, & Ruskoaho, 2018). The early response phase is followed by

the reactivation of fetal gene program including the induction of atrial natriuretic

peptide (ANP) and B-type natriuretic peptide (BNP) expression (Kerkelä, Ulvila,

& Magga, 2015), and a switch to fetal isoforms of contractile sarcomeric proteins

such as cardiac α-actin and α-myosin heavy chain (α-MHC) being replaced by

skeletal α-actin and β-MHC, respectively (Burchfield, Xie, & Hill, 2013; Omura et

al., 2000; Taegtmeyer, Sen, & Vela, 2010).

Despite the treatment available, which currently focus on improving the

prognosis of the patient and relief of symptoms, approximately 50% of patients

suffering from HF die within 5 years of diagnosis. Although there is an ever-

expanding knowledge of the molecular mechanisms involved in LVH and HF, there

is still a need to identify new target molecules in order to develop novel diagnostic

tools and therapies. Previously, DNA microarray techniques have been used to

reveal several novel genes associated with the transition from LVH to HF (Rysa,

Leskinen, Ilves, & Ruskoaho, 2005) as well as novel mechanical stretch responsive

16

genes in cardiomyocytes (Rysä et al., 2018). These dysregulated genes include

transforming growth factor (TGF)-β-stimulated clone 22 (TSC-22), expressed

sequence tag (EST), currently known as retinal degeneration 3-like (Rd3l) and

phosphatase and actin regulator 1 (Phactr1). In addition, several genome-wide

association studies (GWAS)s have associated the PHACTR1 locus with coronary

artery disease (CAD) and MI (Consortium, 2009; Hager et al., 2012; Lu et al., 2012;

Matsuoka et al., 2015; Odonnell et al., 2011; Patel et al., 2012; Jessica van Setten

et al., 2013). The physiological role of these three load-inducible factors in the heart

is still unclear.

The aim of the present study was to investigate the expression of Phactr1, TSC-

22 and Rd3l in cardiac hypertrophy and left ventricular (LV) remodeling. The

expressions of Phactr1 and TSC-22 both at the mRNA and protein level were

studied in vivo in experimental models of pressure overload and myocardial

infarction. Direct myocardial effects of Phactr1 and TSC-22 gene transfer were also

evaluated. In addition, the mechanisms regulating their expression and the effects

of their overexpression were studied in vitro in cultured cardiomyocytes. Finally,

an Rd3l knockout mouse line was generated in order to clarify the function of Rd3l

in the response to pressure overload.

17

2 Review of the literature

2.1 Heart failure

Heart failure is a complex syndrome in which ventricular filling or the ejection of

blood has become impaired as a result of an abnormality in cardiac structure or

function. Impairment of pumping efficiency can result from several causes that

vary in different parts of the world but the main diseases leading to HF are coronary

artery disease with myocardial infarction and chronic hypertension; other causes

are valvular heart disease (VHD), arrhythmias and cardiomyopathies. This chapter

is based on American Heart Association’s and European Society of Cardiology’s

(ESC) guidelines (McMurray et al., 2012; Yancy et al., 2013) for the treatment and

diagnosis of HF unless otherwise stated.

In the Western world, approximately 1-2% of the adult population suffers from

HF; the prevalence increases with age, being ≥ 10% among people 70 years or older.

Although the survival of HF after diagnosis has improved during the past couple of

decades, the mortality rates are still dismal with approximately every second patient

dying within 5 years of diagnosis. (Mozaffarian et al., 2016).

Although disorders of the pericardium, myocardium, endocardium, heart

valves or major blood vessels or some metabolic abnormalities can lead to HF, the

symptoms experienced by most patients with HF result from impaired LV function.

Patients suffering from HF typically display symptoms like shortness of breath and

fatigue, which may limit exercise tolerance, and cause ankle swelling. The

diagnosis of HF can be challenging, especially in the early stages, and should be

made based on a careful history and physical examination. There are several

diagnostic tests to diagnose HF e.g. echocardiography and the determination of the

plasma levels of natriuretic peptides, ANP and BNP.

Left ventricular ejection fraction (LVEF) is important in the diagnosis of HF

with the normal LVEF being ≥50-55%. HF can develop even though LVEF is

preserved or reduced. Depending on guidelines, the definitions of HF with reduced

EF (HFrEF) and HF with preserved EF (HFpEF) have varied. According to ESC’s

present guideline (Ponikowski et al., 2016), HFrEF is commonly defined as HF

with LVEF <40%. HF with preserved EF (HFpEF) is typically considered as ≥50%.

The diagnosis of HFpEF is more difficult and it has been proposed that the

incidence of HFpEF is increasing since more and more patients being hospitalized

with HF have HFpEF. As a group, HFpEF patients are older and more often female

18

and obese with more severe hypertension, anemia and atrial fibrillation than those

with HFrEF. They are also less likely to have coronary heart disease and they enjoy

a better prognosis compared to those with HFrEF. Those patients with an LVEF

ranging from 40 to 49% represent a grey area between HFrEF and HFpEF. In the

European guideline, (Ponikowski et al., 2016), this group is identified as HF with

mid-range EF.

Current treatment options for HF include pharmacological therapy, lifestyle

modifications, implantation of a left or bi-ventricular assist device and heart

transplantation. Angiotensin converting enzyme inhibitors, angiotensin II receptor

blockers and β-blockers improve the prognosis of the patients and relieve their

symptoms. Furthermore, diuretics, aldosterone antagonists and digoxin have also

been used to treat HF (Kemp & Conte, 2012; McMurray et al., 2012). Promisingly,

the combination of neprilysin inhibition with valsartan (sacubitril + valsartan) has

been claimed to exert beneficial effects on congestive heart failure (McMurray et

al., 2014).

2.1.1 Coronary artery disease and myocardial infarction

Coronary artery disease (also known as coronary heart disease) develops when the

heart’s arteries have a build-up of plaque on their inner walls and become hardened

and narrowed. As a consequence, there is a reduction in the flow of blood, nutrients

and oxygen to the myocardium. With time, even as long as decades, some plaques

may become unstable and rupture leading to thrombosis and arterial occlusion

(Libby, 2012). Myocardial infarction is defined as the death of cardiomyocytes due

to prolonged ischemia (Montecucco, Carbone, & Schindler, 2015). Because of their

high sensitivity, troponins T and I are used as specific biochemical markers for

cardiac damage since after a myocardial injury, troponin is released within 2-4

hours and this elevated concentration persists for up to 10-21 days to allow

diagnosis (Korff, Katus, & Giannitsis, 2006). In addition to the elevation in these

biochemical markers, myocardial infarction is characterized by ischemic symptoms

and electrocardiographic changes (Roger, 2007).

In the US, the prevalence of CAD among adults (≥ 20 years of age) is 5.0 %

for women and 7.6 % for men (Mozaffarian et al., 2016). The incidence of MI has

been evaluated in several cohort studies. When estimated according to 100000

adults, the overall incidence has been 205 in the Olmsted County Study (Arciero et

al., 2004; Roger et al., 2002) and 244 in the Worcester Heart Attack Study

(Goldberg, Yarzebski, Lessard, & Gore, 1999). The sex-specific incidence in the

19

Atherosclerosis Risk in Communities study was 190 for women and 410 for men

(Rosamond et al., 1998). The effect of ethnicity has been evaluated in the Corpus

Christi Study (Goff et al., 1997). The incidence in Mexican American women has

been 354 and 486 for men. In non-Hispanic White individuals, the incidence has

been 224 for women and 346 for men. The prevalence of CAD in Finland can be

estimated from drug register data (Stable coronary artery disease: Current Care

Guidelines, 2015); almost 181 000 person were prescribed drugs for the treatment

of stable CAD in Finland in 2013.

CAD is the most common cause of HF and in 63 % of patients with reduced

EF, HF is preceded by CAD (Lee et al., 2009), although hypertension and diabetes

are contributing factors in many cases. Patients with HF and LV systolic

dysfunction after myocardial infarction have a higher risk for cardiac rupture and

arrest, ventricular arrhythmias, recurrent myocardial infarction, stroke, longer

hospitalizations and death than patients without HF and LV systolic dysfunction

post-MI (Minicucci, Azevedo, Polegato, Paiva, & Zornoff, 2011).

Acute MI triggers changes in ventricular geometry and structure, leading to a

rapid decline in systolic function. Prolonged ischemia leads to necrosis and

apoptosis of cardiomyocytes. Since the adult heart has only a limited regenerative

capacity, the infarcted myocardium heals through the formation of a scar. Early

cardiac remodelling is characterized by a progressive ventricular dilation, increased

wall stress and hypertrophy of the remaining viable myocytes. The size of the

infarct and the severity of early remodelling determine the progress of ventricular

dysfunction and the development of chronic HF (Jugdutt, 2012).

Patients who develop signs of HF soon after infarction, tend to have a reduced

tolerance to ischemic injury due to pre-existent comorbidities such as recurrent

ischemia and diabetes. Infarct size, mechanical complications and myocardial

stunning are the major causes of HF during a patient’s hospital stay. Usually

myocyte loss, a hibernating myocardium and ventricular remodeling are related to

the development of HF after the patient has been discharged from the hospital

(Minicucci et al., 2011).

2.1.2 Hypertension

Hypertension is commonly defined as blood pressure (BP) values ≥140/90 mmHg.

The same values are used to diagnose young, middle-aged and elderly subjects,

excluding children and teenagers (Mancia et al., 2013). In the NHANES survey,

the prevalence of hypertension was estimated to be 32.6 % among US adults (≥20

20

years of age). Until 45 years of age, men are more likely than women to have

hypertension. Between the ages of 45 to 54 and 55 to 64 years, there are no

differences between the sexes but from 65 years of age, a higher percentage of

women than men have hypertension. The lifetime risk of HF is twice as high for

people with BP >160/90 mm Hg when compared to those with BP <140/90 mm Hg

(Mozaffarian et al., 2016). In Finland, it is estimated that 2 million individuals in

the adult population suffer from hypertension with approximately one million of

them taking hypertension medication (Hypertension: Current Care Guidelines,

2014).

Hypertension precedes HF in 26 % of patients. In 19 % of HF patients with

reduced EF, hypertension is present before the diagnosis; the corresponding value

for those with preserved EF is 36 % (Lee et al., 2009). However, hypertension can

contribute to HF via indirect mechanisms since it is a risk factor for atherosclerosis

and MI. In particular, in the elderly and in women, hypertension accounts for a

greater proportion of HF cases (Gudmundsdottir, Høieggen, Stenehjem, Waldum,

& Os, 2012).

Chronic hypertension, i.e. a condition present for decades, is a primary

precursor of LVH, which can lead to ventricular dysfunction and eventually to HF.

Diastolic dysfunction has been characterized with suboptimal ventricular filling

caused by insufficient LV relaxation without any change in ventricular compliance.

Hypertension induced diastolic dysfunction can also result from myocardial

fibrosis with collagen accumulation and changes in the shape of the heart’s

chambers (Hill & Olson, 2009).

Hypertension increases the risk for HF in all age groups and the lifetime risk

for HF is double in individuals with BP >140/90 mm Hg when compared to those

with BP <140/90 mm Hg. Therefore, early diagnosis and treatment of hypertension

can effectively prevent further complications.

2.1.3 Other causes for heart failure

LV remodelling occurs in several clinical conditions. In addition to myocardial

infarction and hypertension, valvular heart disease and cardiomyopathies can cause

HF. Valvular heart disease precedes HF in 8% of patients (Lee et al., 2009). Calcific

aortic stenosis and mitral valve regurgitation are the most common types of VHD

in the Western world (Vahanian et al., 2012). Calcific aortic stenosis is a progressive

disease causing a chronic pressure overload that further contributes to LV

21

remodelling (Lindman, Bonow, & Otto, 2013) whereas mitral regurgitation leads

to remodelling by increasing volume overload in LV (Jessup & Brozena, 2003).

Based on the structural and functional changes occurring in the heart, genetic

cardiomyopathies that may cause HF are classified into dilated cardiomyopathy

(DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic cardiomyopathy

and restrictive cardiomyopathy. Although genetic cardiomyopathies cause only a

small proportion of all HF cases, genetic cardiomyopathies are a common source

of morbidity and mortality in children and teenagers, with the prevalence being

approximately 40%. Depending of the type of cardiomyopathy, the genetic

background varies but typically mutations leading to cardiomyopathies are found

in the genes encoding sarcomeric, cell adhesion, transmembrane and nuclear

proteins (Cahill, Ashrafian, & Watkins, 2013).

In addition, arrhythmias and damage to the myocardium as a result of infection,

inflammation or chronic abuse of cardiotoxic substances (e.g. alcohol) can lead to

HF (McMurray et al., 2012; Yancy et al., 2013).

2.2 Cardiac remodelling

The heart has various dynamic mechanisms to help maintain its function in

response to altered demands. Cardiac remodeling is characterized by alterations in

the heart’s size, shape, structure and function which range from the cellular level to

those involving the entire left ventricle; these changes are regulated by mechanical,

neurohormonal and genetic factors. Diverse pathological (e.g. chronic hypertension,

MI, cardiomyopathy or VHD) or physiological stimuli (e.g. growth, pregnancy and

exercise) can increase the workload for the heart, leading to its remodelling and

hypertrophic growth which reduces ventricular wall stress and maintains or even

augments pump function (Mann et al., 2012; Sutton & Sharpe, 2000). Most adult

cardiomyocytes seem to have a limited proliferation capacity, so the heart responds

to increased demands by enlarging the size of the cardiomyocytes. Physiological

cardiac remodeling is a highly orchestrated and reversible process resulting in

decreased cardiac wall stress, increased pumping efficiency and vascularization

with an increase in the number of capillaries (Shimizu & Minamino, 2016).

Pathological remodeling, however, is an irreversible process and when prolonged,

it leads to maladaptive alterations and HF (Fig. 1). In pressure overload, such as in

hypertension or aortic stenosis, a reduction in wall stress is achieved by concentric

hypertrophy which leads to an increase in thickness of the free wall and septum. In

volume overload due to MI or dilated cardiomyopathy, the loss of cardiomyocytes

22

leads to rearrangement of the surviving cardiomyocytes and eccentric hypertrophy

with greater ventricular volumes and subsequent/partial ventricular dilatation.

Pathological stimuli can activate the innate immune system and trigger

inflammation. A short-term inflammatory response can be beneficial for cardiac

repair of damaged tissue but a prolonged inflammatory reaction extends the damage,

induces fibrosis and leads to maladaptive remodeling (Suthahar, Meijers, Silljé, &

de Boer, 2017). The remodeling involves altered gene expression and disturbances

in protein synthesis and cellular metabolism threatening the survival of

cardiomyocytes as well as changes in the ECM and angiogenesis. Ventricular

remodeling is a progressive response; initially it is a compensatory process but

ultimately it becomes detrimental and leads to LV wall thinning and dilatation

(Mann et al., 2012; Wu et al., 2017). Unlike physiological cardiac remodeling,

pathological remodeling is not associated with any increase in myocardial capillary

density proportional to the increased cardiac mass, and capillary rarefaction is one

important feature in the development of HFpEF (Hoenig, Bianchi, Rosenzweig, &

Sellke, 2008; Shimizu & Minamino, 2016).

Cardiac remodeling is accompanied by oxidative stress and the increased

generation of reactive oxygen species as well as disturbed calcium homeostasis

leading to mis- and/or unfolded proteins; this is referred to as endoplasmic

reticulum stress and to cellular autophagy/apoptosis (Wu et al., 2017). During

remodeling, there are also changes in cardiac energy metabolism. In the normal

heart, approximately 70% to 90% of cardiac energy is produced by the oxidation

of fatty acids, with the remaining 10% to 30% mostly originating from the

oxidation of glucose and lactate. Peroxisome proliferator-activated receptors

(PPARs) with estrogen-related receptor are important long-term regulators of

cardiac fatty acid metabolism in the normal heart. Most of the models of cardiac

hypertrophy and failure have revealed reduced cardiac use of fatty acids. However,

there are more variations in glucose use between different models of HF. The stage

of progression and the pathogenesis of the HF may exert an influence on the

observed changes in glucose metabolism (reviewed in Doenst, Nguyen, & Abel,

2013).

Cardiac remodeling involves mitochondrial dysfunction which is characterized

by abnormally high levels of mitochondrial reactive oxygen species and

downregulation of the genes maintaining normal mitochondrial biogenesis, such as

peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)

(Azevedo, Minicucci, Santos, Paiva, & Zornoff, 2013; Schirone et al., 2017).

23

Disturbed calcium homeostasis is a remarkable feature of the compensatory

hypertrophy and heart failure. Cellular calcium cycling is a highly orchestrated,

essential process underpinning normal cardiac contractility being regulated by β-

adrenergic signaling. Calcium-induced calcium release from the sarcoplasmic

reticulum leads to elevated intracellular free Ca2+ concentration and triggers a

contraction of the cardiomyocyte. Relaxation is necessary to allow a new

contraction and therefore most of the cytosolic Ca2+ is pumped back into the

sarcoplasmic reticulum to be released again during a new contraction (Mayourian

et al., 2018). A major cause for impaired Ca2+ signaling and reduced contractility is

the down-regulation of the sarcoplasmic reticulum Ca2+ -ATPase 2 (SERCA2)

which is a key component participating in Ca2+ uptake in sarcoplasmic reticulum

(Barry, Davidson, & Townsend, 2008; Cabassi & Miragoli, 2017).

Fig. 1. Remodeling of cardiac tissue due to pathophysiological stimuli and the

progression to heart failure. Pathological stimuli, such as excessive mechanical load

or myocardial infarction can lead to multiple molecular and cellular processes

contributing to ventricular remodelling. Mechanical stress and neurohumoral activation

trigger hypertrophy of cardiomyocytes. Cardiomyocytes are lost through necrosis,

apoptosis and autophagy. Changes in the ECM leads to fibrosis. Remodeling includes

activation of fetal gene program, angiogenesis and changes in energy metabolism.

Impaired calcium cycling is a hallmark of HF. Modified from Burchfield, Xie, & Hill, 2013.

24

2.2.1 Cardiac fibrosis

Cardiac fibrosis is a process which involves collagen accumulation leading to

structural changes in heart and eventually impaired cardiac function (Krenning,

Zeisberg, & Kalluri, 2010). The cardiac ECM is composed of collagens I and III,

the former being the most abundant type of collagen in the heart,

glycosaminoglycans, glycoproteins and proteoglycans (Kong, Christia, &

Frangogiannis, 2014). Normal ECM provides structural support for

cardiomyocytes and in addition distributes the contractile forces through the

cardiac tissue and mediates electric conduction. The ECM consists of an

endomysium, perimysium and epimysium which surround the myocytes and the

coronary microvasculature. The endomysium is arranged around each individual

myocyte; it supports and connects them to one another. The perimysium envelopes

the group of myocytes forming bundles of fibers that the epimysium surrounds

(Weber, Sun, Bhattacharya, Ahokas, & Gerling, 2013). The composition of the

ECM is very tightly regulated, involving ongoing synthesis and degradation of

ECM proteins and disturbances in this balance precede structural abnormalities and

impaired function of the heart (Marcin Dobaczewski, Gonzalez-Quesada, &

Frangogiannis, 2010). Collagens are synthesized and secreted by fibroblasts and

myofibroblasts and degraded by matrix metalloproteinases (MMPs) (Lindsey, Iyer,

Jung, DeLeon-Pennell, & Ma, 2016).

Several different pathophysiological conditions can lead to cardiac fibrosis.

The death of cardiomyocytes after MI triggers an inflammatory reaction, removal

of injured cells and cell matrix and replacement of dead myocardium with a fibrotic

scar tissue (Fig. 2). In addition, hypertension, aortic stenosis, hypertrophic

cardiomyopathy and diabetes may induce fibrotic remodeling in the heart (Asbun

& Villarreal, 2006; Ashrafian, McKenna, & Watkins, 2011; Berk, Fujiwara, &

Lehoux, 2007; Dweck, Boon, & Newby, 2012). Normal aging can also be

associated with cardiac fibrosis which increases ventricular stiffness and impairs

diastolic function. Several studies suggest that fibrosis in the aging heart is caused

by reduced matrix degradation rather than by increased collagen synthesis

(reviewed in Biernacka & Frangogiannis, 2011).

Various cell types are involved in fibrotic remodeling of the heart. Depending

on the cause of fibrosis, the relative contribution of different cell types to the

fibrotic process varies. The hallmark of the fibrotic response is the trans-

differentiation of fibroblasts into proliferating myofibroblasts that are able to

secrete large amounts of matrix proteins. The contractile activity of myofibroblasts

25

is increased since they are able to express α-smooth muscle actin (α-SMA). There

are also several cell types, including macrophages, mast cells, lymphocytes,

endothelial cells, and cardiomyocytes that participate indirectly in fibrotic

remodeling by secreting fibrogenic mediators, such as cytokines (tumor necrosis

factor, interleukins (IL)s -1β and IL-6, growth factors (including TGF-β) and

vasoactive peptides such as Ang II and endothelin-1 (ET-1) (for Review, see Kong

et al., 2014).

Two types of cardiac fibrosis exist; reparative fibrosis and reactive interstitial

fibrosis (Rai, Sharma, Agrawal, & Agrawal, 2017). Since adult cardiomyocytes

have a limited proliferation capacity, the dead cells after MI are replaced with a

collagen-rich scar. The healing process after myocardial infarction can be divided

into three overlapping phases: inflammatory, proliferative and maturation phases.

The death of cardiomyocytes triggers an intense inflammatory response with

cytokine and chemokine upregulation and infiltration of activated leukocytes into

the infarcted area. The wound is cleansed of dead cells and ECM debris by

phagocytes and this is followed by a suppression of the inflammatory reaction,

leading to the transition to the proliferative phase. During the proliferative phase,

fibroblasts proliferate and differentiate into myofibroblasts that are capable of

secreting large amounts of ECM proteins, such as collagen. This phenomenon is a

key part of the repair process of the infarcted myocardium. As the scar matures, the

majority of infarct myofibroblasts undergo apoptosis and the ECM proteins become

cross-linked to preserve structural integrity (Marcin Dobaczewski et al., 2010;

Nikolaos G. Frangogiannis, 2014).

A diffuse accumulation of ECM can exist at sites unrelated to a focal injury.

Reactive interstitial fibrosis occurs in animal models of left ventricular pressure

overload in which the initial reactive perivascular and interstitial fibrosis is

followed by cardiomyocyte hypertrophy as an adaptive mechanism to preserve

cardiac function and to decrease ventricular wall tension. Eventually, the

cardiomyocytes seem to undergo apoptosis and necrosis and in those areas,

replacement fibrosis is observed (Rai et al., 2017; Rossi, 1998). Interstitial fibrosis

results in increased mechanical stiffness and thereby in diastolic and systolic

dysfunction and this exposes to the patient to arrhythmias when the electrotonic

connectivity between cardiac myocytes is disrupted. Perivascular fibrosis impairs

the coronary blood flow and myocyte oxygen availability and predisposes to

myocardial ischemia (Brown, Ambler, Mitchell, & Long, 2004).

