exercise training attenuates upregulation of p47 phox in...

Research ArticleExercise Training Attenuates Upregulation of p47phox andp67phox in Hearts of Diabetic Rats

Neeru M Sharma1 Brandon Rabeler1 Hong Zheng1

Eugenia Raichlin2 and Kaushik P Patel1

1Department of Cellular amp Integrative Physiology University of Nebraska Medical Center Omaha NE 68198 USA2Division of Cardiology University of Nebraska Medical Center Omaha NE 68198 USA

Correspondence should be addressed to Kaushik P Patel kpatelunmcedu

Received 14 July 2015 Revised 24 September 2015 Accepted 21 December 2015

Academic Editor Jose Luıs Garcıa-Gimenez

Copyright copy 2016 Neeru M Sharma et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Exercise training (ExT) is currently being used as a nonpharmacological strategy to improve cardiac function in diabetic patientsHowever the molecular mechanism(s) underlying its beneficial effects remains poorly understood Oxidative stress is knownto play a key role in the pathogenesis of diabetic cardiomyopathy and one of the enzyme systems that produce reactive oxygenspecies is NADHNADPH oxidase The goal of this study was to investigate the effect of streptozotocin- (STZ-) induced diabeteson expression of p47phox and p67phox key regulatory subunits of NADPH oxidase in cardiac tissues and determine whether ExTcan attenuate these changes Four weeks after STZ treatment expression of p47phox and p67phox increased 23-fold and 16-foldrespectively in left ventricles of diabetic rats and these increases were attenuated with three weeks of ExT initiated 1 week afteronset of diabetes In atrial tissues there was increased expression of p47phox (74) which was decreased by ExT in diabetic ratsFurthermore increased collagen III levels in diabetic hearts (52)were significantly reduced by ExT Taken together ExT attenuatesthe increased expression of p47phox and p67phox in the hearts of diabetic rats which could be an underlyingmechanism for improvingintracardiac matrix and thus cardiac function and prevent cardiac remodeling in diabetic cardiomyopathy

1 Introduction

Diabetes mellitus (DM) is leading metabolic disease withthe highest morbidity and mortality and according to 2014estimate of InternationalDiabetes Federation the disease nowaffects more than 387 million people worldwide DM is asso-ciated with an increased risk of cardiovascular complicationsincluding hypertension and coronary artery disease [1]Thereis growing recognition of diabetic cardiomyopathy a primarymyocardial disease defined as either systolic or diastolic leftventricular dysfunction in otherwise healthy diabetic personThis is one of the most serious complications of diabeteshowever the underlying mechanisms for this dysfunctionare not clearly understood [2 3] Originally Rubler et alin 1972 [4] based on postmortem findings proposed thatdiabetic cardiomyopathy is secondary to underlying hyper-glycemia resulting in a multitude of adverse downstream

effects including impaired myocyte calcium handlingrenin-angiotensin-aldosterone activation microangiopathymyocardial fibrosis and increased oxidative stress [5ndash7]Mechanistically chronic hyperglycemia exerts oxidativestress to cardiomyocytes by the production of reactive oxygenspecies (ROS) culminating in these pathological abnormal-ities [8 9] Indeed a number of studies have reportedincreased ROS formation in cultured cells exposed to highglucose concentrations Similarly in animal models of dia-betes ROS are generated as natural byproducts of oxygenmetabolism and theirmoderate levels are thought to functionas intracellular signaling molecules however high levels ofROS are detrimental to cardiomyocytes and lead to cell deathmitochondrial dysfunction due to mitochondrial fragmenta-tion [10] or impaired insulin signaling [11] ROS contribute toinducing various cardiovascular complications including car-diac dysfunction accelerated by inflammation apoptosis and

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 5868913 11 pageshttpdxdoiorg10115520165868913

2 Oxidative Medicine and Cellular Longevity

fibrosis [12ndash14] Furthermore O2

minus has been shown to impairnitric oxide (NO) dependent vasodilation [15 16] and thusenhance endothelium-dependent vasoconstriction [17 18]and precipitate cardiomyocyte hypertrophy [19]