The origin of the accumulating active myofibroblasts in the fibrotic heart

remains unclear. The most important source might be the activated resident cardiac

26

fibroblasts since numerous proliferating myofibroblasts have been observed in

infarcted hearts (Frangogiannis et al., 1998; Frangogiannis, Michael, & Entman,

2000) and it is believed that these cells sense different pathological stimuli (Ali et

al., 2014; Fredj, Bescond, Louault, & Potreau, 2005; Moore-Morris et al., 2014;

Teekakirikul et al., 2010). Other suggested sources are endothelial cells, epicardial

epithelial cells, pericytes, fibrocytes and hematopoietic bone marrow-derived

progenitor cells (Kong et al., 2014; Travers, Kamal, Robbins, Yutzey, & Blaxall,

2016). However, the contribution of different myofibroblast populations likely

depends on the type of cardiac injury. Unlike in the healing process of the skin

where all myofibroblasts are cleared from the scar, in the heart they can be

identified in the post-MI scar for as long as 17 years after the event (Willems,

Havenith, De Mey, & Daemen, 1994). It has been suggested that the reason for the

maintenance of myofibroblasts in the infarct scar might be to attenuate the

expansion of the scar and prevent the development of HF (van den Borne et al.,

2009).

Fig. 2. Cardiac fibrosis following hypoxic injury involves three overlapping phases:

inflammatory, proliferative, and maturation phases. Inflammatory mediators control

wound healing and removal of death cells which is followed by collagen synthesis and

finally cross-linkage of collagen fibers and scar formation. Time intervals for phases

are suggestive since they vary between species. ECM, extracellular matrix; MMP, matrix

metalloproteinase.

Transforming growth factor β

Transforming growth factor β are pleiotrophic cytokines which perform various

cellular functions, such as the regulation of inflammation, ECM deposition, fibrosis

27

and the control of cell growth, proliferation and differentiation. In mammals, there

exist three structurally similar isoforms, TGF-β1, 2 and 3 that are encoded by three

different genes. TGF-β1 is the most abundantly expressed isoform; this isoform is

found in almost all cell types whereas the other isoforms have more limited

expression patterns in tissues (Bujak & Frangogiannis, 2007; Dobaczewski, Chen,

& Frangogiannis, 2011).

TGF-β is stored in tissues as a latent dimeric complex unable to bind its

receptors. TGF-β is activated by proteolytic cleavage releasing the mature TGF-β

from the complex/inhibitory domain. Several molecules have been described as

TGF-β activators, including proteases, such as plasmin and cathepsin D (Lyons,

Keski-Oja, & Moses, 1988), MMP-2 and MMP-9 (Q. Yu & Stamenkovic, 2000)

and the matricellular protein thrombospondin 1 (Schultz-Cherry, Ribeiro, Gentry,

& Murphy-Ullrich, 1994). In addition, changes in pH can mediate TGF-β activation

(Lyons et al., 1988).

In some animal models of heart failure, upregulated TGF-β levels have been

associated with cardiac hypertrophy and fibrosis. A pressure overload induced by

aortic constriction has been reported to increase TGF-β1 synthesis significantly in

the hypertrophied rat LV (J. M. Li & Brooks, 1997). Ang II signaling correlated to

upregulated TGF-β levels in myocardium after infarction (Sun, Zhang, Zhang, &

Ramires, 1998). TGF-β1 mRNA levels increased also in cultured neonatal rat

ventricular myocytes after treatment with Ang II (Sadoshima & Izumo, 1993) or

norepinephrine (N. Takahashi et al., 1994). Induction of TGF-β1 in the myocardium

could be, at least in part, mediated through Ang II, since treatment of hypertrophied

(Kim et al., 1996) or infarcted rat hearts (Sun et al., 1998; C.-M. Yu, Tipoe, Wing-

Hon Lai, & Lau, 2001) with angiotensin converting enzyme (ACE) inhibitors or

AT1 receptor blockers downregulated TGF-β expression. Moreover, Ang II induced

hypertrophy was abolished in TGF-β1 knockout mice (Schultz Jel et al., 2002).

Elevated cardiac TGF-β1 levels have been detected also in patients with myocardial

hypertrophy due to idiopathic hypertrophic cardiomyopathy (R. K. Li et al., 1997).

In addition to being involved in myocardial remodeling by hypertrophic stimuli,

TGF-β1 is a key molecule in the development of fibrosis. In the healing process of

the infarcted heart, TGF-β regulates the switch from the inflammatory phase to the

proliferative phase and promotes ECM protein synthesis (Bujak & Frangogiannis,

2007). Mice overexpressing TGF-β1 developed a hypertrophic heart accompanied

by interstitial fibrosis in the ventricles when compared with their non-transgenic

controls (Rosenkranz et al., 2002) whereas cardiac-restricted overexpression of

constitutively active mutant human TGF-β1 led to a fibrotic response in the atria

28

but not in the ventricles of the transgenic mice (Nakajima et al., 2000).

Heterozygous TGF-β1 (+/-) deficient mice exhibited attenuated aging-associated

myocardial fibrosis and diastolic dysfunction (Brooks & Conrad, 2000).

TSC-22

The TGF-β stimulated clone 22 (TSC-22) gene sequence was first isolated from

cDNA library of mouse osteoblastic cells treated with TGF-β1 (Shibanuma, Kuroki,

& Nose, 1992). TSC-22 is a highly conserved protein containing a leucine zipper

motif with a molecular mass of 18 kDa. TSC-22 is expressed in various organisms

from Drosophila to humans. The human homologue of the TSC-22 gene was

isolated from a cDNA library extracted from 6 weeks old human embryos (Jay et

al., 1996). At the amino acid level, the human homologue of the TSC-22 is almost

99% identical to the mouse and rat genes. TSC-22 is expressed in several human

adult (including spleen, thymus, prostate, testis, ovary, small intestine, colon, heart,

brain, placenta, lung, liver, skeletal muscle, kidney and pancreas) and fetal

(including brain, lung, liver and kidney) tissues (Jay et al., 1996).

TSC-22 forms homodimers via its leucine zipper domain. It can also

heterodimerize with other transcription factors (Choi et al., 2005; Dobens et al.,

1997; Kester, Blanchetot, Hertog, Van, & Van, 1999; Yan, 2011), such as Smad4

(Choi et al., 2005) and Smad7 (Yan, 2011). TSC-22 has been reported to be

involved in many physiological processes such as the development of Drosophila

melanogaster and mouse (Dobens, Peterson, Treisman, & Raftery, 2000;

Dohrmann, Belaoussoff, & Raftery, 1999; Kester et al., 2000; Treisman, Lai, &

Rubin, 1995), apoptosis (Ohta, Yanagihara, & Nagata, 1997; Yoon et al., 2012) as

well as participating in both cholesterol (Jager et al., 2014) and glucose (Ekim

Üstünel et al., 2016) metabolism. In addition, several studies have focused on the

putative tumor suppressive role of TSC-22 in many cancer cell lines and tumor

tissues (Iida, Anna, Gaskin, Walker, & Devereux, 2007; Kawamata et al., 1998;

Kawamata, Fujimori, & Imai, 2004; Ohta et al., 1997; Shostak et al., 2005; Yoon

et al., 2012; J. Yu et al., 2009). One of the known functions of TSC-22 is to regulate

cellular proliferation via the proto-oncogene, c-MYC. TSC-22 binding inhibits the

suppressive role of c-MYC on cyclin-dependent kinase inhibitors (Zheng, Suzuki,

Nakajo, Nakano, & Kato, 2018).

TSC-22 has been suggested to promote the differentiation of cardiac

myofibroblasts and to contribute to myocardial fibrosis (Yan, 2011). In primary

cardiac fibroblasts isolated from the neonatal rat heart, lentivirus-mediated

29

overexpression of TSC-22 enhanced the TGF-beta-induced expression of several

fibrotic factors e.g. plasminogen activator inhibitor-1 (PAI-1), collagen I and

fibronectin and α-SMA which is a marker for myofibroblast differentiation (Yan,

2011). An upregulation of TSC-22 gene expression in rat heart has been reported

in an experimental model of essential hypertension in spontaneously hypertensive

rats (SHRs) (Rysa, Aro, & Ruskoaho, 2006) as well as after experimental

myocardial infarction (Stanton et al., 2000). The absence of TSC-22 in the heart

has not been studied in detail. TSC-22 knockout mice display a 14% decrease in

the weight of their hearts when compared to wild-type mice (J. Yu et al., 2009).

2.3 Cardiac hypertrophy

The heart responds to diverse physiological and pathological stimuli with

hypertrophic growth (van Berlo et al., 2013). Hypertrophic growth is achieved by

enlarging the existing individual cardiomyocytes since they have only a limited

proliferative capacity. Pregnancy and chronic exercise are two well known

conditions that activate the signalling pathways stimulating myocyte renewal and

reducing cell death resulting in physiological growth with normal cardiac

morphology and function. In physiological hypertrophy, all parts of the ventricle,

chambers, walls and septum enlarge equally and the myocytes gain both width and

length (Maillet, van Berlo, & Molkentin, 2012). Pathological hypertrophy, however,

is initially an adaptive mechanism to normalize wall stress and to maintain cardiac

function but over time, when stress is sustained, these changes become maladaptive

and can progress to HF. Two morphological types of pathological hypertrophy exist.

In concentric hypertrophy, the relative wall and septal thickness increases when the

sarcomeres are added in parallel and the cardiomyocytes mostly increase in width

such that chamber volume can be diminished. Eccentric hypertrophy, in which the

myocytes increase in length rather than width due to the addition of sarcomeres in

series results in an enlargement of the chamber. Relative wall thickness may be

normal, decreased, or increased. Under continuous stress, concentric hypertrophy

can eventually lead to dilated or eccentric hypertrophy. Concentric hypertrophy

often results from hypertension or aortic stenosis whereas eccentric growth is more

common due to volume overload, dilated cardiomyopathy or the situation after

infarction and is characterized with a more advanced cardiac malfunction which

leads to HF (reviewed in Bernardo, Weeks, Pretorius, & McMullen, 2010; Hill &

Olson, 2009; van Berlo et al., 2013).

30

2.3.1 Cardiac hypertrophic stimuli

Cardiac hypertrophic growth is typically initiated either by stretch-sensitive

mechanisms or by certain neuro-endocrine factors and hormones such as Ang II,

ET-1 and TGF-β. These stimuli trigger the induction of complex intracellular

signalling cascades in myocytes that promote hypertrophic gene expression, induce

fibrosis and alter sarcomeric protein organisation (Bernardo et al., 2010; van Berlo

et al., 2013).

Angiotensin II

Angiotensin II is a vasoactive peptide hormone; it is the key effector substance of

the renin-angiotensin aldosterone system which is a major regulatory system of

cardiovascular and renal function. Ang II is produced from angiotensinogen which

is initially cleaved by renin to form angiotensin I (Ang I) which is then activated to

Ang II by ACE (Paul, Poyan Mehr, & Kreutz, 2006). Ang II exerts effects on many

cell types with diverse functions ranging from vasoconstriction, blood pressure

regulation and stimulation of aldosterone release to mediation of growth and

endothelial dysfunction. These cellular events link Ang II to many pathological

cardiovascular processes, such as inflammation, atherosclerosis, thrombosis and

fibrosis that, over time, lead to cardiovascular diseases including MI, stroke and

HF (Mehta & Griendling, 2007).

Several tissues are able to synthesize Ang II locally (Paul et al., 2006). Indeed,

hemodynamic stress can activate a local renin-angiotensin system in the heart

(Lijnen & Petrov, 1999; Yamazaki & Yazaki, 1997). One experiment carried out in

pigs showed that most of the cardiac Ang II peptide was produced from locally

synthesized Ang I (van Kats et al., 1998).

The effects of Ang II in the heart are mediated via Ang II type 1 and Ang II

type 2 receptors (AT1R and AT2R, respectively) that are both G protein-coupled

receptors (GPCRs). Ang II mediates a hypertrophic response in cardiomyocytes

mostly by activating AT1R (Paul et al., 2006). The effects of Ang II mediated via

AT2R on cardiac hypertrophy, however, seem to be complicated since it has been

suggested to have both inducible and inhibitory effects (Lévy, 2004). Transgenic

mice overexpressing AT1R in cardiomyocytes developed cardiomyocyte

hypertrophy and cardiac fibrosis which led to HF without any change in blood

pressure (Paradis, Dali-Youcef, Paradis, Thibault, & Nemer, 2000). This is

evidence that the effects of Ang II occur independently from its actions on blood

31

pressure and furthermore that the local activity of Ang II in cardiomyocytes is

sufficient to induce hypertrophy and remodelling of the heart.

Endothelin-1

Endothelin-1, ET-2 and ET-3 comprise a family of cyclic 21 amino acid peptides

that are encoded by three highly conserved genes. ET-1 is a potent vasoconstrictor

which has autocrine and paracrine regulatory functions in cardiac physiology and

pathology. ET-1 is primarily produced and secreted by endothelin cells and

mediates effects on several cell types in heart. The effects of ET-1 are elicited by

binding to ETA or ETB receptors both of which are GPCRs (Drawnel, Archer, &

Roderick, 2013). Both isoforms are expressed in heart but in cardiomyocytes, the

ETA receptor predominates (B. G. Allen, Phuong, Farhat, & Chevalier, 2003) and

it has higher affinity for ET-1 than the other two family members (Hosoda et al.,

1991).

ET-1 has been shown to have a significant role in cardiomyocyte hypertrophy

and remodelling of the heart. ET-1 transmits a hypertrophic response by activating

different intracellular signalling pathways, such as protein kinase C (PKC) and

mitogen-activated protein kinases (MAPK)s (reviewed in Drawnel et al., 2013). In

cultured cardiomyocytes, ET-1 overexpression induced transcription of various

genes associated with hypertrophy and fetal gene programs (Cullingford et al.,

2008; Ito et al., 1991). The pressure overload induced by aortic banding increased

ET-1 expression levels in rats (Yorikane et al., 1993). Moreover, cardiac-restricted

conditional ET-1 overexpression in transgenic mice caused hypertrophy which

progressed to HF and death (Yang et al., 2004).

2.3.2 Intracellular signalling pathways

Cardiomyocytes undergo cross-talk with the surrounding endothelial cells,

fibroblasts and inflammatory cells as well as integrating signals from ECM, cell

membrane and cellular organelles to translate biomechanical forces into gene

expression. Several factors can initiate hypertrophic stimuli that can activate a

complex network of multiple separate or parallel intracellular signal transduction

pathways that eventually lead to biological responses in cardiomyocytes. These

signalling cascades include protein kinases, such as MAPKs, PKC, and the janus

kinase/signal transducers and activators of transcription (JAK/STAT) pathway and

32

protein phosphatases e.g. the calcineurin/nuclear factor of activated T cells (NFAT)

signalling pathway (Heineke & Molkentin, 2006; Ruwhof & van der Laarse, 2000).

Mitogen-activated protein kinase pathways

Mitogen-activated protein kinases are serine/threonine-specific kinases that relay

extracellular signals that evoke intracellular responses. MAPK pathways are

involved in cell proliferation, differentiation, apoptosis and motility. The activation

of the MAPK signalling cascade is initiated at the cell membrane, where small G

proteins and various protein kinases phosphorylate and activate canonical three-

tiered sequential phosphorylation events. A MAPK kinase kinase activates a

MAPK kinase leading to activation of MAPK through serial phosphorylation. This

canonical activation cascade allows both signal amplification and specificity to

modulate transcription factors that drive context-specific gene expression (Rose,

Force, & Wang, 2010). MAPKs are highly conserved and can been divided into the

three classic MAPK subfamilies i.e. extracellular signal-regulated kinases

(ERK1/2), c-jun N-terminal kinases (JNKs), and p38 kinases. There are some other

atypical MAPKs e.g. ERK3/4, ERK5, ERK7 and Nemo-like kinase. ERK1 and

ERK2 are mainly responsive to stimulation by growth factors, whereas JNKs and

p38 kinases are more specialized transducers of physical, chemical and

physiological stressors, and injury (van Berlo et al., 2013). All three main MAPK

pathways are activated in rodent heart after transverse aortic constriction (TAC)

surgery (Esposito et al., 2001) and in humans suffering from heart failure (Haq et

al., 2001).

The Ras/Raf/MEK1/ERK signalling pathway promotes cardiac hypertrophy.

Transgenic mice with cardiac-specific overexpression of Ras displayed cardiac

hypertrophy (Hunter, Tanaka, Rockman, Ross, & Chien, 1995) and similar results

were observed in transgenic mice overexpressing MEK1 (Bueno et al., 2000).

Cardiac-specific ERK inhibition through the dominant negative form of Raf led to

the situation that the mice became resistant to TAC-induced cardiac hypertrophy

(Harris et al., 2004). ERK1-/- and ERK2+/- mice exhibited a normal cardiac

hypertrophy in response to the pressure overload evoked by TAC (Purcell et al.,

2007).

JNK family members are derived from three genes: JNK1, JNK2 and JNK3.

The activation of JNK is carried out by two MAP kinases, MKK4 and MKK7

(Davis, 1999). The role of JNK pathway in cardiac hypertrophy is somewhat

unclear. JNK1-/-, JNK2-/- and JNK3-/- mice developed cardiac hypertrophy to a

33

similar extent as their wild type counterparts, when subjected to pressure overload

by TAC (Tachibana et al., 2006). However, JNK1-/- mice had reduced LV systolic

function for several weeks after the procedure before returning to normal. These

results suggested that JNK1 was required to maintain cardiac contractility and it

prevented heart failure in the acute phase after pressure overload.

Four p38 MAPKs have been identified: p38α, p38β, p38γ and p38δ, and their

upstream activators are MKK3 and MKK6 (Zarubin & Han, 2005). Overexpression

of MKK3 and MKK6 induced hypertrophic response in cultured cardiomyocytes

(Y. Wang et al., 1998; Zechner, Thuerauf, Hanford, McDonough, & Glembotski,

1997). In vivo, however, activation of p38 was not sufficient to induce hypertrophy.

The cardiac-specific overexpression of MKK3 and MKK6 and the induction of p38

kinase activity did not lead to any significant hypertrophy in transgenic mice when

compared to wild type mice (P. Liao et al., 2001). Instead, the transgenic mice

exhibited increased interstitial fibrosis. The loss of p38 activity in cardiac-specific

p38 DN transgenic mice (S. Zhang et al., 2003) or pressure overload in cardiac-

specific p38 knockout mice had no effect on hypertrophy (Nishida et al., 2004).

The cardiac-specific deletion of p38 resulted in increased apoptosis and fibrosis

(Nishida et al., 2004).

2.3.3 Fetal gene program

The heart responds to the demands posed by overload via an increase in myocyte

mass as well as undergoing changes in gene expression. The first group of genes

activated due to an increased workload is called the immediate-early genes such as

c-fos, c-jun and egr-1; these genes are rapidly and transiently expressed without

any preceding protein synthesis. Many of immediate-early genes encode

transcription factors that are activated by mechanical load or neurohumoral

hypertrophic stimuli (Chien, Knowlton, Zhu, & Chien, 1991; Rysä et al., 2018).

Signaling pathways leading to transcriptional activation of hypertrophic genes are

complex and display significant cross-talk. The key receptors and signaling

cascades involved in hypertrophic growth in adult heart are presented in Fig. 3.

One feature of hypertrophy is that the early response phase is followed by the

activation of a fetal gene program – a set of genes that are normally expressed in

the developing heart but are suppressed in the adult myocardium. ANP and BNP

are synthesized in substantial amounts during embryonic development but their

levels in the healthy adult ventricle are low (Gardner, 2003). Re-expression of ANP

and BNP is induced by a wide variety of load stimuli in several species (Hama et

34

al., 1995; Kerkelä et al., 2015; Ogawa et al., 1991; T. Takahashi, Allen, & Izumo,

1992) and elevated natriuretic peptide plasma levels are used as clinical indicators

of cardiac hypertrophy (Kerkelä et al., 2015). Since natriuretic peptides have

natriuretic, diuretic and vasodilatory effects, their overall function may be to protect

the myocardium by inhibiting the hypertrophic response (Barry et al., 2008).

Other characteristics of the fetal gene program appear to be that the expression

of several sarcomeric proteins becomes switched to fetal isoforms from their adult

counterparts. In the developing mammal heart, β-MHC is the abundant isoform. In

rats and mice, the α-MHC predominates after birth as well as in the mature heart

but in larger mammals, such as humans, the β-MHC isoform dominates also in the

adult ventricle. However, due to the elevated work load, the ratio of the cardiac

MHC isoforms changes in all species. The β-MHC has lower ATPase activity

which leads to a slower and more economical contractility function in the larger

cardiac myocytes (Bernardo et al., 2010; Nadal-Ginard & Mahdavi, 1989). In

addition, the switch from cardiac α-actin to skeletal α-actin, which is the fetal

isoform, has been associated with better contractility in a loaded heart (Hewett,

Grupp, Grupp, & Robbins, 1994; Schwartz et al., 1986).

35

Fig. 3. A simplified diagram of the key signalling pathways involved in cardiac

hypertrophy. Hypertrophic growth of cardiomyocytes is triggered by neurohumoral

activation and biomechanical stress produced by cardiac overload. Ang II, angiotensin

II; ANP, atrial natriuretic peptide; β-MHC, β-myosin heavy chain; BNP, B-type natriuretic

peptide; CAMKII, calcium-calmodulin-dependent kinase; GATA4, GATA binding protein

4; ET-1, endothelin-1; gp130, glycoprotein 130; GPRC, G-protein coupled receptor;

HDACs, histone deacetylases; IP3, inositol triphosphate; JAK, Janus kinase; MAPKs,

mitogen-activated protein kinases; MAPKK, MAPK kinase; MAPKKK, MAPK kinase

kinase; MEF2, myocyte enhancer factor 2; NFAT, nuclear factor of activated T cells; PLC,

phospholipase C; SRF, serum response factor; STAT, signal transducer and activator of

transcription. Modified from Bernardo et al., 2010; Shah & Mann, 2011; Tham, Bernardo,

Ooi, Weeks, & McMullen, 2015.

2.3.4 Actin proteins

The contraction unit in cardiac muscle is composed of two important filaments,

actin and myosin. Actin is essential for several basic functions in organisms,

including cell motility, maintaining cell shape, endo- and exocytosis, cell division

36

and muscle contraction (Perrin & Ervasti, 2010; Tondeleir, Vandamme,

Vandekerckhove, Ampe, & Lambrechts, 2009). Actin is highly conserved between

species and is expressed in all eukaryotic cells (Vandekerckhove & Weber, 1978).

In mammals, there are six different actin isoforms: tissue-restricted skeletal α-actin,

cardiac α-actin, α-SMA and γ-SMA, and ubiquitously expressed cytoplasmic β-

cytoplasmic actin and γ-cytoplasmic actin. The isoforms differ only by few amino

acids but they seem to have unique expression patterns and specific cellular

functions in the tissues in which they predominate (Vandekerckhove & Weber,

1978).

Along with myosin, cardiac α-actin and skeletal α-actin are important

components of the contraction unit in striated heart and skeletal muscle. Both types

exist in cardiac muscle but their expression patterns differ spatially and temporally.

During cardiac development, skeletal α-actin predominates but after birth, the adult

isoform, cardiac α-actin, is the main thin filament component. In normal adult

rodent heart, the expression levels of skeletal α-actin are low but during the

remodeling process in hypertrophied and failing hearts, skeletal α-actin expression

is re-induced (Omura et al., 2000; Schwartz et al., 1986). The switch to the fetal

actin isoform can be induced both in vivo due to pressure load or myocardial

infarction (Nadal-Ginard & Mahdavi, 1989; Omura et al., 2000; Schwartz et al.,

1986) and in vitro in cultured cardiomyocytes by mechanical load (Komuro et al.,

1991; Sadoshima, Jahn, Takahashi, Kulik, & Izumo, 1992).

All actin isoforms seem to have specialized functions since knockout mouse

models of actin isoforms have distinct phenotypes. Most of the cardiac α-actin null

mice die prenatally, and those that survive until birth, die within two 2 weeks

(Kumar et al., 1997). Both the homozygous and heterozygous cardiac α-actin mice

expressed increased levels of skeletal α-actin and α-SMA in their hearts but this

was insufficient to restore the deficient myofibrillar function in these knockout

mice. In addition, cardiac α-actin knockout mice were able to reach adulthood when

γ-SMA was overexpressed under the control of cardiac α-myosin heavy chain

promoter. However, the hearts of these transgenic mice are hypodynamic, enlarged

and hypertrophic (Kumar et al., 1997). Skeletal α-actin deficient mice appear

healthy at birth but die within 9 days after birth with reduced muscle strength and

growth deficits (Crawford et al., 2002). However, the transgenic expression of

cardiac α-actin sufficiently rescued the lethality and prevented the reduction in

muscle function associated with the loss of skeletal α-actin (Nowak et al., 2009).

In BALB/c mice, a perturbation to the cardiac α-actin locus resulted in hearts

that contained up to 50 % of the skeletal α-actin. The hearts of these mice had better

37

contractility (Garner, Minty, Alonso, Barton, & Buckingham, 1986; Hewett et al.,

1994). Normal hearts of large mammals such as humans expressed higher levels of

skeletal α-actin mRNA than small mammals such as rodents (Boheler et al., 1991);

this has been postulated to account for their better contractility due to the abundant

amounts of skeletal α-actin (Chaponnier & Gabbiani, 2004; Yin, Ren, & Guo, 2015).

Overall, these observations from mouse models emphasize the compensatory

expression of muscle actin isoforms and the importance of the balance in actin

levels in order to meet the requirements of cell specific function.

There are a few mutations in cardiac α-actin that have been associated with

DCM and HCM (Arad et al., 2005; Mogensen et al., 2004; Mogensen et al., 1999;

T M Olson, Michels, Thibodeau, Tai, & Keating, 1998; T M Olson et al., 2000; Van

Driest et al., 2003). It has been suggested that in patients with DCM, mutations in

cardiac α-actin affect the force transmission from the sarcomere to the surrounding

syncytium (Mogensen et al., 1999). However, mutations associated with HCM

have been proposed to affect sarcomere contraction due to an impaired formation

of actin filaments or due to interference with the actin-myosin interaction

(Mogensen et al., 2004; T M Olson et al., 2000).

2.3.5 Phosphatase and actin regulatory proteins

The Phactr family of phosphatase and actin regulatory proteins comprises four

members: Phactr1, Phactr2, Phactr3/Scaponin and Phactr4. They act as modulators

of protein phosphatase 1 (PP1) and bind to actin. In rats, Phactrs are expressed

widely in the central nervous system with lower expression levels being found in

lung, heart, kidney and testis (P. B. Allen, Greenfield, Svenningsson, Haspeslagh,

& Greengard, 2004). Depending on the cell type and cell cycle, different subcellular

localization patterns of Phactrs have been reported from nucleus to cytosol and

plasma membrane (P. B. Allen et al., 2004; Huet et al., 2013; Itoh, Uchiyama,

Taniguchi, & Sagara, 2014; Sagara et al., 2003; Sagara, Arata, & Taniguchi, 2009;

Y. Zhang, Kim, & Niswander, 2012).

Phactr proteins are conserved, 75 kDa in size with each Phactr protein

containing one N-terminal RPEL motif and a C-terminal triple RPEL repeat

followed by the PP1-binding domain. Phactrs have been implicated in several

biological processes including the regulation of actin cytoskeleton dynamics (Fils-

Aime et al., 2013; Wiezlak et al., 2012), the modulation of cell motility and

migration (Sagara et al., 2009), axon elongation (Farghaian et al., 2011) and

angiogenesis (Allain et al., 2012; Jarray et al., 2011).

38

Phactrs have been linked to some human diseases such as coronary artery

disease and myocardial infarction, cancers and Parkinson’s disease (Bankovic et al.,

2010; Consortium, 2009; Solimini et al., 2013; Wider et al., 2009).

Phosphatase and actin regulator 1

The PHACTR1 locus has been associated with CAD and MI in several

genome-wide association studies (GWAS) (Consortium, 2009; Hager et al., 2012;

Lu et al., 2012; Matsuoka et al., 2015; Odonnell et al., 2011; Paquette, Dufour, &

Baass, 2018; Patel et al., 2012; Jessica van Setten et al., 2013). PHACTR1 has also

been linked to migraine without aura (Freilinger et al., 2012), cervical artery

dissection (Debette et al., 2015), and fibromuscular dysplasia (Kiando et al., 2016)

in GWAS (Allain et al., 2012). Phactr1 has been reported to modulate vascular

calcification in murine embryonic stem cell-derived smooth muscle cells

(Aherrahrou, Aherrahrou, Schunkert, & Erdmann, 2017). In a DNA microarray

study using cultured rat cardiomyocytes, Phactr1 was shown to be activated by

mechanical stretch (Rysä et al., 2018).

In human tissues, PHACTR1 has been detected in the brain and aorta as well

as in normal and infarcted heart (Beaudoin et al., 2015). PHACTR1 controls tube

formation and cell survival in Human Umbilical Vascular Endothelial Cells

(HUVECs) since PHACTR1 depletion by siRNA induced apoptosis (Jarray et al.,

2011). The same research group found also that down-regulation of PHACTR1

disrupted the control of actin polymerization and prevented the depolymerization

in HUVECs.

In human melanoma cells, PHACTR1 seems to be required for actomyosin

assembly and the invasive behavior of these malignant cells (Wiezlak et al., 2012).

Binding to PP1 was inhibited by the interaction of G-actin with the C-terminal

RPEL repeat of PHACTR1 (Wiezlak et al., 2012). In human breast cancer cells, a

PHACTR1 knockdown impaired actin dynamics (polymerization and

depolymerization) which is needed for cell motility and migration (Fils-Aime et al.,

2013). PHACTR1 was discovered to be a negatively regulated downstream target

of a potential tumor suppressor miRNA-584 the levels of which were down-

regulated by TGF-β during cell migration of breast cancer cells. Thus, increased

expression of PHACTR1 seems to be required for TGF-β-induced cell migration

(Fils-Aime et al., 2013). These reports highlight the importance of PHACTR1 in

actin related cellular processes.

39

2.3.6 Transcription factors in cardiac hypertrophy

Several transcription factors that are active during the development of the heart

reappear in a stressed adult heart as a part of the fetal gene program. The cardiac-

expressed transcription factors that are involved in cardiac hypertrophy, such as

GATA-4, Nkx2.5, Smads, serum response factor (SRF), myocardin-related

transcription factors (MRTFs) and myocyte enhancer factor-2 (MEF-2) have been

reviewed by Kohli et al. (Kohli, Ahuja, & Rani, 2011) and Oka et al. (Oka, Xu, &

Molkentin, 2007). Here the focus will be placed on SRF and the MRTFs that act as

SRF co-factors and have a regulatory role in gene expression of actin proteins.

Serum response factor

Serum response factor (SRF) belongs to a family of proteins that contain a MADS

(Mcm1 and Arg80 in yeast, Agamous and Deficient in plants, and SRF in animals)

box domain and which act as transcription factors. SRF binds to serum response

elements, more specifically to the CArG box DNA consensus CC(A/T)6GG which

is found in the promoters of SRF target genes (E. N. Olson & Nordheim, 2010).

SRF is ubiquitously expressed but through recruitment of many cell-type specific

cofactors, it can adopt specialized roles in different cellular environments

(Mikhailov & Torrado, 2012). SRF is important in cardiac development and growth

when it acts as a pivotal transcriptional regulator of the numerous genes necessary

for cardiac function and contraction, such as immediate early transcription factors

and actin proteins that are re-activated in the adult heart as part of the cardiac

hypertrophic response (Parmacek, 2007).

The development of SRF knockout mouse embryos ceased before mesoderm

formation and the embryos displayed reduced expression levels of both the early

response genes c-fos and Egr-1 and α-actin, i.e. well-known SRF target genes

(Arsenian, Weinhold, Oelgeschläger, Rüther, & Nordheim, 1998). Similar results

were obtained by cardiac-specific ablation of SRF which led to failed cardiogenesis

and down-regulation of SRF-targets α-actin and SM22α (Niu et al., 2005). SRF

deficient cardiomyocytes exhibited impaired Z-disc and stress fiber formation with

mislocalization and reduced expression of sarcomeric proteins, evident signs of a

defective contractile apparatus structure and function (Balza & Misra, 2006).

The regulatory role of SRF is not restricted to development as revealed in

studies with adult transgenic mice. Transgenic mice with cardiac-specific SRF

overexpression suffered from cardiac hypertrophy with fibrosis (X. Zhang et al.,

40

2001). Cardiac specific Cre-mediated loss of SRF in adult mice led to dilated

cardiomyopathy with impaired contractility and LV function that rapidly

progressed to heart failure (Parlakian et al., 2005). Gene expression of cardiac

alpha-actin was markedly downregulated and changes in the expression of the

genes involved in calcium-handling and energy flux were also detected. Since both

the cardiac-specific SRF overexpression and deletion have been associated with a

cardiomyopathic state, the activation and function of SRF has to be strictly

regulated.

Myocardin-related transcription factors

Myocardin-related transcription factors (MRTFs), including myocardin, MRTF-

A/MKL1/MAL and MRTF-B/MKL2, comprise a family of proteins that function

as powerful coactivators of SRF. MRTFs bind to SRF and synergistically activate

the transcription of the genes involved in cytoskeletal organization and muscle cell

differentiation and function (Parmacek, 2007). Myocardin is expressed primarily

in cardiac and smooth muscle cells whereas MRTF-A and MRTF-B display more

widespread expression patterns (D.-Z. Wang et al., 2002). MRTFs exhibit strong

homology and in the N-terminus they have RPEL domains that in MRTF-A and

MRTF-B are able to bind monomeric G-actin which via Rho signaling regulate

MRTF-mediated transcription of the SRF-dependent genes encoding contractile

and cytoskeletal proteins (Parmacek, 2007).

Myocardin is necessary for smooth muscle cell differentiation since myocardin

deficient mice embryos die early and fail to develop vascular smooth muscle cells

while cardiogenesis occurs normally (S. Li, Wang, Wang, Richardson, & Olson,

2003). Cardio-restricted ablation of myocardin in adult mice leads to DCM with

disruptions in sarcomeric organization, increased cardiomyocyte apoptosis and

depressed systolic function (Huang et al., 2009). Different hypertrophic signals

such as phenylephrine, ET-1 (Xing et al., 2006), aldosterone and isoproterenol (K.

Wang, Long, Zhou, & Li, 2010) have induced myocardin expression in neonatal

cardiomyocytes; in hypoxia, the elevated expression of myocardin is apparently

mediated via Ang II and the ERK pathway (Chiu, Wang, Chung, & Shyu, 2010).

Overexpression of myocardin induced hypertrophy and expression of fetal genes

(Xing et al., 2006).

MRTF-A null mice are viable and show no obvious organ abnormalities.

However, knockout females are unable to productively nurse their offspring due a

failure in mammary myoepithelial cell differentiation, evidence that MRTF-A

41

could be a regulator of myoepithelial cell development (S. Li, Chang, Qi,

Richardson, & Olson, 2006; Sun et al., 2006). Furthermore, Sun et al. have shown

that 35% of MRTF-A-/- mouse embryos suffered lethal dilatation of cardiac

chambers although the rest of the embryos exhibited no obvious abnormalities (Sun

et al., 2006). Based on these studies, it seems that MRTF-A has an unclear role in

cardiovascular development. Although homozygous MRTF-A null adult mice have

no obvious cardiac deficiencies under basal conditions, they display an abnormal

adaptation to cardiac overload.

Adult mice lacking the MRTF-A gene show an attenuated hypertrophic

response both to the acute (1h) and chronic pressure (3 weeks) overload induced

by thoracic aortic banding. The expression levels of BNP, skeletal α-actin and

smooth muscle α-actin genes were significantly up-regulated in wild-type mice but

the increases were significantly attenuated in MRTF-A null mice. The expression

of BNP and skeletal α-actin was also weaker in MRTF-A-/- mice than in their wild-

type counterparts when the pressure overload was induced by chronic Ang II

treatment (Kuwahara et al., 2010).

In cultured neonatal rat cardiomyocytes, the overexpression of MRTF-A

induced hypertrophic growth and the expression of cardiac hypertrophy marker

genes. In addition, the expression of MRTF-A was up-regulated by hypertrophic

signals, whereas inhibition of endogenous MRTF-A expression by dominant-

negative mutant or siRNA reduced the abilities of phenylephrine, Ang II and TGF-

β toinduce hypertrophy in cultured cardiomyocytes (X.-H. Liao et al., 2011).

In contrast to MRTF-A knockout mice, MRTF-B knockout embryos have a

lethal phenotype and cardiovascular defects, including a thin-walled myocardium,

ventricular septal defects, an abnormal patterning of the branchial arch arteries and

truncus arteriosus (J. Li et al., 2005; Oh, Richardson, & Olson, 2005; Wei, Che, &

Chen, 2007). However, mice with a cardiomyocyte-specific deletion of MRTF-B

are viable and display a normal phenotype at least until the age of one year

(Mokalled et al., 2015). The group of Mokalled bred these mice with constitutive

MRTF-A knockout mice to generate mice lacking both MRTF-A and MRTF-B in

the heart. Most of these double knockout mice died within 2 weeks after birth and

the rest succumbed before the age of 13 weeks. Neonatal MRTF-A/B null mice had

defects in sarcomeric organization and function whereas those few mice reaching

early adulthood had reduced cardiac contractility and adult onset heart failure.

42

2.4 Identifying genetic susceptibility and molecular markers for

heart failure

Human genetics provides a powerful and unbiased tool to discover causal

mediators of pathophysiology of cardiac diseases. The Human Genome Project and

the development of economical and reliable DNA technology arrays applicable to

high-throughput and large-scale data generation have enabled genome wide

analyses. In addition, the availability of large population data with hundreds or

thousands of subjects is needed to provide sufficient statistical power to detect true

associations of modest genetic effects (Manolio, 2010).

2.4.1 Genome-wide association studies

Genome-wide association studies (GWAS) involve genotyping a large set of

genetic variants and comparison of allele distributions between cases with a disease

and controls without the disease of interest (J. G. Smith & Newton-Cheh, 2015).

Unlike conventional methods that concentrate on one or a few genes or genetic

regions, the GWAS investigate the whole genome. Scanning of the genome can be

used in several study designs, such as cohort studies, case-control studies and

clinical trials (Manolio, 2010). The most common forms of DNA variation are

single nucleotide polymorphisms (SNPs) which are defined as variants with an over

5% prevalence in the population. For comparison, single base mutations occur with

a frequency of < 1% whereas variants observed at an intermediate frequency >1%

but <5% are considered rare variants (Dubé & Hegele, 2013).

GWAS usually focus on the associations between SNPs and traits like major

diseases of question. GWAS can also be carried out from the aspect of phenotype-

first. Subjects known to differ from a particular quantitative trait, such as blood

pressure or ejection fraction are classified initially by their clinical manifestation

and this is followed by genomic association studies. In GWAS, the frequency or

intensity of a biallelic genetic variant between case and control subjects is

compared in SNP arrays, and a allele that is more frequently observed in subjects

with the disease is considered to be associated with the disease and is defined as

the risk allele (Dubé & Hegele, 2013). The associations need to be tested with a

large number of statistical analyses and require extreme levels of statistical

significance (Zeller, Blankenberg, & Diemert, 2012). The position of the variant

in the genome need not necessarily be located in the coding regions, in fact the vast

majority (>80 %) of SNP-trait associations reside in non-coding regions (Hindorff

43

et al., 2009). Genetic variants identified to a certain locus of human genome and

associated with a disease in GWAS cannot, on their own, specify which genes are

causal. They might rather be in linkage disequilibrium with a causal variant located

nearby on the same chromosome (Kessler, Vilne, & Schunkert, 2016) and for that

reason, additional investigations are needed to identify exact mechanisms.

GWAS are a powerful technique for identifying genetic variants and relating

these to complex disorders. However, GWAS also have their own limitations and

challenges. GWAS require large sample sizes in order that they can fulfil the strict

thresholds of statistical significance (P < 5×10-8) and they have the potential to

produce false-positive results and genotyping errors. They are insensitive to rare

variants and structural variants, and the selection of control and case needs to be

performed carefully to avoid biases (Pearson & Manolio, 2008). Nonetheless,

GWAS can help to detect genetic loci for multifactorial diseases. However, since

the majority of the associated loci are found in non-coding regions, these

associations lack any link to specific genes and to pathophysiological mechanisms

through which these variants predispose to disease and thus additional molecular

studies are needed to identify the variants causing the disease of interest (Manolio,

2010; Zeller et al., 2012).

Genome-wide association studies in heart failure

To date, genome-wide association studies have identified 184 SNPs associated

with HF, corresponding to two studies. Table 1 represents the 20 GWAS SNPs with

the strongest (smallest p-value) association with heart failure (M. J. Li et al., 2012).

Since non-congenital HF is a multifactorial syndrome which can develop slowly as

a result of conditions such as CAD and hypertension, most GWAS in HF have been

designed to investigate those SNPs associated with these risk factors. Blood

pressure and dyslipidemia are important quantitative traits that predispose to HF. A

multi-stage meta-analysis of 200 000 individuals of European ancestry has

identified a total of 16 loci regulating blood pressure (International Consortium for

Blood Pressure Genome-Wide Association Studies et al., 2011). In a similar

population sample of over 100 000 participants, 95 common variants associated

with dyslipidemia were screened and also found to correlate with CAD (Teslovich

et al., 2010). Interestingly, some studies have found new loci associated with CAD

without affecting traditional risk factors (Peden et al., 2011; Schunkert et al., 2011).

A couple of new SNPs associated with early-onset MI have also been identified in

a multi-staged meta-analysis (Consortium, 2009).

44

The meta-analysis of cohort studies examined nearly 21000 European-ancestry

individuals and almost 3000 African-ancestry individuals who were followed-up

for incident heart failure events. One locus from both ancestry groups was

identified, rs10519210 (nearest gene Ubiquitin carboxyl-terminal hydrolase 3) in

European-ancestry participants and rs11172782 (nearest gene Leucine-rich repeats

and immunoglobulin-like domains protein 3) among African-ancestry participants

(N. L. Smith et al., 2010). Another meta-analysis of the same cohort identified one

loci, rs12638540 (in an intron of CKLF-like MARVEL transmembrane domain

containing 7) that was significantly associated with all-cause mortality in

individuals of European-ancestry with HF (Morrison et al., 2010). However, these

findings will need to be replicated. Another case control study included almost

1600 Caucasian patients with heart failure and nearly 600 controls (Cappola et al.,

2010). In this study, about 50000 SNPs in 2000 genes linked to cardiovascular

disorders were analysed. Of these, two SNPs, rs1739843 (heat shock protein family,

member 7) and rs6787362 (FERM domain-containing protein 4B), were associated

with advanced heart failure.

45

Table 2. The 20 most significant loci for heart failure loaded from GWAS database.

SNP id pubmedid/

dbGaP id

Chromo-

some

p-value Gene Function

rs12638540 20400778 3 0.0000003 CMTM7 B-1a B cell differentiation

rs729201 pha002884 16 0.000000785 CDH15 calcium ion binding

rs2125623 20400778 15 0.000001 OTUD7A DNA binding

rs6868223 20400778 5 0.000002 ADAMTS12 metalloendopeptidase activity

rs2241032 pha002884 16 0.00000353 GAS8 epithelial cilium movement

rs2731968 pha002884 5 0.00000376 EGFLAM calcium ion binding

rs7687921 20400778 4 0.000004 GPM6A neuron migration

rs11880198 20445134 19 0.000006 GNA15 G-protein coupled receptor binding

rs7120489 20400778 11 0.000007 PARVA sprouting angiogenesis

rs17121271 pha002885 13 0.00000709 TUBGCP3 structural molecule activity

rs2964519 pha002884 5 0.00000723 RANBP17 GTP binding

rs3805455 pha002884 5 0.0000076 GABRP extracellular ligand-gated ion channel

activity

rs17159640 20400778 7 0.000009 IFRD1 nucleus

rs11582563 pha002885 1 0.0000128 TRIM33 negative regulation of transcription

by RNA polymerase II

rs1565862 pha002885 12 0.0000132 CPM carboxypeptidase activity

rs885479 pha002884 16 0.0000142 MC1R melanocortin receptor activity

rs1372173 pha002885 8 0.00003 CNGB3 photoreceptor outer segment

rs10506584 pha002884 12 0.0000303 CNOT2 negative regulation of transcription

by RNA polymerase II

rs2035645 pha002884 15 0.0000342 ST8SIA2 Golgi membrane

rs10483611 pha002885 14 0.0000348 GPR137C integral component of membrane

Function inferred from Gene Ontology database top hit.

2.4.2 Genome-wide gene expression profiling studies

Advances in the genome projects and in computational biology led to the

development of high-throughput methods for genome-wide gene expression

analyses i.e. transcriptomics. At first, DNA microarrays containing predetermined

sequences were widely used (Trevino, Falciani, & Barrera-Saldaña, 2007). In DNA

microarrays, a collection of DNA probes are attached to the surface as spots.

Purified nucleic acid from the cells or tissue to be studied is labelled with a

fluorophore and hybridized to the array. The image is scanned and the intensity of

the signal is analysed to determine the relative abundance of bound molecules

(Goodwin, McPherson, & McCombie, 2016). Currently, whole next-generation

sequencing (NGS) technologies (also known as high-throughput sequencing) that

46

make it possible to examine the whole sequence, are increasingly being used in

biomedical research (Goodwin et al., 2016; Mardis, 2013). In humans,

transcriptomic studies e.g. in heart failure (Margulies et al. 2005) and different

cardiomyopathies (Hannenhalli et al., 2006) have been performed, and gene

expression profiles have been related in different pathological conditions e.g. in the

prognosis of heart failure (Heidecker et al., 2008) and with respect to the

atherosclerosis burden of coronary and carotid arteries (Ma & Liew, 2003; Patino

et al., 2005). In animal studies, transcriptomics has been widely utilized to screen

gene expression levels in various experimental models of cardiovascular diseases

(Harpster et al., 2006; Rysa et al., 2005). At the level of individual cardiac cells,

transcriptomic profiles of cardiomyocytes, fibroblasts, leukocytes, and endothelial

cells from infarcted and non-infarcted neonatal and adult mouse hearts (Quaife-

Ryan et al., 2017) as well as sex-specific transcriptomic profiles of adult rat

ventricular myocytes have been characterized (Trexler, Odell, Jeong, Dowell, &

Leinwand, 2017). Ultimately, the integration of genotyping and expression data can

be used to reveal the cardiovascular disease-associated genes. Figure 4 represents

epigenetic regulatory factors, genomic variants and cellular modifications leading

to a certain phenotype as well as the different “omic” technologies used to explore

the actions and relationships between these molecules.

47

Fig. 4. Schematic presentation of the relationship between different “-ome/-omics”

components, epigenetic regulatory factors, gene transcription, post-translational

modification, protein localization and function, contributing to different functions in

cells and tissues, and eventually leading to certain phenotype. The genome integrates

intrinsic and environmental signals. Kinases and phosphatases modulate the cellular

activities of the proteins in the responses to external stimuli. The environment can

interact directly with the genome to cause reversible epigenetic changes and thus affect

gene expression and silencing. “Omics”-based approaches provide opportunities to

discover biomarkers for diseases. Modified from Pasipoularides, 2017.

48

49

3 Aims of the research

The aim of the study was to characterize the role of novel load-inducible genes in

cardiac hypertrophy and LV remodeling in different experimental models of cardiac

hypertrophy and failure. In addition, an Rd3l knockout mouse line was generated

to investigate the function of Rd3l in the overloaded heart. Specifically the

objectives were:

1. To examine the expression of Phactr1 in LV remodeling post-MI and to

investigate the effects of adenovirus-mediated Phactr1 overexpression in vivo

on cardiac function and in vitro in cultured cardiomyocytes.

2. To study the cardiac TSC-22 expression in vivo during different experimental

models of cardiac load.

3. To determine Rd3l expression in the heart during cardiac load and to generate

an Rd3l knockout mouse line to clarify the function of Rd3l in the animals’

response to the pressure overload in the heart.

50

51

4 Materials and methods

The experimental models and the average number of animals used in the models

are summarized in Table 2. The methods used in original publications are listed in

Table 3. Detailed descriptions with references can be found in the original

publications I-III.

Table 3. Summary of the experimental models and methods.

Original publication Experimental models Average number of animals

used/model

I Adenovirus-mediated gene transfer in vivo and

in vitro

150

Myocardial infarction 60

Cell culture

II Adenovirus-mediated gene transfer in vivo and

in vitro

26

Myocardial infarction 42

Pressure overload (Arginine8-vasopressin and

Ang II)

90

Losartan infusion 26

Cell culture

III Pressure overload (Ang II) 56

Myocardial infarction 42

Establishment of a knockout mouse line

Pressure overload (TAC) 40

Animals used in the establishment of a knockout mouse line and the neonatal pups used for in vitro cell

culture experiments are excluded

52

Table 4. Summary of the methods used in each publication.

Original publication Method

I Production of adenoviral vectors

Antibody production

RNA isolation and real-time quantitative RT-PCR

Protein extraction

Western blot

EMSA

Echocardiography

Histological analysis

Statistical analysis

II Antibody production

RNA isolation, Northern blot and real-time quantitative RT-PCR

Protein extraction

Western blot

Echocardiography

Histological analysis

Statistical analysis

III DNA cloning

Antibody production

Southern blot

RNA isolation, Northern blot and real-time quantitative RT-PCR

Echocardiography

Histological analysis

Statistical analysis

53

5 Results and discussion

The most relevant findings from the original articles are presented and discussed

here. The original publications are referred to by the Roman numerals I-III and

figures with the corresponding numbers as in the original articles.

5.1 Phactr1 expression is rapidly reduced in experimental MI (I)

Although PHACTR1 has been associated with MI and CAD in GWAS (Consortium,

2009; Hager et al., 2012; Lu et al., 2012; Matsuoka et al., 2015; Odonnell et al.,

2011; Patel et al., 2012; van Setten et al., 2013), the function of PHACTR1 in the

heart is unknown. First, the effect of post-infarction myocardial remodeling on

Phactr1 expression in adult rats in a model of acute MI induced by ligation of left

anterior descending coronary artery was investigated. Rapid and significant

reductions both in Phactr1 mRNA and protein levels (0.4-fold and 0.9-fold,

respectively) were observed already at 1 day in response to MI (I, Fig 1A-B). The

expression levels returned to basal levels in two weeks. This suggests that Phactr1

could have a role in the early remodeling process after infarction.

Stretching of cardiomyocytes occurs in the remodeling heart after acute MI

(Force, Michael, Kilter, & Haq, 2002). Mechanical stretch activates intracellular

signalling pathways, including the p38 MAPK pathway and leads to changes in

gene expression (Rohini, Agrawal, Koyani, & Singh, 2010; Rose et al., 2010).

Phactr1 mRNA levels were downregulated both in stretched cardiomyocytes and

after p38 overexpression in cultured neonatal rat ventricular myocytes (NRVMs) (I,

Fig 1C-D). This suggests that cardiac overload post-infarction down-regulates

Phactr1 expression in the heart via the p38 MAPK pathway triggered by direct

mechanical myocyte stretch. However, to clarify the precise function of

endogenous p38 in regulation of Phactr1, p38 MAPK inhibition experiments will

be needed.

5.2 Phactr1 regulates skeletal α-actin to cardiac α-actin switch (I)

An adenoviral gene transfer protocol was used to examine the direct myocardial

effects of Phactr1 in the normal adult rat LV and in hearts during the post-infarction

remodelling process. Significantly increased and sustained Phactr1 mRNA and

protein levels were detected for up to 2 weeks when compared to control animals

54

receiving virus expressing LacZ also at a virus dose of 5×108 infectious units (I,

Fig 2 C-D, 7A-B).

One feature of the remodelling process of the stressed heart is the appearance

of the fetal gene program (Burchfield et al., 2013; Taegtmeyer et al., 2010). The

early response includes transcription of c-fos, c-jun and early growth response -1

(EGR-1), followed by reprogramming of cardiac gene expression including up-

regulation of natriuretic peptides and a switch to fetal isoforms of contractile

proteins such as cardiac α-actin being substituted by skeletal α-actin (Frey, Katus,

Olson, & Hill, 2004; Izumo, Nadal-Ginard, & Mahdavi, 1988; Taegtmeyer et al.,

2010). Normally, only cardiac α-actin is expressed in the adult rat heart but the

expression of the fetal form, skeletal α-actin, is reactivated in the pathologically

stressed heart (Nadal-Ginard & Mahdavi, 1989). Mechanical loading induces the

expression of skeletal α-actin also in vitro in cultured cardiomyocytes (Komuro et

al., 1991; Sadoshima et al., 1992). Since Phactrs are a family of actin regulatory

proteins, the effect of Phactr1 overexpression on contractility proteins was studied.

The key finding emerging from this study is that Phactr1 overexpression changed

the skeletal α-actin to cardiac α-actin ratio both in vivo and in vitro. Phactr1

overexpression significantly elevated the skeletal α-actin to cardiac α-actin ratio

(1.5-fold, P<0.05) 3 days after Phactr1 gene delivery but the ratio was reduced (0.6-

fold, P<0.05) at 2 weeks in normal adult rat hearts (I Fig 3A and E). A similar effect

was seen in infarcted rat hearts where the skeletal to cardiac α-actin ratio was 50%

lower at 2 weeks after the MI (P<0.05) (Fig 7C). In agreement with in vivo studies

at 3 days, the skeletal α-actin to cardiac α-actin ratio significantly increased (1.8-

fold, P<0.001) in response to adenovirus-mediated Phactr1 overexpression for 48

hours in cultured NRVMs (I, Fig 9B). Phactr1 mRNA expression levels post-MI

and the effect of Phactr1overexpression on the skeletal α-actin to cardiac α-actin

ratio at 2 weeks in normal adult rat hearts and at 2 weeks after MI are shown in

Figure 5. The extractions were done from the whole LV anterior wall in order to

investigate mRNA and protein levels in the infarcted hearts. More detailed data of

the gene expression could have been gathered if the infarct region and the remote

non-infarcted area would have been separated, since the gene expression levels may

vary between these distinct areas.

55

Fig. 5. A) Phactr1 mRNA levels from the LV tissue samples 1 day, 2 weeks and 4 weeks

after MI. B) The effect of Phactr1 gene transfer on the skeletal α-actin (skα-A) to cardiac

α-actin (caα-A) ratio 2 weeks after Phactr1 gene transfer and C) 2 weeks after Phactr1

gene delivery and MI. mRNA levels are measured by RT-PCR. The results are expressed

as mean ± SEM. *P<0.05, **P<0.01 versus sham, LacZ or MI+LacZ (Student’s t-test).

56

5.3 SRF DNA binding activity is enhanced due to Phactr1

overexpression in vitro (I)

Increased DNA binding activity of SRF (1.7-fold, P<0.05) was observed due to

Phactr1 overexpression in cultured NRVMs (I, Fig 9D). SRF is a transcription

factor that binds to serum response elements (SREs) and is an important regulator

of many of the genes involved in cardiac function, including ANP and c-fos

(Argentin et al., 1994) as well as contractile proteins such as skeletal α-actin and

cardiac α-actin (Davey, Kelly, & Wildeman, 1995; Gustafson & Kedes, 1989).

Since Phactr1 overexpression increased DNA binding activity of SRF in cultured

NRVMs, SRF could be a potential mechanism triggering the change in the skeletal

α-actin to cardiac α-actin ratio. Moreover, other SRF target genes expressed and

known to have a function in heart, were investigated at the mRNA level. In addition

to the skeletal α-actin to cardiac α-actin ratio and the β-MHC to α-MHC ratio, c-

fos and corin mRNA levels changed due to Phactr1 overexpression in cultured

NRVMS (I, Fig 9C and 9E). Other analysed SRF target genes did not become

altered due to overexpression indicating that factors other than SRF mediate the

effects of overexpression on cardiac function. It is also possible that SRF interacts

with coactivators/corepressors that modify its regulatory function. MRTFs act as

activators of SRF (Mikhailov & Torrado, 2012). The protein levels of MRTF-A

from cultured NRVMs after Phactr1 overexpression were measured using Western

blot analysis (data not shown). However, Phactr1 overexpression induced no

significant changes in the MRTF-A protein levels. Therefore, more mechanistical

studies will be needed to clarify the effects of Phactr1 overexpression on actin

regulation in heart.

The immunohistochemical evaluation revealed the presence of strong

cytoplasmic and nuclear Phactr1 staining that localized to cardiomyocytes in

normal adult rat hearts (I Fig 2E) which is in agreement with previous findings

showing various subcellular localization patterns for Phactrs (P. B. Allen et al.,

2004; Huet et al., 2013; Sagara et al., 2003, 2009; Y. Zhang et al., 2012). Allain et

al. (2012) have reported increased PHACTR1 expression levels in HUVECs after

treatment with the vascular endothelial growth factor (VEGF) isoform (VEGF-

A165) (Allain et al., 2012), in fact, PHACTR1 seems to be essential for endothelial

cell function in HUVECs since PHACTR1 depletion increased apoptosis and

impaired tube formation (Jarray et al., 2011). In our study, however, Phactr1 did

not localize to the endocardial endothelium in rat hearts when evaluated by

immunohistochemistry. Furthermore, Phactr1 overexpression in normal adult rat

57

hearts exerted no influence on capillary density during the 2 week follow-up period

(I, Fig 5D). In addition, Phactr1 gene overexpression did not result in increased

apoptosis or cell proliferation as determined by TUNEL and Ki-67 stainings,

respectively (I Fig 5B-C) with stained apoptotic bodies and cells being counted

from the hot spot areas chosen from high power fields (40x) in each sample.

5.4 The effects of Phactr1 gene delivery on cardiac function and

structure in normal and infarcted hearts (I)

Next, the possible effects of local myocardial Phactr1 gene delivery on cardiac

function and structure were evaluated. Interestingly, Phactr1 gene transfer had no

statistically significant effects on LVEF or fractional shortening (FS) (I, Fig 6A-C)

when assessed by echocardiography 3 days, 1 week or 2 weeks after Phactr1 gene

delivery (I, summary of the data in Table 2). In addition, there was no difference in

the thickness of the interventricular septum (IVS) during diastole between LacZ-

and Phactr1-treated groups (I, Fig 6D). However, the IVS during systole was

slightly but statistically significantly (P<0.05) thicker in the Phactr1-treated group

at 3 days after Phactr1 overexpression when compared to the LacZ-treated group

(I, Fig 6E). There were no significant differences in EF, FS or LV dimensions in

Phactr1-treated animals after MI when compared to LacZ-treated group (I, Fig 6F-

I). There were no statistically significant changes observed in the extent of

myocardial fibrosis, apoptosis, cell proliferation and angiogenesis in normal rat

heart LV tissues after Phactr1 gene delivery (I, Fig 5A-D). In addition, Phactr1 gene

transfer after MI had no influence on fibrosis or apoptosis (I, Fig 7E-F).

Since Phactr1 acts as an actin regulator, some changes in LV structure and

function could be expected. However, adenoviral gene delivery resulted in only a

transient Phactr1 overexpression and limited the study to investigating the early

remodeling process. To monitor longer-term effects on cardiac function, adeno-

associated virus induced overexpression would be a better model. In addition,

transgenic or knockout animal models or gene silencing methods are needed.

5.5 MI associated PHACTR1 allele is not associated with cardiac

dysfunction in humans (I)

Since PHACTR1 SNP rs12526453 had been associated with early-onset MI

(Consortium, 2009), the effect of the human PHACTR1 risk allele on cardiac

function was studied. No significant association was observed between PHACTR1

58

SNP rs12526453 and diastolic dysfunction in age and sex adjusted additive model

where the genotypic distribution of rs12526453 was compared between subjects

having normal diastolic function or mild diastolic dysfunction and those with

significant diastolic dysfunction. In addition, the association between rs12526453

and systolic function was insignificant. This data is consistent with the results from

animal experiments. However, in end-stage failing hearts, PHACTR1 expression

was up-regulated at the protein level (2.8-fold, P<0.01) (I, Fig 1E), indicating that

further studies will be required to clarify in more detail the mechanisms regulating

PHACTR1 expression in human subjects.

Overall, in this study, MI associated PHACTR1 allele was not associated

significantly with cardiac function. However, PHACTR1 SNP rs12526453 has

earlier been associated with early-onset MI and the present study demonstrated that

Phactr1 regulates the skeletal α-actin to cardiac α-actin ratio in both healthy and

infarcted heart of adult rodents. Therefore, further studies with larger populations

will be needed to investigate the association of this allele with cardiac function.

Figure 6 is a schematic presentation of the potential mechanisms regulating

Phactr1 expression and the downstream targets of Phactr1.

59

Fig. 6. Schematic presentation of the potential mechanisms regulating Phactr1

expression and the downstream targets of Phactr1.

5.6 TSC-22 is up-regulated by multiple hypertrophic stimuli and

post-MI (II)

In original publication II, the expression of TSC-22 was examined in different

models of cardiac hypertrophy and heart failure. Since previous studies have shown

increased TSC-22 mRNA levels in old spontaneously hypertensive rats (SHRs)

(Rysa et al., 2005), the TSC-22 protein levels were measured in 12-to-20-month-

old SHR and their age-matched normotensive Wistar Kyoto (WKY) rats. TSC-22

protein levels were significantly higher in SHRs (1.6-fold, P<0.05) (II, Fig 1A).

60

Then, the effect of post-infarction myocardial remodeling on TSC-22 expression

was investigated in a model of acute MI. Induction of TSC-22 in the post-MI

remodeling process is rapid and persistent since LV TSC-22 mRNA levels were up-

regulated already on day 1 following the MI (4.1-fold, P<0.001) and a significant

increase was seen also 4 weeks after MI (1.9-fold, P<0.5) when compared to sham-

operated animals (II, Fig 1B). These results are in agreement with previous gene

expression profiling studies (Stanton et al., 2000). A similar induction was seen in

TSC-22 protein levels when assessed by Western blot analysis (II, Fig 1C).

Next, the effect of acute and chronic pressure overload on TSC-22 gene

expression was studied. Ang II infusion significantly increased LV TSC-22 mRNA

levels at 6 h (12.0-fold, P<0.001) and mRNA levels remained up-regulated for two

weeks. Administration of arginine vasopressin induced a significant increase in

TSC-22 already at 1 h, and after 4 h, TSC-22 mRNA levels were markedly elevated

(1.6-fold, P<0.05 and 5.5-fold, P<0.001, respectively) (II, Fig 2A-B). The Ang II-

induced changes in TSC-22 gene expression were completely abolished by the Ang

II type 1-receptor blocker, losartan (II, Fig 2C). A similar rapid up-regulation in

response to hemodynamic stress has been seen in the expression pattern of

immediate-early genes such as c-fos, c-jun and Egr-1 (Hoshijima & Chien, 2002).

The results are summarized in Figure 7.

61

Fig. 7. A) TSC-22 total protein levels analysed by Western blot from 12 and 20 months

old WKY and SHR hearts. *P<0.05 versus WKY (Student’s t-test). The effect of MI on rat

LV tissue TSC-22 mRNA levels B) measured by RT-PCR or Northern blot and TSC-22

protein levels C) analysed by Western blot. *P<0.05, **P<0.01, ***P<0.001 versus sham

(Student’s t-test). D) The effect of Ang II administration on rat LV TSC-22 mRNA levels

as assessed by Northern blot analysis. The results are expressed as mean ± SEM.

***P<0.001 versus control (Student’s t-test). Representative Western blots and Northern

blots are shown.

Altogether, these data in conjunction with the previous findings (Rysa et al., 2005;

Stanton et al., 2000) imply that TSC-22 up-regulation is rapid and sustainable

throughout the time course of cardiac remodeling. Despite the fact that TSC-22 was

also expressed in human hearts (data not shown) both the mRNA and protein levels

remained unchanged in failing hearts when compared to the control hearts. One

possible explanation, in addition to differences between the species, might be that

elevated TSC-22 levels are needed for controlling transcriptional activity during

the remodeling process but are no longer evident in the failing heart.

62

5.7 Reduced TSC-22 expression levels after long-term treatment of

heart failure with metoprolol and hypertension with metoprolol

or losartan (II)

Beta-blockers and AT1R antagonists are important groups of drugs in the treatment

of hypertensive hypertrophy and myocardial infarction induced heart failure.

Increased TGF-β expression levels have been associated with cardiac hypertrophy

and fibrosis in different animal models of HF (J. M. Li & Brooks, 1997; Yao Sun,

Zhang, Zhang, & Ramires, 1998). The association between TGF-β1 and both the

renin-angiotensin-aldosterone and the beta-adrenergic systems have been

suggested in several studies (Brand & Schneider, 1995; Bujak & Frangogiannis,

2007; Rosenkranz, 2004). The hypertrophic response to Ang II treatment was

abolished in TGF-β1-deficient mice, indicating that the effects of Ang II on cardiac

remodelling are at least partly mediated by TGF-β1 (Schultz et al., 2002).

In original article II, post-infarction treatment with metoprolol improved LV

function and attenuated LV remodelling in rats as previously described (Mustonen

et al., 2010). The LV TSC-22 gene expression levels significantly increased after

MI (2.8-fold, P<0.001) and metoprolol treatment for 2 weeks reduced TSC-22

mRNA levels by 32% (II, Fig 3A). In addition, both the beta-blocker metoprolol

and the AT1R antagonist losartan reduced TSC-22 gene expression in rats in a

chronic pressure overload model (II, Fig 3B), which could be a result of the

beneficial effects of these drugs on the development of cardiac fibrosis (Brouri et

al., 2004; De Carvalho Frimm, Sun, & Weber, 1997; Schieffer et al., 1994). By

activating the AT1R, endogenous Ang II plays a role in the formation of fibrosis

partly by stimulating the synthesis of TGF-β1 (Brand & Schneider, 1995; Bujak &

Frangogiannis, 2007; Rosenkranz, 2004). In SHRs, cardiac hypertrophy has been

associated with increased fibrosis (Boluyt et al., 2005; Kuoppala et al., 2003).

However, it is likely that effects of losartan on TSC-22 expression are mediated by

multiple mechanisms since losartan reduced TSC-22 expression also in response to

acute pressure overload.

5.8 Hypertrophic stimuli inducible signalling pathways regulating

TSC-22 gene expression (II)

The mechanisms and signalling pathways regulating cardiac TSC-22 expression

have not been extensively studied. In this study, the hypertrophic agonist ET-1

increased both the TSC-22 mRNA and protein levels in cultured NRVMs. TSC-22

63

mRNA levels were significantly up-regulated at 4 h and were still up-regulated at

24 h. A significant increase in TSC-22 protein levels was detected at 12 h with

maximal expression level at 24 h (II, Fig 4A-B). Since MAPK pathways are known

to be activated in response to hypertrophic stimuli, including ET-1 (Rose et al.,

2010), cultured NRVMs were treated with ET-1 for 1 h and exposed to two MAPK

inhibitors, PD98059 or SB203580. Neither the ERK inhibitor PD98059 nor the

inhibitor of both p38α and p38β, SB203580, exerted any significant effect on ET-1

–induced up-regulation of TSC-22 mRNA levels (II, Fig 4C-D). However,

SB203580 alone was able to increase TSC-22 mRNA levels (II, Fig 4D).

In addition, adenoviruses expressing constitutively active MAP kinase kinase

3b(E) (MKK3bE) and wild-type p38α which cause p38 MAPK-overexpression

were injected into the LV free wall of adult Sprague-Dawley rats in order to

evaluate whether p38 MAPKs regulate cardiac TSC-22 gene expression in vivo.

Overexpression of p38 MAPK significantly increased TSC-22 expression both at

the mRNA and protein level in vivo (II, Fig 4E-F), which suggests that TSC-22 is

up-regulated in the heart via the p38 MAPK pathway in cardiac remodelling after

MI and in hypertensive hypertrophy. The interpretation of the results is difficult

since the p38 inhibitor SB203580 did not have any effect on ET-1 induced TSC-22

up-regulation in vitro, which might be a consequence of the fact that SB203580

alone was able to induce an elevation of TSC-22 gene expression.

5.9 The effect of TSC-22 overexpression on cardiac gene

expression in vivo (II)

Next, the in vivo gene transfer protocol was established in order to investigate the

direct myocardial effects of TSC-22 on cardiac gene expression. Adenovirus

mediated gene transfer locally increased TSC-22 mRNA levels in the adult rat LV,

starting at three days and lasting up to 2 weeks (7.7-fold, P<0.001 and 1.7-fold,

P<0.01, respectively) following the injections (II, Fig 6A). TSC-22 protein levels

were significantly up-regulated at 3 days (76.1-fold, P<0.001) after gene transfer

when compared to LacZ-injected control LVs (II, Fig 6B). No significant changes

in LV dimensions or cardiac function were seen at either 3 days or 2 weeks after

TSC-22 gene delivery was assessed by echocardiography (II, table 2).

TSC-22 and LacZ overexpressing hearts were immunostained to evaluate the

localization of TSC-22. In agreement with a previous study (Kato et al., 2010),

TSC-22 overexpressing hearts displayed both the nuclear and cytoplasmic

64

positivity in TSC-22 immunostaining (II, Fig 6C), indicating that TSC-22 could act

at the transcriptional level.

One key finding emerging from the study was that TSC-22 gene delivery in the

adult rat heart was able to regulate the synthesis of collagen. TSC-22

overexpression induced a significant up-regulation of Col3a1 gene expression and

a similar trend was seen in Col1a1 mRNA levels (II, Fig 6D). In previous studies,

TSC-22 has been associated with collagen expression since in cultured rat

fibroblasts, lentivirus-mediated TSC-22 overexpression enhanced TGF-β –induced

expression of collagen I (Yan, 2011), and in kidney cells, col1a2 has been shown

to be regulated by TSC-22 (Kato et al., 2010). Fibrosis is associated with altered

synthesis and degradation of type I and III collagens. This study, in conjunction

with previous findings (Kato et al., 2010; Yan, 2011), suggests that TSC-22 has a

role as a regulator of collagen synthesis during cardiac remodelling. According to

Yan, TSC-22 increases also the expression of PAI-1 and fibronectin in rat

fibroblasts (Yan, 2011). However, in this study, neither PAI-1 nor fibronectin

expression changed 2 weeks after TSC-22 gene delivery, indicating there might be

cell type specific differences in the regulation of TSC-22 gene expression.

There are some limitations in the study that need to be considered. The results

of TSC-22 gene delivery on collagen expression were assessed at the level of

transcriptional expression. Since gene expression is regulated by several

posttranscriptional and posttranslational mechanisms, the details of the expression

at the protein level need to be clarified. It would also be interesting to study the

influence of TSC-22 gene delivery on fibrosis by performing an

immunohistochemical analysis. Overall, here the molecular mechanisms of cardiac

hypertrophy and heart failure were investigated, and consequently may help in

finding new therapeutic interventions for these diseases. However, it has to be

recalled that the key findings of the study were derived from animal models, and

the translation of this kind of data to the clinic is not straightforward.

5.10 Rd3l gene expression and tissue distribution (III)

In a previous DNA microarray study, Rd3l was one of the ESTs whose expression

levels changed significantly with the transition from LVH to diastolic HF in

hypertrophied hearts of SHRs (Rysa et al., 2005). In agreement with the results

obtained by the gene expression profiling study, 12-month-old SHRs showed

higher Rd3l mRNA levels (2.1-fold, P<0.01) when compared to age-matched

normotensive WKY rats but reduced to the levels of WKY rats at the age of 20-

65

months (III, Fig 2A). When the Rd3l gene expression in Sprague-Dawley rat tissues

was examined, the highest Rd3l gene expression levels were detected in heart, eye,

thymus and brain as measured by quantitative RT-PCR analysis.

5.11 Rd3l expression levels in experimental models of hypertension

and MI in rats (III)

Cardiac Rd3l expression was studied in animal models of cardiac remodelling.

After an Ang II infusion, Rd3l expression levels were reduced starting at 6 h and

remaining low for up to 2 weeks, with the lowest levels (0.3-fold, P<0.001) being

detected at 12 h after Ang II administration (III, Fig 3A). Rd3l mRNA levels were

significantly reduced at day 1 and at 2 weeks (0.5-fold, P<0.01 and 0.6-fold,

P<0.05, respectively) post-MI (III, Fig 3B). Altogether, these results suggest that

Rd3l could have a role in LV remodelling process.

5.12 Generation of the Rd3l knockout mouse line (III)

The function of Rd3l is completely unknown. A conventional Rd3l knockout mouse

line was generated in order to characterize Rd3l in more detail. The exons 2 and 3

of the Rd3l gene and the intron between them were replaced by the LacZ-neo

cassette (III, Fig 1A) by homologous recombination in ES cells. Southern blot

analysis (III, Fig 1B) and PCR (III, Fig 1C) were used to confirm the correct

targeting of the Rd3l gene. There were no obvious phenotypic differences observed

in heterozygous or knockout mice relative to wild-type mice. Ear biopsies from the

mice were genotyped by PCR and confirmed in a few samples by Southern blot

analysis (III, Fig 1B-C).

5.13 The effects of hemodynamic pressure overload on Rd3l-

deficient mice (III)

Next, the absence of any effects attributable to Rd3l on cardiac function and

structure was evaluated in a model of hemodynamic pressure overload. Six months

old Rd3l knockout mice and their age-matched wild-type mice, which were used

as a control, were subjected to TAC for 4 weeks and their cardiac structure and

function were assessed by echocardiography. A loss of the Rd3l gene induced

abnormalities in the function of the heart in response to pressure overload. Pressure

overload induced a reduction of the LV ejection fraction both in wild type (15%)

66

and knockout (11%) mice, although this was only statistically significant in wild

type mice (P<0.05) (III, Fig 4A). The TAC procedure did not result in a significant

reduction in FS in either of the groups (III, Fig 4B). An increase in interventricular

septum (IVS) dimension during diastole due to TAC was significant both in wild

type and Rd3l knockout mice although the increase was smaller in Rd3l null mice

(46%, P<0.001 vs. 21%, P<0.01, respectively) (III, Fig 4C). The left ventricular

posterior wall (LVPW) dimension during diastole increased both in wild type and

Rd3l knockout mice due to the TAC operation although this increase was greater

in the Rd3l knockout mice (29%, P<0.01 vs. 40%, P<0.001) (III, Fig 4D). There

were no significant changes in LV volume during diastole between the groups due

to TAC (III, Fig 4E). Rd3l null mice had a shorter isovolumic relaxation time (IVRT)

(III, Fig 4F).

The inactivation of Rd3l gene affected the morphology of the heart when

animals were subjected to TAC induced pressure overload. TAC induced a

significant increase (P<0.05) in the cardiomyocyte size in LV but not in the septum

of the Rd3l knockout mice when compared to sham operated wild-type and

knockout controls (III, Fig 5A-B). In addition, there was a trend for increased LV

wall thickness and fibrosis in Rd3l mice subjected to TAC but the changes were not

statistically significant (III, Fig 5C-D). These results indicate that the absence of

Rd3l induces cardiomyocyte hypertrophy and fibrosis in loaded heart.

Finally, the effect of pressure overload on cardiac gene expression was

analysed. LV Rd3l mRNA levels were reduced significantly (50%, P<0.05) in wild

type mice due to the TAC procedure whereas Rd3l mRNA levels were undetectable

in Rd3l-knockout mice when analysed by RT-PCR (III, Fig 1D). Consistent with

previous study (Szabo et al., 2014), both ANP and BNP levels were significantly

up-regulated (6.3-fold, P<0.05 and 2.4-fold, P<0.01, respectively) in the wild-type

TAC group when compared to the wild-type sham group. Interestingly, these

increases were not significant (ANP) or were more moderate (BNP) (1.9-fold,

P<0.05) in the Rd3l knockout TAC group when compared to the sham groups (III,

Fig 6A-B). The skeletal α-actin to cardiac α-actin ratio increased significantly

(23.4-fold, P<0.01) in the wild-type TAC group (III, Fig 6C, E, G). However, this

change was not significant in the knockout TAC group. TAC resulted in an elevated

β-MHC to α-MHC ratio in both the wild-type (3.8-fold, P<0.01) and knockout (3.4-

fold, P<0.01) groups (III, Fig 6D, F and H). These results suggest that a loss of

Rd3l gene is associated with dysregulated activation of the fetal gene program in

the heart.

67

Natriuretic peptides ANP and BNP are cardioprotective hormones that cause

vasodilatation and exert hypotensive actions (Pandey, 2011; Ruskoaho et al., 1991).

Hemodynamic overload and cardiac remodelling induce the synthesis and secretion

of natriuretic peptides in the heart and they are used as diagnostic markers in HF

(Kerkelä et al., 2015). ANP binds to guanylyl cyclase/natriuretic peptide receptor-

A (GC-A/NPRA) which induces an elevation of intracellular cyclic guanosine

monophosphate (cGMP) and an inhibition of cardiac hypertrophy and fibrosis

(Pandey, 2011). The inhibition of cGMP hydrolysis by phosphodiesterase 5A

(PDE5A) has been shown to exert a positive impact on cardiac function (Kass,

2012). The PDE5 inhibitor, sildenafil, was able to suppress hypertrophy in mice

with TAC induced pressure-overload (Takimoto et al., 2005). PDE5 overexpression

led to adverse LV remodelling post-MI (Pokreisz et al., 2009). In addition,

myocyte-specific PDE5 overexpression impaired cardiac function and increased

fibrosis when the mice were subjected to TAC induced pressure overload but the

effects were reversed by PDE5 inhibition (M. Zhang et al., 2010).

RD3 is an important paralog of Rd3l. RD3 proteins are involved in the

regulation of guanylyl cyclase activity and cGMP levels in retina (Peshenko et al.,

2011). Since the ANP response in Rd3l null mice was attenuated, our results

suggest that the absence of Rd3l may induce a smaller increase in the levels of

cGMP in response to pressure overload. A loss of Rd3l function in pressure

overloaded heart could lead to increased myocyte hypertrophy and cardiac

dysfunction due to reduced cGMP levels in myocardium. However, further studies

will be required to clarify the function of Rd3l in cardiac hypertrophy.

In addition, the limitations of gene modification technology must be taken into

consideration. The phenotype produced by genetic manipulation through

constitutive knockout may not truly reflect the functional role of target gene in a

studied pathological condition. Secondary, compensatory, or off-target effects can

lead to misinterpretations. Non-coding intron regions, when located between the

target exons, are also replaced by the targeting construct and these might contain

important regulatory areas for other genes.

68

69

6 Summary and conclusions

This study characterized the roles of three novel factors, Phactr1, TSC-22 and Rd3l,

in different experimental models of cardiac hypertrophy and failure.

1. Phactr1 expression was rapidly down-regulated post-MI in the left ventricles,

likely due to the direct mechanical stretching of cardiac myocytes. Phactr1

overexpression changed the skeletal α-actin to cardiac α-actin ratio in vivo both

in healthy and in infarcted rat hearts, as well as in vitro in cultured

cardiomyocytes. Phactr1 could regulate the switch in the actin isoform due to

SRF since overexpression of Phactr1 led to enhanced SRF DNA binding

activity.

2. A myocardial injury and multiple hypertrophic stimuli induced a rapid up-

regulation of TSC-22 in rat heart. TSC-22 overexpression increased col3a1

gene expression in adult rat heart, suggesting that TSC-22 could regulate

collagen synthesis in heart and could be a target for preventing the formation

of cardiac fibrosis.

3. Hypertrophied hearts of SHRs expressed lower levels of Rd3l mRNA with

aging. Rd3l expression was also reduced in Ang II –induced pressure overload

and post-MI. The absence of Rd3l led to increased cardiomyocyte hypertrophy

and cardiac dysfunction in response to the TAC-induced pressure overload,

suggesting that Rd3l may be involved in the pathological hypertrophic

response in the heart.

70

71

References

Aherrahrou, R., Aherrahrou, Z., Schunkert, H., & Erdmann, J. (2017). Coronary artery disease associated gene Phactr1 modulates severity of vascular calcification in vitro. Biochemical and Biophysical Research Communications, 491(2), 396–402. http://doi.org/10.1016/j.bbrc.2017.07.090

Ali, S. R., Ranjbarvaziri, S., Talkhabi, M., Zhao, P., Subat, A., Hojjat, A., … Ardehali, R. (2014). Developmental Heterogeneity of Cardiac Fibroblasts Does Not Predict Pathological Proliferation and Activation. Circulation Research, 115(7), 625–635. http://doi.org/10.1161/CIRCRESAHA.115.303794

Allain, B., Jarray, R., Borriello, L., Leforban, B., Dufour, S., Liu, W. Q., … Lepelletier, Y. (2012). Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells. Cellular Signalling, 24(1), 214–223. http://doi.org/10.1016/j.cellsig.2011.09.003; 10.1016/j.cellsig.2011.09.003

Allen, B. G., Phuong, L. L., Farhat, H., & Chevalier, D. (2003). Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes. Canadian Journal of Physiology and Pharmacology, 81(2), 95–104. http://doi.org/10.1139/y02-155

Allen, P. B., Greenfield, A. T., Svenningsson, P., Haspeslagh, D. C., & Greengard, P. (2004). Phactrs 1-4: A family of protein phosphatase 1 and actin regulatory proteins. Proceedings of the National Academy of Sciences, 101(18), 7187–7192.

Arad, M., Penas-Lado, M., Monserrat, L., Maron, B. J., Sherrid, M., Ho, C. Y., … Seidman, C. E. (2005). Gene mutations in apical hypertrophic cardiomyopathy. Circulation, 112(18), 2805–11. http://doi.org/10.1161/CIRCULATIONAHA.105.547448

Arciero, T. J., Jacobsen, S. J., Reeder, G. S., Frye, R. L., Weston, S. A., Killian, J. M., & Roger Vr, V. éronique L. (2004). Temporal trends in the incidence of coronary disease. The American Journal of Medicine, 117(4), 228–33. http://doi.org/10.1016/j.amjmed.2004.04.008

Argentin, S., Ardati, A., Tremblay, S., Lihrmann, I., Robitaille, L., Drouin, J., & Nemer, M. (1994). Developmental stage-specific regulation of atrial natriuretic factor gene transcription in cardiac cells. Molecular and Cellular Biology, 14(1), 777–90. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8264645

Arsenian, S., Weinhold, B., Oelgeschläger, M., Rüther, U., & Nordheim, A. (1998). Serum response factor is essential for mesoderm formation during mouse embryogenesis. The EMBO Journal, 17(21), 6289–99. http://doi.org/10.1093/emboj/17.21.6289

Asbun, J., & Villarreal, F. J. (2006). The Pathogenesis of Myocardial Fibrosis in the Setting of Diabetic Cardiomyopathy. Journal of the American College of Cardiology, 47(4), 693–700. http://doi.org/10.1016/j.jacc.2005.09.050

Ashrafian, H., McKenna, W. J., & Watkins, H. (2011). Disease Pathways and Novel Therapeutic Targets in Hypertrophic Cardiomyopathy. Circulation Research, 109(1), 86–96. http://doi.org/10.1161/CIRCRESAHA.111.242974

72

Azevedo, P. S., Minicucci, M. F., Santos, P. P., Paiva, S. A. R., & Zornoff, L. A. M. (2013). Energy Metabolism in Cardiac Remodeling and Heart Failure. Cardiology in Review, 21(3), 135–140. http://doi.org/10.1097/CRD.0b013e318274956d

Balza, R. O., & Misra, R. P. (2006). Role of the serum response factor in regulating contractile apparatus gene expression and sarcomeric integrity in cardiomyocytes. The Journal of Biological Chemistry, 281(10), 6498–510. http://doi.org/ 10.1074/jbc.M509487200

Bankovic, J., Stojsic, J., Jovanovic, D., Andjelkovic, T., Milinkovic, V., Ruzdijic, S., & Tanic, N. (2010). Identification of genes associated with non-small-cell lung cancer promotion and progression. Lung Cancer, 67(2), 151–159. http://doi.org/ 10.1016/j.lungcan.2009.04.010

Barry, S. P., Davidson, S. M., & Townsend, P. A. (2008). Molecular regulation of cardiac hypertrophy. The International Journal of Biochemistry & Cell Biology, 40(10), 2023–2039. http://doi.org/10.1016/j.biocel.2008.02.020; 10.1016/j.biocel.2008.02.020

Beaudoin, M., Gupta, R. M., Won, H.-H., Lo, K. S., Do, R., Henderson, C. A., … Lettre, G. (2015). Myocardial Infarction-Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary Arteries. Arteriosclerosis, Thrombosis, and Vascular Biology, 35(6), 1472–9. http://doi.org/10.1161/ATVBAHA.115.305534

Berk, B. C., Fujiwara, K., & Lehoux, S. (2007). ECM remodeling in hypertensive heart disease. The Journal of Clinical Investigation, 117(3), 568–75. http://doi.org/10.1172/JCI31044

Bernardo, B. C., Weeks, K. L., Pretorius, L., & McMullen, J. R. (2010). Molecular distinction between physiological and pathological cardiac hypertrophy: Experimental findings and therapeutic strategies. Pharmacology and Therapeutics, 128, 191–227. http://doi.org/10.1016/j.pharmthera.2010.04.005

Biernacka, A., & Frangogiannis, N. G. (2011). Aging and Cardiac Fibrosis. Aging and Disease, 2(2), 158–173. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/21837283

Boheler, K. R., Carrier, L., de la Bastie, D., Allen, P. D., Komajda, M., Mercadier, J. J., & Schwartz, K. (1991). Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. Journal of Clinical Investigation, 88(1), 323–330. http://doi.org/10.1172/JCI115295

Boluyt, M. O., Robinson, K. G., Meredith, A. L., Sen, S., Lakatta, E. G., Crow, M. T., … Bing, O. H. (2005). Heart failure after long-term supravalvular aortic constriction in rats. American Journal of Hypertension, 18(2 Pt 1), 202–212. http://doi.org/S0895-7061(04)01017-9 [pii]

Brand, T., & Schneider, M. D. (1995). The TGF beta superfamily in myocardium: ligands, receptors, transduction, and function. Journal of Molecular and Cellular Cardiology, 27(1), 5–18. http://doi.org/S0022-2828(08)80003-X [pii]

Brooks, W. W., & Conrad, C. H. (2000). Myocardial fibrosis in transforming growth factor beta(1)heterozygous mice. Journal of Molecular and Cellular Cardiology, 32(2), 187–95. http://doi.org/10.1006/jmcc.1999.1065

73

Brouri, F., Hanoun, N., Mediani, O., Saurini, F., Hamon, M., Vanhoutte, P. M., & Lechat, P. (2004). Blockade of β1- and desensitization of β 2-adrenoceptors reduce isoprenaline-induced cardiac fibrosis. European Journal of Pharmacology, 485, 227–234. http://doi.org/10.1016/j.ejphar.2003.11.063

Brown, R. D., Ambler, S. K., Mitchell, M. D., & Long, C. S. (2004). THE CARDIAC FIBROBLAST: Therapeutic Target in Myocardial Remodeling and Failure. http://dx.doi.org/10.1146/annurev.pharmtox.45.120403.095802.

Bueno, O. F., De Windt, L. J., Tymitz, K. M., Witt, S. A., Kimball, T. R., Klevitsky, R., … Molkentin, J. D. (2000). The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. The EMBO Journal, 19(23), 6341–6350. http://doi.org/10.1093/emboj/19.23.6341

Bujak, M., & Frangogiannis, N. G. (2007). The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovascular Research, 74(2), 184–195. http://doi.org/S0008-6363(06)00439-1 [pii]

Burchfield, J. S., Xie, M., & Hill, J. A. (2013). Pathological Ventricular Remodeling: Mechanisms: Part 1 of 2. Circulation, 128(4), 388–400. http://doi.org/10.1161/CIRCULATIONAHA.113.001878

Cabassi, A., & Miragoli, M. (2017). Altered Mitochondrial Metabolism and Mechanosensation in the Failing Heart: Focus on Intracellular Calcium Signaling. International Journal of Molecular Sciences, 18(7), 1487. http://doi.org/ 10.3390/ijms18071487

Cahill, T. J., Ashrafian, H., & Watkins, H. (2013). Genetic cardiomyopathies causing heart failure. Circulation Research, 113(6), 660–75. http://doi.org/10.1161/ CIRCRESAHA.113.300282

Cappola, T. P., Li, M., He, J., Ky, B., Gilmore, J., Qu, L., … Dorn, G. W. (2010). Common variants in HSPB7 and FRMD4B associated with advanced heart failure. Circulation. Cardiovascular Genetics, 3(2), 147–54. http://doi.org/10.1161/ CIRCGENETICS.109.898395

Chaponnier, C., & Gabbiani, G. (2004). Pathological situations characterized by altered actin isoform expression. The Journal of Pathology, 204(4), 386–395. http://doi.org/ 10.1002/path.1635

Chien, K. R., Knowlton, K. U., Zhu, H., & Chien, S. (1991). Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB Journal�: Official Publication of the Federation of American Societies for Experimental Biology, 5(15), 3037–46. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1835945

Chiu, C.-Z., Wang, B.-W., Chung, T.-H., & Shyu, K.-G. (2010). Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes. Clinical Science (London, England�: 1979), 119(7), 273–82. http://doi.org/10.1042/CS20100084

Choi, S. J., Moon, J. H., Ahn, Y. W., Ahn, J. H., Kim, D. U., & Han, T. H. (2005). Tsc-22 enhances TGF-beta signaling by associating with Smad4 and induces erythroid cell differentiation. Molecular and Cellular Biochemistry, 271(1-2), 23–28.

74

Consortium, M. I. G. (2009). Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants. Nature Genetics [Nat.Genet.].Vol.41, 41(3), 334–341.

Crawford, K., Flick, R., Close, L., Shelly, D., Paul, R., Bove, K., … Lessard, J. (2002). Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and die during the neonatal period. Molecular and Cellular Biology, 22(16), 5887–96. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12138199

Cullingford, T. E., Markou, T., Fuller, S. J., Giraldo, A., Pikkarainen, S., Zoumpoulidou, G., … Sugden, P. (2008). Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes. Genome Biology, 9(2), R32. http://doi.org/10.1186/gb-2008-9-2-r32

Davey, H. W., Kelly, J. K., & Wildeman, A. G. (1995). The nucleotide sequence, structure, and preliminary studies on the transcriptional regulation of the bovine alpha skeletal actin gene. DNA and Cell Biology, 14(7), 609–618.

Davis, R. J. (1999). Signal transduction by the c-Jun N-terminal kinase. Biochemical Society Symposium, 64, 1–12. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10207617

De Carvalho Frimm, C., Sun, Y., & Weber, K. T. (1997). Angiotensin II receptor blockade and myocardial fibrosis of the infarcted rat heart. The Journal of Laboratory and Clinical Medicine, 129, 439–446.

Debette, S., Kamatani, Y., Metso, T. M., Kloss, M., Chauhan, G., Engelter, S. T., … CADISP group. (2015). Common variation in PHACTR1 is associated with susceptibility to cervical artery dissection. Nature Genetics, 47(1), 78–83. http://doi.org/10.1038/ng.3154

Dobaczewski, M., Chen, W., & Frangogiannis, N. G. (2011). Transforming growth factor (TGF)-beta signaling in cardiac remodeling. Journal of Molecular and Cellular Cardiology, 51(4), 600–606. http://doi.org/10.1016/j.yjmcc.2010.10.033 [doi]

Dobaczewski, M., Gonzalez-Quesada, C., & Frangogiannis, N. G. (2010). The extracellular matrix as a modulator of the inflammatory and reparative response following myocardial infarction. Journal of Molecular and Cellular Cardiology, 48, 504–11. http://doi.org/10.1016/j.yjmcc.2009.07.015

Dobens, L. L., Hsu, T., Twombly, V., Gelbart, W. M., Raftery, L. A., & Kafatos, F. C. (1997). The Drosophila bunched gene is a homologue of the growth factor stimulated mammalian TSC-22 sequence and is required during oogenesis. Mechanisms of Development, 65(1-2), 197–208.

Dobens, L. L., Peterson, J. S., Treisman, J., & Raftery, L. A. (2000). Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary. Development (Cambridge, England), 127(4), 745–54. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/10648233

Doenst, T., Nguyen, T. D., & Abel, E. D. (2013). Cardiac metabolism in heart failure: implications beyond ATP production. Circulation Research, 113(6), 709–24. http://doi.org/10.1161/CIRCRESAHA.113.300376

75

Dohrmann, C. E., Belaoussoff, M., & Raftery, L. A. (1999). Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development. Mechanisms of Development, 84(1-2), 147–51. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10473130

Drawnel, F. M., Archer, C. R., & Roderick, H. L. (2013). The role of the paracrine/autocrine mediator endothelin-1 in regulation of cardiac contractility and growth. British Journal of Pharmacology, 168(2), 296–317. http://doi.org/10.1111/j.1476-5381.2012.02195.x

Dubé, J. B., & Hegele, R. A. (2013). Genetics 100 for cardiologists: basics of genome-wide association studies. The Canadian Journal of Cardiology, 29(1), 10–7. http://doi.org/10.1016/j.cjca.2012.10.011

Dweck, M. R., Boon, N. A., & Newby, D. E. (2012). Calcific Aortic Stenosis: A Disease of the Valve and the Myocardium. Journal of the American College of Cardiology, 60(19), 1854–1863. http://doi.org/10.1016/j.jacc.2012.02.093

Ekim Üstünel, B., Friedrich, K., Maida, A., Wang, X., Krones-Herzig, A., Seibert, O., … Herzig, S. (2016). Control of diabetic hyperglycaemia and insulin resistance through TSC22D4. Nature Communications, 7, 13267. http://doi.org/10.1038/ncomms13267

Esposito, G., Prasad, S. V, Rapacciuolo, A., Mao, L., Koch, W. J., & Rockman, H. A. (2001). Cardiac overexpression of a G(q) inhibitor blocks induction of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase activity in in vivo pressure overload. Circulation, 103(10), 1453–8. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/11245652

Farghaian, H., Chen, Y., Fu, A. W., Fu, A. K., Ip, J. P., Ip, N. Y., … Cole, A. R. (2011). Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm. The Journal of Biological Chemistry, 286(22), 19724–19734. http://doi.org/10.1074/jbc.M110.205781 [doi]

Fils-Aime, N., Dai, M., Guo, J., El-Mousawi, M., Kahramangil, B., Neel, J. C., & Lebrun, J. J. (2013). MicroRNA-584 and the protein phosphatase and actin regulator 1 (PHACTR1), a new signaling route through which transforming growth factor-beta Mediates the migration and actin dynamics of breast cancer cells. The Journal of Biological Chemistry, 288(17), 11807–11823. http://doi.org/10.1074/jbc.M112.430934; 10.1074/jbc.M112.430934

Force, T., Michael, A., Kilter, H., & Haq, S. (2002). Stretch-activated pathways and left ventricular remodeling. Journal of Cardiac Failure, 8, S351–S358. http://doi.org/10.1054/jcaf.2002.129272

Frangogiannis, N. G. (2014). The inflammatory response in myocardial injury, repair, and remodelling. Nature Reviews Cardiology, 11(5), 255–265. http://doi.org/10.1038/ nrcardio.2014.28

Frangogiannis, N. G., Michael, L. H., & Entman, M. L. (2000). Myofibroblasts in reperfused myocardial infarcts express the embryonic form of smooth muscle myosin heavy chain (SMemb). Cardiovascular Research, 48(1), 89–100. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11033111

76

Frangogiannis, N. G., Perrard, J. L., Mendoza, L. H., Burns, A. R., Lindsey, M. L., Ballantyne, C. M., … Entman, M. L. (1998). Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion. Circulation, 98(7), 687–98. http://doi.org/10.1161/01.cir.98.7.687

Fredj, S., Bescond, J., Louault, C., & Potreau, D. (2005). Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation. Journal of Cellular Physiology, 202(3), 891–899. http://doi.org/10.1002/jcp.20197

Freilinger, T., Anttila, V., de Vries, B., Malik, R., Kallela, M., Terwindt, G. M., … Consortium, I. H. G. (2012). Genome-wide association analysis identifies susceptibility loci for migraine without aura. Nature Genetics, 44(7), 777–782. http://doi.org/10.1038/ng.2307; 10.1038/ng.2307

Frey, N., Katus, H. A., Olson, E. N., & Hill, J. A. (2004). Hypertrophy of the heart: a new therapeutic target? Circulation, 109(13), 1580–1589. http://doi.org/10.1161/ 01.CIR.0000120390.68287.BB

Gardner, D. G. (2003). Natriuretic peptides: markers or modulators of cardiac hypertrophy? Trends in Endocrinology & Metabolism, 14(9), 411–416. http://doi.org/10.1016/S1043-2760(03)00113-9

Garner, I., Minty, A. J., Alonso, S., Barton, P. J., & Buckingham, M. E. (1986). A 5’ duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue. The EMBO Journal, 5(10), 2559–67. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/3023046

Goff, D. C., Nichaman, M. Z., Chan, W., Ramsey, D. J., Labarthe, D. R., & Ortiz, C. (1997). Greater incidence of hospitalized myocardial infarction among Mexican Americans than non-Hispanic whites. The Corpus Christi Heart Project, 1988-1992. Circulation, 95(6), 1433–40. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9118510

Goldberg, R. J., Yarzebski, J., Lessard, D., & Gore, J. M. (1999). A two-decades (1975 to 1995) long experience in the incidence, in-hospital and long-term case-fatality rates of acute myocardial infarction: a community-wide perspective. Journal of the American College of Cardiology, 33(6), 1533–9. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/10334419

Goodwin, S., McPherson, J. D., & McCombie, W. R. (2016). Coming of age: ten years of next-generation sequencing technologies. Nature Reviews. Genetics, 17(6), 333–51. http://doi.org/10.1038/nrg.2016.49

Gudmundsdottir, H., Høieggen, A., Stenehjem, A., Waldum, B., & Os, I. (2012). Hypertension in women: latest findings and clinical implications. Therapeutic Advances in Chronic Disease, 3(3), 137–46. http://doi.org/10.1177/2040622312438935

Gustafson, T. A., & Kedes, L. (1989). Identification of multiple proteins that interact with functional regions of the human cardiac alpha-actin promoter. Molecular and Cellular Biology, 9(8), 3269–3283.

77

Hager, J., Kamatani, Y., Cazier, J. B., Youhanna, S., Ghassibe-Sabbagh, M., Platt, D. E., … Consortium, F. (2012). Genome-wide association study in a Lebanese cohort confirms PHACTR1 as a major determinant of coronary artery stenosis. PloS One, 7(6), e38663. http://doi.org/10.1371/journal.pone.0038663; 10.1371/journal.pone.0038663

Hama, N., Itoh, H., Shirakami, G., Nakagawa, O., Suga, S., Ogawa, Y., … Hashimoto, Y. (1995). Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction. Circulation, 92(6), 1558–64. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7664440

Hannenhalli, S., Putt, M. E., Gilmore, J. M., Wang, J., Parmacek, M. S., Epstein, J. A., … Cappola, T. P. (2006). Transcriptional genomics associates FOX transcription factors with human heart failure. Circulation, 114(12), 1269–76. http://doi.org/10.1161/CIRCULATIONAHA.106.632430

Haq, S., Choukroun, G., Lim, H., Tymitz, K. M., del Monte, F., Gwathmey, J., … Molkentin, J. D. (2001). Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation, 103(5), 670–7. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11156878

Harpster, M. H., Bandyopadhyay, S., Thomas, D. P., Ivanov, P. S., Keele, J. A., Pineguina, N., … Stayton, M. M. (2006). Earliest changes in the left ventricular transcriptome postmyocardial infarction. Mammalian Genome�: Official Journal of the International Mammalian Genome Society, 17(7), 701–15. http://doi.org/10.1007/s00335-005-0120-1

Harris, I. S., Zhang, S., Treskov, I., Kovacs, A., Weinheimer, C., & Muslin, A. J. (2004). Raf-1 Kinase Is Required for Cardiac Hypertrophy and Cardiomyocyte Survival in Response to Pressure Overload. Circulation, 110(6), 718–723. http://doi.org/10.1161/ 01.CIR.0000138190.50127.6A

Heidecker, B., Kasper, E. K., Wittstein, I. S., Champion, H. C., Breton, E., Russell, S. D., … Hare, J. M. (2008). Transcriptomic biomarkers for individual risk assessment in new-onset heart failure. Circulation, 118(3), 238–46. http://doi.org/10.1161/ CIRCULATIONAHA.107.756544

Heineke, J., & Molkentin, J. D. (2006). Regulation of cardiac hypertrophy by intracellular signalling pathways. Nature Reviews. Molecular Cell Biology, 7(8), 589–600. http://doi.org/10.1038/nrm1983

Hewett, T. E., Grupp, I. L., Grupp, G., & Robbins, J. (1994). Alpha-skeletal actin is associated with increased contractility in the mouse heart. Circulation Research, 74(4), 740–746.

Hill, J. A., & Olson, E. N. (2009). Cardiac Plasticity. http://dx.doi.org/10.1056/ NEJMra 072139.

Hindorff, L. A., Sethupathy, P., Junkins, H. A., Ramos, E. M., Mehta, J. P., Collins, F. S., & Manolio, T. A. (2009). Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proceedings of the National Academy of Sciences, 106(23), 9362–9367. http://doi.org/10.1073/pnas.0903103106

78

Hoenig, M. R., Bianchi, C., Rosenzweig, A., & Sellke, F. W. (2008). The cardiac microvasculature in hypertension, cardiac hypertrophy and diastolic heart failure. Current Vascular Pharmacology, 6(4), 292–300. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18855717

Hoshijima, M., & Chien, K. R. (2002). Mixed signals in heart failure: cancer rules. The Journal of Clinical Investigation, 109(7), 849–855. http://doi.org/10.1172/JCI15380 [doi]

Hosoda, K., Nakao, K., Hiroshi-Arai, Suga, S., Ogawa, Y., Mukoyama, M., … Imura, H. (1991). Cloning and expression of human endothelin-1 receptor cDNA. FEBS Letters, 287(1-2), 23–6. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1652463

Huang, J., Min Lu, M., Cheng, L., Yuan, L.-J., Zhu, X., Stout, A. L., … Parmacek, M. S. (2009). Myocardin is required for cardiomyocyte survival and maintenance of heart function. Proceedings of the National Academy of Sciences of the United States of America, 106(44), 18734–9. http://doi.org/10.1073/pnas.0910749106

Huet, G., Rajakyla, E. K., Viita, T., Skarp, K. P., Crivaro, M., Dopie, J., & Vartiainen, M. K. (2013). Actin-regulated feedback loop based on Phactr4, PP1 and cofilin maintains the actin monomer pool. Journal of Cell Science, 126(Pt 2), 497–507. http://doi.org/10.1242/jcs.113241; 10.1242/jcs.113241

Hunter, J. J., Tanaka, N., Rockman, H. A., Ross, J., & Chien, K. R. (1995). Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice. The Journal of Biological Chemistry, 270(39), 23173–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7559464

Hypertension: Current Care Guidelines. (2014). Working group set up by the Finnish Medical Society Duodecim and the Finnish Cardiac Society. Helsinki: The Finnish Medical Society Duodecim. Retrieved June 30, 2018, from www.kaypahoito.fi

Iida, M., Anna, C. H., Gaskin, N. D., Walker, N. J., & Devereux, T. R. (2007). The putative tumor suppressor Tsc-22 is downregulated early in chemically induced hepatocarcinogenesis and may be a suppressor of Gadd45b. Toxicological Sciences�: An Official Journal of the Society of Toxicology, 99(1), 43–50. http://doi.org/kfm138 [pii]

International Consortium for Blood Pressure Genome-Wide Association Studies, Ehret, G. B., Munroe, P. B., Rice, K. M., Bochud, M., Johnson, A. D., … Johnson, T. (2011). Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk. Nature, 478(7367), 103–9. http://doi.org/10.1038/nature10405

Ito, H., Hirata, Y., Hiroe, M., Tsujino, M., Adachi, S., Takamoto, T., … Marumo, F. (1991). Endothelin-1 induces hypertrophy with enhanced expression of muscle-specific genes in cultured neonatal rat cardiomyocytes. Circulation Research, 69(1), 209–15. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2054934

Itoh, A., Uchiyama, A., Taniguchi, S., & Sagara, J. (2014). Phactr3/scapinin, a member of protein phosphatase 1 and actin regulator (phactr) family, interacts with the plasma membrane via basic and hydrophobic residues in the N-terminus. PloS One, 9(11), e113289. http://doi.org/10.1371/journal.pone.0113289

79

Izumo, S., Nadal-Ginard, B., & Mahdavi, V. (1988). Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proceedings of the National Academy of Sciences of the United States of America, 85(2), 339–343.

Jager, J., Greiner, V., Strzoda, D., Seibert, O., Niopek, K., Sijmonsma, T. P., … Herzig, S. (2014). Hepatic transforming growth factor-beta 1 stimulated clone-22 D1 controls systemic cholesterol metabolism. Molecular Metabolism, 3(2), 155–166. http://doi.org/10.1016/j.molmet.2013.12.007 [doi]

Jarray, R., Allain, B., Borriello, L., Biard, D., Loukaci, A., Larghero, J., … Raynaud, F. (2011). Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis. Biochimie, 93(10), 1668–1675. http://doi.org/10.1016/j.biochi.2011.07.010 [doi]

Jay, P., Ji, J. W., Marsollier, C., Taviaux, S., Bergé-Lefranc, J. L., & Berta, P. (1996). Cloning of the human homologue of the TGF beta-stimulated clone 22 gene. Biochemical and Biophysical Research Communications, 222(3), 821–6. http://doi.org/10.1006/bbrc.1996.0825

Jessup, M., & Brozena, S. (2003). Heart failure. The New England Journal of Medicine, 348(20), 2007–2018. http://doi.org/10.1056/NEJMra021498

Jugdutt, B. I. (2012). Ischemia/Infarction. Heart Failure Clinics, 8(1), 43–51. http://doi.org/10.1016/j.hfc.2011.08.006

Kass, D. A. (2012). Cardiac role of cyclic-GMP hydrolyzing phosphodiesterase type 5: from experimental models to clinical trials. Current Heart Failure Reports, 9(3), 192–9. http://doi.org/10.1007/s11897-012-0101-0

Kato, M., Wang, L., Putta, S., Wang, M., Yuan, H., Sun, G., … Natarajan, R. (2010). Post-transcriptional up-regulation of Tsc-22 by Ybx1, a target of miR-216a, mediates TGF-{beta}-induced collagen expression in kidney cells. The Journal of Biological Chemistry, 285(44), 34004–34015. http://doi.org/10.1074/jbc.M110.165027 [doi]

Kawamata, H., Fujimori, T., & Imai, Y. (2004). TSC-22 (TGF-beta stimulated clone-22): a novel molecular target for differentiation-inducing therapy in salivary gland cancer. Current Cancer Drug Targets, 4(6), 521–529.

Kawamata, H., Nakashiro, K., Uchida, D., Hino, S., Omotehara, F., Yoshida, H., & Sato, M. (1998). Induction of TSC-22 by treatment with a new anti-cancer drug, vesnarinone, in a human salivary gland cancer cell. British Journal of Cancer, 77(1), 71–78.

Kemp, C. D., & Conte, J. V. (2012). The pathophysiology of heart failure. Cardiovascular Pathology�: The Official Journal of the Society for Cardiovascular Pathology, 21(5), 365–71. http://doi.org/10.1016/j.carpath.2011.11.007

Kerkelä, R., Ulvila, J., & Magga, J. (2015). Natriuretic Peptides in the Regulation of Cardiovascular Physiology and Metabolic Events. Journal of the American Heart Association, 4(10), e002423. http://doi.org/10.1161/JAHA.115.002423

Kessler, T., Vilne, B., & Schunkert, H. (2016). The impact of genome-wide association studies on the pathophysiology and therapy of cardiovascular disease. EMBO Molecular Medicine. http://doi.org/10.15252/emmm.201506174

80

Kester, H. A., Blanchetot, C., Hertog, J., Van, der S., & Van, der B. (1999). Transforming Growth Factor- beta -stimulated Clone-22 Is a Member of a Family of Leucine Zipper Proteins That Can Homo- and Heterodimerize and Has Transcriptional Repressor Activity. Journal of Biological Chemistry [J.Biol.Chem.].Vol.274, 274(39), 27439–27447.

Kester, H. A., Ward-Van Oostwaard, T. M. J., Goumans, M. J., Van Rooijen, M. A., Van Der Saag, P. T., Van Der Burg, B., & Mummery, C. L. (2000). Expression of TGF-? stimulated clone-22 (TSC-22) in mouse development and TGF-? signalling. Developmental Dynamics, 218(4), 563–572. http://doi.org/10.1002/1097-0177(2000)9999:9999<::AID-DVDY1021>3.0.CO;2-Q

Kiando, S. R., Tucker, N. R., Castro-Vega, L.-J., Katz, A., D’Escamard, V., Tréard, C., … Bouatia-Naji, N. (2016). PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance. PLOS Genetics, 12(10), e1006367. http://doi.org/10.1371/journal.pgen.1006367

Kim, S., Ohta, K., Hamaguchi, A., Yukimura, T., Miura, K., & Iwao, H. (1996). Effects of an AT1 receptor antagonist, an ACE inhibitor and a calcium channel antagonist on cardiac gene expressions in hypertensive rats. British Journal of Pharmacology, 118(3), 549–556. http://doi.org/10.1111/j.1476-5381.1996.tb15437.x

Kohli, S., Ahuja, S., & Rani, V. (2011). Transcription factors in heart: promising therapeutic targets in cardiac hypertrophy. Current Cardiology Reviews, 7(4), 262–71. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/22758628

Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., … Yazaki, Y. (1991). Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. Possible role of protein kinase C activation. The Journal of Biological Chemistry, 266(2), 1265–1268.

Kong, P., Christia, P., & Frangogiannis, N. G. (2014). The pathogenesis of cardiac fibrosis. Cellular and Molecular Life Sciences, 71(4), 549–574. http://doi.org/10.1007/s00018-013-1349-6

Korff, S., Katus, H. A., & Giannitsis, E. (2006). Differential diagnosis of elevated troponins. Heart (British Cardiac Society), 92(7), 987–93. http://doi.org/10.1136/hrt.2005.071282

Krenning, G., Zeisberg, E. M., & Kalluri, R. (2010). The origin of fibroblasts and mechanism of cardiac fibrosis. Journal of Cellular Physiology, 225(3), 631–7. http://doi.org/10.1002/jcp.22322

Kumar, A., Crawford, K., Close, L., Madison, M., Lorenz, J., Doetschman, T., … Lessard, J. L. (1997). Rescue of cardiac alpha-actin-deficient mice by enteric smooth muscle gamma-actin. Proceedings of the National Academy of Sciences of the United States of America, 94(9), 4406–4411.

Kuoppala, A., Shiota, N., Lindstedt, K. A., Rysä, J., Leskinen, H. K., Luodonpää, M., … Kokkonen, J. O. (2003). Expression of bradykinin receptors in the left ventricles of rats with pressure overload hypertrophy and heart failure. Journal of Hypertension, 21, 1729–1736. http://doi.org/10.1097/00004872-200309000-00023

81

Kuwahara, K., Kinoshita, H., Kuwabara, Y., Nakagawa, Y., Usami, S., Minami, T., … Nakao, K. (2010). Myocardin-related transcription factor A is a common mediator of mechanical stress- and neurohumoral stimulation-induced cardiac hypertrophic signaling leading to activation of brain natriuretic peptide gene expression. Molecular and Cellular Biology, 30(17), 4134–48. http://doi.org/10.1128/MCB.00154-10

Lee, D. S., Gona, P., Vasan, R. S., Larson, M. G., Benjamin, E. J., Wang, T. J., … Levy, D. (2009). Relation of disease pathogenesis and risk factors to heart failure with preserved or reduced ejection fraction: insights from the framingham heart study of the national heart, lung, and blood institute. Circulation, 119(24), 3070–7. http://doi.org/10.1161/CIRCULATIONAHA.108.815944

Lévy, B. I. (2004). Can angiotensin II type 2 receptors have deleterious effects in cardiovascular disease? Implications for therapeutic blockade of the renin-angiotensin system. Circulation, 109(1), 8–13. http://doi.org/10.1161/01.CIR.0000096609. 73772.C5

Li, J. M., & Brooks, G. (1997). Differential protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in cardiomyocytes during pressure overload-induced hypertrophy. Journal of Molecular and Cellular Cardiology, 29(8), 2213–24. http://doi.org/10.1006/jmcc.1997.0457

Li, J., Zhu, X., Chen, M., Cheng, L., Zhou, D., Lu, M. M., … Parmacek, M. S. (2005). Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development. Proceedings of the National Academy of Sciences of the United States of America, 102(25), 8916–21. http://doi.org/10.1073/pnas.0503741102

Li, M. J., Wang, P., Liu, X., Lim, E. L., Wang, Z., Yeager, M., … Wang, J. (2012). GWASdb: a database for human genetic variants identified by genome-wide association studies. Nucleic Acids Research, 40(D1), D1047–D1054. http://doi.org/10.1093/nar/gkr1182

Li, R. K., Li, G., Mickle, D. A., Weisel, R. D., Merante, F., Luss, H., … Williams, W. G. (1997). Overexpression of transforming growth factor-beta1 and insulin-like growth factor-I in patients with idiopathic hypertrophic cardiomyopathy. Circulation, 96(3), 874–81. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9264495

Li, S., Chang, S., Qi, X., Richardson, J. A., & Olson, E. N. (2006). Requirement of a myocardin-related transcription factor for development of mammary myoepithelial cells. Molecular and Cellular Biology, 26(15), 5797–808. http://doi.org/10.1128/ MCB.00211-06

Li, S., Wang, D.-Z., Wang, Z., Richardson, J. A., & Olson, E. N. (2003). The serum response factor coactivator myocardin is required for vascular smooth muscle development. Proceedings of the National Academy of Sciences of the United States of America, 100(16), 9366–70. http://doi.org/10.1073/pnas.1233635100

Liao, P., Georgakopoulos, D., Kovacs, A., Zheng, M., Lerner, D., Pu, H., … Wang, Y. (2001). The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy. Proceedings of the National Academy of Sciences, 98(21), 12283–12288. http://doi.org/10.1073/pnas.211086598

82

Liao, X.-H., Wang, N., Liu, Q.-X., Qin, T., Cao, B., Cao, D.-S., & Zhang, T.-C. (2011). Myocardin-related transcription factor-A induces cardiomyocyte hypertrophy. IUBMB Life, 63(1), 54–61. http://doi.org/10.1002/iub.415

Libby, P. (2012). Inflammation in atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 32(9), 2045–51. http://doi.org/10.1161/ATVBAHA.108.179705

Lijnen, P., & Petrov, V. (1999). Renin-angiotensin system, hypertrophy and gene expression in cardiac myocytes. Journal of Molecular and Cellular Cardiology, 31(5), 949–70. http://doi.org/10.1006/jmcc.1999.0934

Lindman, B. R., Bonow, R. O., & Otto, C. M. (2013). Current management of calcific aortic stenosis. Circulation Research, 113(2), 223–37. http://doi.org/10.1161/ CIRCRESAHA.111.300084

Lindsey, M. L., Iyer, R. P., Jung, M., DeLeon-Pennell, K. Y., & Ma, Y. (2016). Matrix metalloproteinases as input and output signals for post-myocardial infarction remodeling. Journal of Molecular and Cellular Cardiology, 91, 134–140. http://doi.org/10.1016/j.yjmcc.2015.12.018

Lu, X., Wang, L., Chen, S., He, L., Yang, X., Shi, Y., … Gu, D. (2012). Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease. Nature Genetics, 44(8), 890–894. http://doi.org/10.1038/ng.2337; 10.1038/ng.2337

Lyons, R. M., Keski-Oja, J., & Moses, H. L. (1988). Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium. The Journal of Cell Biology, 106(5), 1659–65. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/2967299

Ma, J., & Liew, C.-C. (2003). Gene profiling identifies secreted protein transcripts from peripheral blood cells in coronary artery disease. Journal of Molecular and Cellular Cardiology, 35(8), 993–8. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/12878486

Maillet, M., van Berlo, J. H., & Molkentin, J. D. (2012). Molecular basis of physiological heart growth: fundamental concepts and new players. Nature Reviews Molecular Cell Biology, 14(1), 38–48. http://doi.org/10.1038/nrm3495

Mancia, G., Fagard, R., Narkiewicz, K., Redon, J., Zanchetti, A., Böhm, M., … Wood, D. A. (2013). 2013 ESH/ESC guidelines for the management of arterial hypertension: the Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). European Heart Journal, 34(28), 2159–219. http://doi.org/10.1093/eurheartj/eht151

Mann, D. L., Barger, P. M., & Burkhoff, D. (2012). Myocardial recovery and the failing heart: myth, magic, or molecular target? Journal of the American College of Cardiology, 60(24), 2465–2472. http://doi.org/10.1016/j.jacc.2012.06.062 [doi]

Manolio, T. A. (2010). Genomewide Association Studies and Assessment of the Risk of Disease. http://dx.doi.org/10.1056/NEJMra0905980.

Mardis, E. R. (2013). Next-generation sequencing platforms. Annual Review of Analytical Chemistry (Palo Alto, Calif.), 6(1), 287–303. http://doi.org/10.1146/annurev-anchem-062012-092628

83

Matsuoka, R., Abe, S., Tokoro, F., Arai, M., Noda, T., Watanabe, S., … Yamada, Y. (2015). Association of six genetic variants with myocardial infarction. International Journal of Molecular Medicine, 35(5), 1451–9. http://doi.org/10.3892/ijmm.2015.2115

Mayourian, J., Ceholski, D. K., Gonzalez, D. M., Cashman, T. J., Sahoo, S., Hajjar, R. J., & Costa, K. D. (2018). Physiologic, Pathologic, and Therapeutic Paracrine Modulation of Cardiac Excitation-Contraction Coupling. Circulation Research, 122(1), 167–183. http://doi.org/10.1161/CIRCRESAHA.117.311589

McMurray, J. J. V, Adamopoulos, S., Anker, S. D., Auricchio, A., Böhm, M., Dickstein, K., … Zeiher, A. (2012). ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart. European Heart Journal, 33(14), 1787–847. http://doi.org/10.1093/eurheartj/ehs104

McMurray, J. J. V., Packer, M., Desai, A. S., Gong, J., Lefkowitz, M. P., Rizkala, A. R., … PARADIGM-HF Investigators and Committees. (2014). Angiotensin–Neprilysin Inhibition versus Enalapril in Heart Failure. New England Journal of Medicine, 371(11), 993–1004. http://doi.org/10.1056/NEJMoa1409077

Mehta, P. K., & Griendling, K. K. (2007). Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. American Journal of Physiology. Cell Physiology, 292(1), C82–97. http://doi.org/10.1152/ajpcell.00287.2006

Mikhailov, A. T., & Torrado, M. (2012). In search of novel targets for heart disease: myocardin and myocardin-related transcriptional cofactors. Biochemistry Research International, 2012, 973723. http://doi.org/10.1155/2012/973723 [doi]

Minicucci, M. F., Azevedo, P. S., Polegato, B. F., Paiva, S. A. R., & Zornoff, L. A. M. (2011). Heart failure after myocardial infarction: clinical implications and treatment. Clinical Cardiology, 34(7), 410–4. http://doi.org/10.1002/clc.20922

Mogensen, J., Klausen, I. C., Pedersen, A. K., Egeblad, H., Bross, P., Kruse, T. A., … Holmes, K. (1999). α-cardiac actin is a novel disease gene in familial hypertrophic cardiomyopathy. Journal of Clinical Investigation, 103(10), R39–R43. http://doi.org/10.1172/JCI6460

Mogensen, J., Perrot, A., Andersen, P. S., Havndrup, O., Klausen, I. C., Christiansen, M., … Børglum, A. D. (2004). Clinical and genetic characteristics of cardiac actin gene mutations in hypertrophic cardiomyopathy. Journal of Medical Genetics, 41(1), 10e–10. http://doi.org/10.1136/jmg.2003.010447

Mokalled, M. H., Carroll, K. J., Cenik, B. K., Chen, B., Liu, N., Olson, E. N., & Bassel-Duby, R. (2015). Myocardin-related transcription factors are required for cardiac development and function. Developmental Biology, 406(2), 109–16. http://doi.org/10.1016/j.ydbio.2015.09.006

Montecucco, F., Carbone, F., & Schindler, T. H. (2015). Pathophysiology of ST-segment elevation myocardial infarction: novel mechanisms and treatments. European Heart Journal. http://doi.org/10.1093/eurheartj/ehv592

84

Moore-Morris, T., Guimarães-Camboa, N., Banerjee, I., Zambon, A. C., Kisseleva, T., Velayoudon, A., … Zambon, A. (2014). Resident fibroblast lineages mediate pressure overload–induced cardiac fibrosis. Journal of Clinical Investigation, 124(7), 2921–2934. http://doi.org/10.1172/JCI74783

Morrison, A. C., Felix, J. F., Cupples, L. A., Glazer, N. L., Loehr, L. R., Dehghan, A., … Smith, N. L. (2010). Genomic Variation Associated With Mortality Among Adults of European and African Ancestry With Heart Failure: The Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium. Circulation: Cardiovascular Genetics, 3(3), 248–255. http://doi.org/10.1161/CIRCGENETICS.109.895995

Mozaffarian, D., Benjamin, E. J., Go, A. S., Arnett, D. K., Blaha, M. J., Cushman, M., … Turner, M. B. (2016). Executive Summary: Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation, 133(4), 447–54. http://doi.org/10.1161/CIR.0000000000000366

Mustonen, E., Leskinen, H., Aro, J., Luodonpää, M., Vuolteenaho, O., Ruskoaho, H., & Rysä, J. (2010). Metoprolol treatment lowers thrombospondin-4 expression in rats with myocardial infarction and left ventricular hypertrophy. Basic and Clinical Pharmacology and Toxicology, 107, 709–717. http://doi.org/10.1111/j.1742-7843.2010.00564.x

Nadal-Ginard, B., & Mahdavi, V. (1989). Molecular basis of cardiac performance. Plasticity of the myocardium generated through protein isoform switches. The Journal of Clinical Investigation, 84(6), 1693–1700. http://doi.org/10.1172/JCI114351 [doi]

Nakajima, H., Nakajima, H. O., Salcher, O., Dittiè, A. S., Dembowsky, K., Jing, S., & Field, L. J. (2000). Atrial but not ventricular fibrosis in mice expressing a mutant transforming growth factor-beta(1) transgene in the heart. Circulation Research, 86(5), 571–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10720419

Nishida, K., Yamaguchi, O., Hirotani, S., Hikoso, S., Higuchi, Y., Watanabe, T., … Otsu, K. (2004). p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload. Molecular and Cellular Biology, 24(24), 10611–20. http://doi.org/10.1128/MCB.24.24.10611-10620.2004

Niu, Z., Yu, W., Zhang, S. X., Barron, M., Belaguli, N. S., Schneider, M. D., … Schwartz, R. J. (2005). Conditional mutagenesis of the murine serum response factor gene blocks cardiogenesis and the transcription of downstream gene targets. The Journal of Biological Chemistry, 280(37), 32531–8. http://doi.org/10.1074/jbc.M501372200

Nowak, K. J., Ravenscroft, G., Jackaman, C., Filipovska, A., Davies, S. M., Lim, E. M., … Laing, N. G. (2009). Rescue of skeletal muscle alpha-actin-null mice by cardiac (fetal) alpha-actin. The Journal of Cell Biology, 185(5), 903–15. http://doi.org/10.1083/ jcb.200812132

Odonnell, C. J., Kavousi, M., Smith, A. V., Kardia, S. L. R., Feitosa, M. F., Hwang, S. J., … Witteman, J. C. M. (2011). Genome-wide association study for coronary artery calcification with follow-up in myocardial infarction. Circulation, 124, 2855–2864. http://doi.org/10.1161/CIRCULATIONAHA.110.974899

85

Ogawa, Y., Nakao, K., Mukoyama, M., Hosoda, K., Shirakami, G., Arai, H., … Imura, H. (1991). Natriuretic peptides as cardiac hormones in normotensive and spontaneously hypertensive rats. The ventricle is a major site of synthesis and secretion of brain natriuretic peptide. Circulation Research, 69(2), 491–500. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1830518

Oh, J., Richardson, J. A., & Olson, E. N. (2005). Requirement of myocardin-related transcription factor-B for remodeling of branchial arch arteries and smooth muscle differentiation. Proceedings of the National Academy of Sciences of the United States of America, 102(42), 15122–7. http://doi.org/10.1073/pnas.0507346102

Ohta, S., Yanagihara, K., & Nagata, K. (1997). Mechanism of apoptotic cell death of human gastric carcinoma cells mediated by transforming growth factor beta. The Biochemical Journal, 324 ( Pt 3(Pt 3), 777–782.

Oka, T., Xu, J., & Molkentin, J. D. (2007). Re-employment of developmental transcription factors in adult heart disease. Seminars in Cell & Developmental Biology, 18(1), 117–131. http://doi.org/S1084-9521(06)00123-6 [pii]

Olson, E. N., & Nordheim, A. (2010). Linking actin dynamics and gene transcription to drive cellular motile functions. Nature reviews.Molecular Cell Biology, 11(5), 353–365. http://doi.org/10.1038/nrm2890 [doi]

Olson, T. M., Doan, T. P., Kishimoto, N. Y., Whitby, F. G., Ackerman, M. J., & Fananapazir, L. (2000). Inherited and de novo Mutations in the Cardiac Actin Gene Cause Hypertrophic Cardiomyopathy. Journal of Molecular and Cellular Cardiology, 32(9), 1687–1694. http://doi.org/10.1006/jmcc.2000.1204

Olson, T. M., Michels, V. V, Thibodeau, S. N., Tai, Y. S., & Keating, M. T. (1998). Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science (New York, N.Y.), 280(5364), 750–2. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/9563954

Omura, T., Yoshiyama, M., Takeuchi, K., Hanatani, A., Kim, S., Yoshida, K., … Yoshikawa, J. (2000). Differences in time course of myocardial mRNA expression in non-infarcted myocardium after myocardial infarction. Basic Research in Cardiology, 95(4), 316–323.

Pandey, K. N. (2011). Guanylyl cyclase / atrial natriuretic peptide receptor-A: role in the pathophysiology of cardiovascular regulation. Canadian Journal of Physiology and Pharmacology, 89(8), 557–73. http://doi.org/10.1139/y11-054

Paquette, M., Dufour, R., & Baass, A. (2018). PHACTR1 genotype predicts coronary artery disease in patients with familial hypercholesterolemia. Journal of Clinical Lipidology. http://doi.org/10.1016/j.jacl.2018.04.012

Paradis, P., Dali-Youcef, N., Paradis, F. W., Thibault, G., & Nemer, M. (2000). Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling. Proceedings of the National Academy of Sciences of the United States of America, 97(2), 931–6. http://doi.org/10.1073/pnas.97.2.931

86

Parlakian, A., Charvet, C., Escoubet, B., Mericskay, M., Molkentin, J. D., Gary-Bobo, G., … Li, Z. (2005). Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart. Circulation, 112(19), 2930–9. http://doi.org/10.1161/CIRCULATIONAHA.105.533778

Parmacek, M. S. (2007). Myocardin-related transcription factors: critical coactivators regulating cardiovascular development and adaptation. Circulation Research, 100(5), 633–644. http://doi.org/100/5/633 [pii]

Pasipoularides, A. (2017). Genomic translational research: Paving the way to individualized cardiac functional analyses and personalized cardiology. International Journal of Cardiology, 230, 384–401. http://doi.org/10.1016/j.ijcard.2016.12.097

Patel, R. S., Morris, A. A., Ahmed, Y., Kavtaradze, N., Sher, S., Su, S., … Quyyumi, A. A. (2012). A genetic risk variant for myocardial infarction on chromosome 6p24 is associated with impaired central hemodynamic indexes. American Journal of Hypertension, 25(7), 797–803. http://doi.org/10.1038/ajh.2012.41

Patino, W. D., Mian, O. Y., Kang, J.-G., Matoba, S., Bartlett, L. D., Holbrook, B., … Hwang, P. M. (2005). Circulating transcriptome reveals markers of atherosclerosis. Proceedings of the National Academy of Sciences of the United States of America, 102(9), 3423–8. http://doi.org/10.1073/pnas.0408032102

Paul, M., Poyan Mehr, A., & Kreutz, R. (2006). Physiology of local renin-angiotensin systems. Physiological Reviews, 86(3), 747–803. http://doi.org/10.1152/ physrev.00036.2005

Pearson, T. A., & Manolio, T. A. (2008). How to interpret a genome-wide association study. JAMA, 299(11), 1335–44. http://doi.org/10.1001/jama.299.11.1335

Peden, J. F., Hopewell, J. C., Saleheen, D., Chambers, J. C., Hager, J., Soranzo, N., … Anand, S. S. (2011). A genome-wide association study in Europeans and South Asians identifies five new loci for coronary artery disease. Nature Genetics, 43(4), 339–344. http://doi.org/10.1038/ng.782

Perrin, B. J., & Ervasti, J. M. (2010). The actin gene family: Function follows isoform. Cytoskeleton, 67(10), 630–634. http://doi.org/10.1002/cm.20475

Peshenko, I. V, Olshevskaya, E. V, Azadi, S., Molday, L. L., Molday, R. S., & Dizhoor, A. M. (2011). Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins. Biochemistry, 50(44), 9511–9. http://doi.org/10.1021/bi201342b

Pokreisz, P., Vandenwijngaert, S., Bito, V., Van den Bergh, A., Lenaerts, I., Busch, C., … Janssens, S. P. (2009). Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice. Circulation, 119(3), 408–16. http://doi.org/10.1161/CIRCULATIONAHA.108.822072

Ponikowski, P., Voors, A. A., Anker, S. D., Bueno, H., Cleland, J. G. F., Coats, A. J. S., … Document Reviewers. (2016). 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. European Journal of Heart Failure, 18(8), 891–975. http://doi.org/10.1002/ejhf.592

87

Purcell, N. H., Wilkins, B. J., York, A., Saba-El-Leil, M. K., Meloche, S., Robbins, J., & Molkentin, J. D. (2007). Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo. Proceedings of the National Academy of Sciences, 104(35), 14074–14079. http://doi.org/10.1073/ pnas.0610906104

Quaife-Ryan, G. A., Sim, C. B., Ziemann, M., Kaspi, A., Rafehi, H., Ramialison, M., … Porrello, E. R. (2017). Multicellular Transcriptional Analysis of Mammalian Heart Regeneration. Circulation, 136(12), 1123–1139. http://doi.org/10.1161/ CIRCULATIONAHA.117.028252

Rai, V., Sharma, P., Agrawal, S., & Agrawal, D. K. (2017). Relevance of mouse models of cardiac fibrosis and hypertrophy in cardiac research. Molecular and Cellular Biochemistry, 424(1-2), 123–145. http://doi.org/10.1007/s11010-016-2849-0

Roger, V. L. (2007). Epidemiology of myocardial infarction. The Medical Clinics of North America, 91(4), 537–52; ix. http://doi.org/10.1016/j.mcna.2007.03.007

Roger, V. L., Jacobsen, S. J., Weston, S. A., Goraya, T. Y., Killian, J., Reeder, G. S., … Frye, R. L. (2002). Trends in the incidence and survival of patients with hospitalized myocardial infarction, Olmsted County, Minnesota, 1979 to 1994. Annals of Internal Medicine, 136(5), 341–8. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/11874305

Rohini, A., Agrawal, N., Koyani, C. N., & Singh, R. (2010). Molecular targets and regulators of cardiac hypertrophy. Pharmacological Research�: The Official Journal of the Italian Pharmacological Society, 61(4), 269–280. http://doi.org/10.1016/j.phrs.2009.11.012 [doi]

Rosamond, W. D., Chambless, L. E., Folsom, A. R., Cooper, L. S., Conwill, D. E., Clegg, L., … Heiss, G. (1998). Trends in the incidence of myocardial infarction and in mortality due to coronary heart disease, 1987 to 1994. The New England Journal of Medicine, 339(13), 861–7. http://doi.org/10.1056/NEJM199809243391301

Rose, B. A., Force, T., & Wang, Y. (2010). Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale. Physiological Reviews, 90(4), 1507–1546. http://doi.org/10.1152/physrev.00054.2009 [doi]

Rosenkranz, S. (2004). TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovascular Research, 63(3), 423–432. http://doi.org/10.1016/j.cardiores.2004.04.030 [doi]

Rosenkranz, S., Flesch, M., Amann, K., Haeuseler, C., Kilter, H., Seeland, U., … Bohm, M. (2002). Alterations of beta-adrenergic signaling and cardiac hypertrophy in transgenic mice overexpressing TGF-beta(1). American Journal of physiology.Heart and Circulatory Physiology, 283(3), H1253–62. http://doi.org/10.1152/ ajpheart.00578.2001 [doi]

Rossi, M. A. (1998). Pathologic fibrosis and connective tissue matrix in left ventricular hypertrophy due to chronic arterial hypertension in humans. Journal of Hypertension, 16(7), 1031–41. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9794745

88

Ruskoaho, H., Kinnunen, P., Mäntymaa, P., Uusimaa, P., Taskinen, T., Vuolteenaho, O., & Leppäluoto, J. (1991). Cellular signals regulating the release of ANF. Canadian Journal of Physiology and Pharmacology, 69(10), 1514–24. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1838021

Ruwhof, C., & van der Laarse, A. (2000). Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways. Cardiovascular Research, 47(1), 23–37. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10869527

Rysa, J., Aro, J., & Ruskoaho, H. (2006). Early left ventricular gene expression profile in response to increase in blood pressure. Blood Pressure, 15(6), 375–383.

Rysa, J., Leskinen, H., Ilves, M., & Ruskoaho, H. (2005). Distinct upregulation of extracellular matrix genes in transition from hypertrophy to hypertensive heart failure. Hypertension, 45(5), 927–933. http://doi.org/01.HYP.0000161873.27088.4c [pii]

Rysä, J., Tokola, H., & Ruskoaho, H. (2018). Mechanical stretch induced transcriptomic profiles in cardiac myocytes. Scientific Reports, 8(1), 4733. http://doi.org/ 10.1038/s41598-018-23042-w

Sadoshima, J., & Izumo, S. (1993). Molecular characterization of angiotensin II--induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Critical role of the AT1 receptor subtype. Circulation Research, 73(3), 413–23. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8348686

Sadoshima, J., Jahn, L., Takahashi, T., Kulik, T. J., & Izumo, S. (1992). Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy. The Journal of Biological Chemistry, 267(15), 10551–10560.

Sagara, J., Arata, T., & Taniguchi, S. (2009). Scapinin, the protein phosphatase 1 binding protein, enhances cell spreading and motility by interacting with the actin cytoskeleton. PloS One, 4(1), e4247. http://doi.org/10.1371/journal.pone.0004247 [doi]

Sagara, J., Higuchi, T., Hattori, Y., Moriya, M., Sarvotham, H., Shima, H., … Taniguchi, S. (2003). Scapinin, a putative protein phosphatase-1 regulatory subunit associated with the nuclear nonchromatin structure. The Journal of Biological Chemistry, 278(46), 45611–45619. http://doi.org/10.1074/jbc.M305227200 [doi]

Schieffer, B., Wirger, A., Meybrunn, M., Seitz, S., Holtz, J., Riede, U. N., & Drexler, H. (1994). Comparative effects of chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on cardiac remodeling after myocardial infarction in the rat. Circulation, 89, 2273–2282. http://doi.org/10.1161/ 01.CIR.89.5.2273

Schirone, L., Forte, M., Palmerio, S., Yee, D., Nocella, C., Angelini, F., … Frati, G. (2017). A Review of the Molecular Mechanisms Underlying the Development and Progression of Cardiac Remodeling. Oxidative Medicine and Cellular Longevity, 2017, 3920195. http://doi.org/10.1155/2017/3920195

Schultz Jel, J., Witt, S. A., Glascock, B. J., Nieman, M. L., Reiser, P. J., Nix, S. L., … Doetschman, T. (2002). TGF-beta1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II. The Journal of Clinical Investigation, 109(6), 787–796. http://doi.org/10.1172/JCI14190 [doi]

89

Schultz, J. E. J., Witt, S. A., Glascock, B. J., Nieman, M. L., Reiser, P. J., Nix, S. L., … Doetschman, T. (2002). TGF-beta1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II. The Journal of Clinical Investigation, 109(6), 787–96. http://doi.org/10.1172/JCI14190

Schultz-Cherry, S., Ribeiro, S., Gentry, L., & Murphy-Ullrich, J. E. (1994). Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system. The Journal of Biological Chemistry, 269(43), 26775–82. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7929413

Schunkert, H., Konig, I. R., Kathiresan, S., Reilly, M. P., Assimes, T. L., Holm, H., … Samani, N. J. (2011). Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nature Genetics, 43(4), 333–338. http://doi.org/10.1038/ng.784; 10.1038/ng.784

Schwartz, K., de la Bastie, D., Bouveret, P., Oliviero, P., Alonso, S., & Buckingham, M. (1986). Alpha-skeletal muscle actin mRNA’s accumulate in hypertrophied adult rat hearts. Circulation Research, 59(5), 551–555.

Shah, A. M., & Mann, D. L. (2011). In search of new therapeutic targets and strategies for heart failure: recent advances in basic science. Lancet (London, England), 378(9792), 704–12. http://doi.org/10.1016/S0140-6736(11)60894-5

Shibanuma, M., Kuroki, T., & Nose, K. (1992). Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors. The Journal of Biological Chemistry, 267(15), 10219–10224.

Shimizu, I., & Minamino, T. (2016). Physiological and pathological cardiac hypertrophy. Journal of Molecular and Cellular Cardiology, 97, 245–62. http://doi.org/10.1016/ j.yjmcc.2016.06.001

Shostak, K. O., Dmitrenko, V. V, Vudmaska, M. I., Naidenov, V. G., Beletskii, A. V, Malisheva, T. A., … Kavsan, V. M. (2005). Patterns of expression of TSC-22 protein in astrocytic gliomas. Experimental Oncology, 27(4), 314–318. http://doi.org/24/464 [pii]

Smith, J. G., & Newton-Cheh, C. (2015). Genome-wide association studies of late-onset cardiovascular disease. Journal of Molecular and Cellular Cardiology, 83, 131–141. http://doi.org/10.1016/j.yjmcc.2015.04.004

Smith, N. L., Felix, J. F., Morrison, A. C., Demissie, S., Glazer, N. L., Loehr, L. R., … Vasan, R. S. (2010). Association of Genome-Wide Variation With the Risk of Incident Heart Failure in Adults of European and African Ancestry: A Prospective Meta-Analysis From the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. Circulation: Cardiovascular Genetics, 3(3), 256–266. http://doi.org/10.1161/CIRCGENETICS.109.895763

Solimini, N. L., Liang, A. C., Xu, C., Pavlova, N. N., Xu, Q., Davoli, T., … Elledge, S. J. (2013). STOP gene Phactr4 is a tumor suppressor. Proceedings of the National Academy of Sciences of the United States of America, 110(5), E407–14. http://doi.org/10.1073/pnas.1221385110; 10.1073/pnas.1221385110

90

Stable coronary artery disease: Current Care Guidelines. (2015). Working group set up by the Finnish Medical Society Duodecim and the Finnish Cardiac Society. Helsinki: The Finnish Medical Society Duodecim. Retrieved June 30, 2018, from www.kaypahoito.fi

Stanton, L. W., Garrard, L. J., Damm, D., Garrick, B. L., Lam, A., Kapoun, A. M., … White, R. T. (2000). Altered patterns of gene expression in response to myocardial infarction. Circulation Research, 86(9), 939–945.

Sun, Y., Boyd, K., Xu, W., Ma, J., Jackson, C. W., Fu, A., … Morris, S. W. (2006). Acute myeloid leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function. Molecular and Cellular Biology, 26(15), 5809–26. http://doi.org/ 10.1128/MCB.00024-06

Sun, Y., Zhang, J. Q., Zhang, J., & Ramires, F. J. (1998). Angiotensin II, transforming growth factor-beta1 and repair in the infarcted heart. Journal of Molecular and Cellular Cardiology, 30(8), 1559–69. http://doi.org/10.1006/jmcc.1998.0721

Sun, Y., Zhang, J. Q., Zhang, J., & Ramires, F. J. A. (1998). Angiotensin II, Transforming Growth Factor-β1and Repair in the Infarcted Heart. Journal of Molecular and Cellular Cardiology, 30(8), 1559–1569. http://doi.org/10.1006/jmcc.1998.0721

Suthahar, N., Meijers, W. C., Silljé, H. H. W., & de Boer, R. A. (2017). From Inflammation to Fibrosis-Molecular and Cellular Mechanisms of Myocardial Tissue Remodelling and Perspectives on Differential Treatment Opportunities. Current Heart Failure Reports, 14(4), 235–250. http://doi.org/10.1007/s11897-017-0343-y

Sutton, M. G., & Sharpe, N. (2000). Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation, 101(25), 2981–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10869273

Szabo, Z., Magga, J., Alakoski, T., Ulvila, J., Piuhola, J., Vainio, L., … Kerkela, R. (2014). Connective Tissue Growth Factor Inhibition Attenuates Left Ventricular Remodeling and Dysfunction in Pressure Overload-Induced Heart Failure. Hypertension, 63(6), 1235–1240. http://doi.org/10.1161/HYPERTENSIONAHA.114.03279

Tachibana, H., Perrino, C., Takaoka, H., Davis, R. J., Naga Prasad, S. V., & Rockman, H. A. (2006). JNK1 is required to preserve cardiac function in the early response to pressure overload. Biochemical and Biophysical Research Communications, 343(4), 1060–1066. http://doi.org/10.1016/j.bbrc.2006.03.065

Taegtmeyer, H., Sen, S., & Vela, D. (2010). Return to the fetal gene program. Annals of the New York Academy of Sciences, 1188(1), 191–198. http://doi.org/10.1111/j.1749-6632.2009.05100.x

Takahashi, N., Calderone, A., Izzo, N. J., Mäki, T. M., Marsh, J. D., & Colucci, W. S. (1994). Hypertrophic stimuli induce transforming growth factor-beta 1 expression in rat ventricular myocytes. The Journal of Clinical Investigation, 94(4), 1470–6. http://doi.org/10.1172/JCI117485

Takahashi, T., Allen, P. D., & Izumo, S. (1992). Expression of A-, B-, and C-type natriuretic peptide genes in failing and developing human ventricles. Correlation with expression of the Ca(2+)-ATPase gene. Circulation Research, 71(1), 9–17. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1535030

91

Takimoto, E., Champion, H. C., Li, M., Belardi, D., Ren, S., Rodriguez, E. R., … Kass, D. A. (2005). Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nature Medicine, 11(2), 214–22. http://doi.org/10.1038/nm1175

Teekakirikul, P., Eminaga, S., Toka, O., Alcalai, R., Wang, L., Wakimoto, H., … Parikka, V. (2010). Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-β. Journal of Clinical Investigation, 120(10), 3520–3529. http://doi.org/10.1172/JCI42028

Teslovich, T. M., Musunuru, K., Smith, A. V, Edmondson, A. C., Stylianou, I. M., Koseki, M., … Kathiresan, S. (2010). Biological, clinical and population relevance of 95 loci for blood lipids. Nature, 466(7307), 707–13. http://doi.org/10.1038/nature09270

Tham, Y. K., Bernardo, B. C., Ooi, J. Y. Y., Weeks, K. L., & McMullen, J. R. (2015). Pathophysiology of cardiac hypertrophy and heart failure: signaling pathways and novel therapeutic targets. Archives of Toxicology, 89(9), 1401–1438. http://doi.org/10.1007/s00204-015-1477-x

Tondeleir, D., Vandamme, D., Vandekerckhove, J., Ampe, C., & Lambrechts, A. (2009). Actin isoform expression patterns during mammalian development and in pathology: Insights from mouse models. Cell Motility and the Cytoskeleton, 66(10), 798–815. http://doi.org/10.1002/cm.20350

Travers, J. G., Kamal, F. A., Robbins, J., Yutzey, K. E., & Blaxall, B. C. (2016). Cardiac Fibrosis. Circulation Research, 118(6), 1021–1040. http://doi.org/10.1161/ CIRCRESAHA.115.306565

Treisman, J. E., Lai, Z. C., & Rubin, G. M. (1995). Shortsighted acts in the decapentaplegic pathway in Drosophila eye development and has homology to a mouse TGF-beta-responsive gene. Development (Cambridge, England), 121(9), 2835–45. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7555710

Trevino, V., Falciani, F., & Barrera-Saldaña, H. A. (2007). DNA microarrays: a powerful genomic tool for biomedical and clinical research. Molecular Medicine (Cambridge, Mass.), 13(9-10), 527–41. http://doi.org/10.2119/2006-00107.Trevino

Trexler, C. L., Odell, A. T., Jeong, M. Y., Dowell, R. D., & Leinwand, L. A. (2017). Transcriptome and Functional Profile of Cardiac Myocytes Is Influenced by Biological Sex. Circulation. Cardiovascular Genetics, 10(5), e001770. http://doi.org/10.1161/CIRCGENETICS.117.001770

Vahanian, A., Alfieri, O., Andreotti, F., Antunes, M. J., Barón-Esquivias, G., Baumgartner, H., … Zembala, M. (2012). Guidelines on the management of valvular heart disease (version 2012): the Joint Task Force on the Management of Valvular Heart Disease of the European Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery (EACTS). European Journal of Cardio-Thoracic Surgery�: Official Journal of the European Association for Cardio-Thoracic Surgery, 42(4), S1–44. http://doi.org/10.1093/ejcts/ezs455

van Berlo, J. H., Maillet, M., & Molkentin, J. D. (2013). Signaling effectors underlying pathologic growth and remodeling of the heart. The Journal of Clinical Investigation, 123(1), 37–45. http://doi.org/10.1172/JCI62839

92

van den Borne, S. W. M., van de Schans, V. A. M., Strzelecka, A. E., Vervoort-Peters, H. T. M., Lijnen, P. M., Cleutjens, J. P. M., … Frangogiannis, N. (2009). Mouse strain determines the outcome of wound healing after myocardial infarction. Cardiovascular Research, 84(2), 273–82. http://doi.org/10.1093/cvr/cvp207

Van Driest, S. L., Ellsworth, E. G., Ommen, S. R., Tajik, A. J., Gersh, B. J., & Ackerman, M. J. (2003). Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy. Circulation, 108(4), 445–51. http://doi.org/10.1161/01.CIR.0000080896.52003.DF

van Kats, J. P., Danser, A. H., van Meegen, J. R., Sassen, L. M., Verdouw, P. D., & Schalekamp, M. A. (1998). Angiotensin production by the heart: a quantitative study in pigs with the use of radiolabeled angiotensin infusions. Circulation, 98(1), 73–81. http://doi.org/10.1161/01.cir.98.1.73

van Setten, J., Isgum, I., Smolonska, J., Ripke, S., de Jong, P. A., Oudkerk, M., … de Bakker, P. I. (2013). Genome-wide association study of coronary and aortic calcification implicates risk loci for coronary artery disease and myocardial infarction. Atherosclerosis, 228(2), 400–405. http://doi.org/10.1016/j.atherosclerosis.2013.02.039; 10.1016/j.atherosclerosis.2013.02.039

van Setten, J., Isgum, I., Smolonska, J., Ripke, S., de Jong, P. A., Oudkerk, M., … de Bakker, P. I. W. (2013). Genome-wide association study of coronary and aortic calcification implicates risk loci for coronary artery disease and myocardial infarction. Atherosclerosis, 228(2), 400–5. http://doi.org/10.1016/j.atherosclerosis.2013.02.039

Vandekerckhove, J., & Weber, K. (1978). At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. Journal of Molecular Biology, 126(4), 783–802. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/745245

Wang, D.-Z., Li, S., Hockemeyer, D., Sutherland, L., Wang, Z., Schratt, G., … Olson, E. N. (2002). Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proceedings of the National Academy of Sciences of the United States of America, 99(23), 14855–60. http://doi.org/10.1073/pnas.222561499

Wang, K., Long, B., Zhou, J., & Li, P.-F. (2010). miR-9 and NFATc3 regulate myocardin in cardiac hypertrophy. The Journal of Biological Chemistry, 285(16), 11903–12. http://doi.org/10.1074/jbc.M109.098004

Wang, Y., Huang, S., Sah, V. P., Ross Jr, J., Brown, J. H., Han, J., & Chien, K. R. (1998). Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. The Journal of Biological Chemistry, 273(4), 2161–2168.

Weber, K. T., Sun, Y., Bhattacharya, S. K., Ahokas, R. A., & Gerling, I. C. (2013). Myofibroblast-mediated mechanisms of pathological remodelling of the heart. Nature Reviews Cardiology, 10(1), 15–26. http://doi.org/10.1038/nrcardio.2012.158

Wei, K., Che, N., & Chen, F. (2007). Myocardin-related transcription factor B is required for normal mouse vascular development and smooth muscle gene expression. Developmental Dynamics�: An Official Publication of the American Association of Anatomists, 236(2), 416–25. http://doi.org/10.1002/dvdy.21041

93

Wider, C., Lincoln, S. J., Heckman, M. G., Diehl, N. N., Stone, J. T., Haugarvoll, K., … Ross, O. A. (2009). Phactr2 and Parkinson’s disease. Neuroscience Letters, 453(1), 9–11. http://doi.org/10.1016/j.neulet.2009.02.009

Wiezlak, M., Diring, J., Abella, J., Mouilleron, S., Way, M., McDonald, N. Q., & Treisman, R. (2012). G-actin regulates the shuttling and PP1 binding of the RPEL protein Phactr1 to control actomyosin assembly. Journal of Cell Science, 125(Pt 23), 5860–5872. http://doi.org/10.1242/jcs.112078; 10.1242/jcs.112078

Willems, I. E., Havenith, M. G., De Mey, J. G., & Daemen, M. J. (1994). The alpha-smooth muscle actin-positive cells in healing human myocardial scars. The American Journal of Pathology, 145(4), 868–75. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/7943177

Wu, Q.-Q., Xiao, Y., Yuan, Y., Ma, Z.-G., Liao, H.-H., Liu, C., … Tang, Q.-Z. (2017). Mechanisms contributing to cardiac remodelling. Clinical Science (London, England�: 1979), 131(18), 2319–2345. http://doi.org/10.1042/CS20171167

Xing, W., Zhang, T.-C., Cao, D., Wang, Z., Antos, C. L., Li, S., … Wang, D.-Z. (2006). Myocardin induces cardiomyocyte hypertrophy. Circulation Research, 98(8), 1089–97. http://doi.org/10.1161/01.RES.0000218781.23144.3e

Yamazaki, T., & Yazaki, Y. (1997). Is there major involvement of the renin-angiotensin system in cardiac hypertrophy? Circulation Research, 81(5), 639–42. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9351435

Yan, X. (2011). TSC-22 promotes transforming growth factor beta-mediated cardiac myofibroblast differentiation by antagonizing Smad7 activity. Molecular & Cellular Biology, 31(18), 3700–3709.

Yancy, C. W., Jessup, M., Bozkurt, B., Butler, J., Casey, D. E., Drazner, M. H., … American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. (2013). 2013 ACCF/AHA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation, 128(16), e240–e327. http://doi.org/10.1161/CIR.0b013e31829e8776

Yang, L. L., Gros, R., Kabir, M. G., Sadi, A., Gotlieb, A. I., Husain, M., & Stewart, D. J. (2004). Conditional cardiac overexpression of endothelin-1 induces inflammation and dilated cardiomyopathy in mice. Circulation, 109(2), 255–61. http://doi.org/10.1161/ 01.CIR.0000105701.98663.D4

Yin, Z., Ren, J., & Guo, W. (2015). Sarcomeric protein isoform transitions in cardiac muscle: a journey to heart failure. Biochimica et Biophysica Acta, 1852(1), 47–52. http://doi.org/10.1016/j.bbadis.2014.11.003

Yoon, C. H., Rho, S. B., Kim, S. T., Kho, S., Park, J., Jang, I. S., … Lee, S. H. (2012). Crucial role of TSC-22 in preventing the proteasomal degradation of p53 in cervical cancer. PloS One, 7(8), e42006. http://doi.org/10.1371/journal.pone.0042006 [doi]

Yorikane, R., Sakai, S., Miyauchi, T., Sakurai, T., Sugishita, Y., & Goto, K. (1993). Increased production of endothelin-1 in the hypertrophied rat heart due to pressure overload. FEBS Letters, 332(1-2), 31–34. http://doi.org/10.1016/0014-5793(93)80476-B

94

Yu, C.-M., Tipoe, G. L., Wing-Hon Lai, K., & Lau, C.-P. (2001). Effects of combination of angiotensin-converting enzyme inhibitor and angiotensin receptor antagonist on inflammatory cellular infiltration and myocardial interstitial fibrosis after acute myocardial infarction. Journal of the American College of Cardiology, 38(4), 1207–1215. http://doi.org/10.1016/S0735-1097(01)01518-2

Yu, J., Ershler, M., Yu, L., Wei, M., Hackanson, B., Yokohama, A., … Caligiuri, M. A. (2009). TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia. Blood, 113(22), 5558–5567. http://doi.org/10.1182/blood-2009-02-205732 [doi]

Yu, Q., & Stamenkovic, I. (2000). Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis. Genes & Development, 14(2), 163–176. http://doi.org/10.1101/GAD.14.2.163

Zarubin, T., & Han, J. (2005). Activation and signaling of the p38 MAP kinase pathway. Cell Research, 15(1), 11–8. http://doi.org/10.1038/sj.cr.7290257

Zechner, D., Thuerauf, D. J., Hanford, D. S., McDonough, P. M., & Glembotski, C. C. (1997). A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression. The Journal of Cell Biology, 139(1), 115–27. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/9314533

Zeller, T., Blankenberg, S., & Diemert, P. (2012). Genomewide Association Studies in Cardiovascular Disease--An Update 2011. Clinical Chemistry, 58(1), 92–103. http://doi.org/10.1373/clinchem.2011.170431

Zhang, M., Takimoto, E., Hsu, S., Lee, D. I., Nagayama, T., Danner, T., … Kass, D. A. (2010). Myocardial remodeling is controlled by myocyte-targeted gene regulation of phosphodiesterase type 5. Journal of the American College of Cardiology, 56(24), 2021–30. http://doi.org/10.1016/j.jacc.2010.08.612

Zhang, S., Weinheimer, C., Courtois, M., Kovacs, A., Zhang, C. E., Cheng, A. M., … Muslin, A. J. (2003). The role of the Grb2–p38 MAPK signaling pathway in cardiac hypertrophy and fibrosis. Journal of Clinical Investigation, 111(6), 833–841. http://doi.org/ 10.1172/JCI16290

Zhang, X., Azhar, G., Chai, J., Sheridan, P., Nagano, K., Brown, T., … Wei, J. Y. (2001). Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor. American Journal of Physiology. Heart and Circulatory Physiology, 280(4), H1782–92. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11247792

Zhang, Y., Kim, T. H., & Niswander, L. (2012). Phactr4 regulates directional migration of enteric neural crest through PP1, integrin signaling, and cofilin activity. Genes & Development, 26(1), 69–81. http://doi.org/10.1101/gad.179283.111 [doi]

Zheng, L., Suzuki, H., Nakajo, Y., Nakano, A., & Kato, M. (2018). Regulation of c-MYC transcriptional activity by transforming growth factor-beta 1-stimulated clone 22. Cancer Science, 109(2), 395–402. http://doi.org/10.1111/cas.13466

95

Original articles

I Kelloniemi A., Szabo Z., Serpi R., Näpänkangas J., Ohukainen P., Tenhunen O., Kaikkonen L., Koivisto E., Bagyura Z., Kerkelä R., Leosdottir M., Hedner T., Melander O., Ruskoaho H.*, Rysä J.* (2015). The early-onset myocardial infarction associated PHACTR1 gene regulates skeletal and cardiac alpha-actin gene expression. PLoS ONE 10(6). http://doi.org/10.1371/journal.pone.0130502.

II Kelloniemi A., Aro J., Näpänkangas J., Koivisto E., Mustonen E., Ruskoaho H., Rysä J. (2015). TSC-22 up-regulates collagen 3a1 gene expression in the rat heart. BMC Cardiovascular Disorders, 15(1). http://doi.org/10.1186/s12872-015-0121-2.

III Kelloniemi A., Szabo Z., Serpi R., Aro J., Soininen R., Ruskoaho H.*, Rysä J.* Genetic retinal degeneration 3-like (Rd3l) deficiency induces cardiac dysfunction in response to pressure overload. Manuscript.

Reprinted with permission from PLOS (I) and BioMed Central (II) under the

Creative Commons Attribution License.

Original publications are not included in the electronic version of the dissertation.

96

A C T A U N I V E R S I T A T I S O U L U E N S I S

Book orders:Granum: Virtual book storehttp://granum.uta.fi/granum/

S E R I E S D M E D I C A

1467. Tikanmäki, Marjaana (2018) Preterm birth and parental and pregnancy relatedfactors in association with physical activity and fitness in adolescence and youngadulthood

1468. Juvonen-Posti, Pirjo (2018) Work-related rehabilitation for strengthening workingcareers : a multiperspective and mixed methods study of its mechanisms

1469. Palaniswamy, Saranya (2018) Vitamin D status and its association with leukocytetelomere length, obesity and inflammation in young adults : a Northern FinlandBirth Cohort 1966 study

1470. Bur, Hamid (2018) Biological prognostic and predictive markers in Hodgkinlymphoma

1471. Haapanen, Henri (2018) Preconditioning against ischemic injury of the centralnervous system in aortic surgery : an experimental study in a porcine model withremote ischemic preconditioning and diazoxide

1472. Mahlman, Mari (2018) Genetic background and antenatal risk factors ofbronchopulmonary dysplasia

1473. Lantto, Ulla (2018) Etiology and outcome of PFAPA (periodic fever, aphthousstomatitis, pharyngitis and adenitis) syndrome among patients operated withtonsillectomy in childhood

1474. Hintsala, Heidi (2018) Cardiovascular responses to cold exposure in untreatedhypertension

1475. Alakortes, Jaana (2018) Social-emotional and behavioral development problems in1 to 2-year-old children in Northern Finland : reports of mothers, fathers andhealthcare professionals

1476. Puhto, Teija (2018) The burden of healthcare-associated infections in primary andtertiary healthcare wards and the cost of procedure-related prosthetic jointinfections

1477. Honkila, Minna (2018) Chlamydia trachomatis infections in neonates and infants

1478. Krooks, Laura (2018) Malocclusions in relation to facial soft tissue characteristics,facial aesthetics and temporomandibular disorders in the Northern Finland BirthCohort 1966

1479. Hoque Apu, Ehsanul (2018) Migration and invasion pattern analysis of oral cancercells in vitro

UNIVERSITY OF OULU P .O. Box 8000 F I -90014 UNIVERSITY OF OULU FINLAND

A C T A U N I V E R S I T A T I S O U L U E N S I S

University Lecturer Tuomo Glumoff

University Lecturer Santeri Palviainen

Postdoctoral research fellow Sanna Taskila

Professor Olli Vuolteenaho

University Lecturer Veli-Matti Ulvinen

Planning Director Pertti Tikkanen

Professor Jari Juga

University Lecturer Anu Soikkeli

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-952-62-2028-4 (Paperback)ISBN 978-952-62-2029-1 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1481

AC

TAA

nnina Kelloniem

i

OULU 2018

D 1481

Annina Kelloniemi

NOVEL FACTORS REGULATING CARDIAC REMODELING IN EXPERIMENTAL MODELSOF CARDIAC HYPERTROPHY AND FAILURE

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;BIOCENTER OULU