ROS is generated by all cell types within the heartincluding cardiomyocytes endothelial cells vascular smoothmuscle cells (VSMCs) fibroblasts and infiltrating inflam-matory cells [20] Nicotinamide adenine dinucleotide phos-phate (NADPH) oxidases (NOXs) are membrane-associatedenzymes which are the primary physiological producersof O2

minus [21] and ROS [20 22] NOXs consist of 4 majorsubunits a plasma membrane-spanning cytochrome b

558

composed of a large gp91phox subunit and a smaller p22phox

subunit whereas 2 cytosolic subunits are p47phox and p67phoxEndothelial cells (ECs) andmyocytes express all four compo-nents of NADPHoxidase [23 24] while VSMCs express all ofthe subunits with the exception of p67phox subunit [25] Thelowmolecular weight G protein rac2 (in some cells rac1) par-ticipates in the assembly of the active complex and a secondG protein rap1A is thought to be involved in the deactivationof NADPH oxidase enzyme activity

DM is associated with increased levels of Ang II [26ndash30] and numerous studies have shown that Ang II upsurgesproduction of O

2

minus by activating NADPH oxidases [31ndash34]in VSMCs [35ndash37] adventitial fibroblasts [32 38] ECs [3339] and cardiomyocytes [34] Additionally hyperglycemiaa key clinical manifestation of diabetes also produces ROSvia NADPH oxidase activation by glycation end products[40] while blocking of ROS formation is known to preventhyperglycemic damage [41] In rat ventricularmyocytes highglucose conditions induced cardiac contractile dysfunctionand cardiomyopathy occurs via Ang II type 1 receptor (AT

1R)

mediated activation of NADPH oxidase [27 28 30] Highglucose conditions may also increase the activity of NADPHoxidase by increasing the expression of its various subunitsresulting in increased production of ROS [8 42] Infusionof Ang II has also been shown to increase NADPH oxidaseactivity and O

2

minus production by increasing expression ofp67phox and gp91phox in aortic adventitial cells [32] Exposureof ventricularmyocytes to high glucose also increases expres-sion of p47phox and production of ROS and these increaseswere blocked by AT

1R antagonist L-158809 [42] Addition-

ally Hink et al [43] found increasedNADPH oxidase activityand a 7-fold increase in gp91phox mRNA in aortic tissue fromrats with streptozotocin- (STZ-) induced diabetes

Aerobic exercise of moderate intensity and frequencyis one of a number of cardiac rehabilitation programs pre-scribed to patients with chronic heart failure [44] Exercisetraining (ExT) reduces blood glucose body fat and insulinresistance and improves glycemic control lipid metabolismand baroreflex sensitivity in diabetes [45ndash48] AdditionallyExT lowers plasma Ang II levels [49ndash51] and reduces renalsympathetic nerve activity and arterial pressure responses toAng II [50ndash53] Previous studies have also shown that ExTincreases expression of superoxide dismutase (SOD) cata-lase and glutathione in cardiac tissues [49 54ndash56]The abilityof these antioxidant enzymes to reduce ROS has also beenwell documented [57 58] However the impact of ExT on

NADPHoxidase activity and expression of its subunits in dia-betes is not entirely clear These findings have led us to inves-tigate whether diabetes-induced cardiac dysfunction may bedue in part to increased expression of p47phox and p67phox andfurther whether ExT could improveattenuate the increasedexpression of these subunits induced by diabetes

2 Materials and Methods

21 Induction andVerification of Experimental Diabetes Ani-mals used for this study were approved by the Animal Careand Use Committee of the University of Nebraska MedicalCenter and all experiments were conducted according to theAPSrsquos Guiding Principles for Research Involving Animals andHuman Beings and the National Institutes of Health Guidefor the Care and Use of Laboratory Animals Male Sprague-Dawley rats weighing between 180 and 190 g were purchasedfrom Sasco Breeding Laboratories (Omaha NE) After 1week of acclimatization in housing facilities diabetes wasinduced by a single intraperitoneal injection (65mgkg) ofstreptozotocin (STZ Sigma) in a 2 solution of cold 01Mcitrate buffer (pH 45) Onset of diabetes was identified bypolydipsia polyuria and blood glucose levels gt250mgdLControl animals were injected with citrate buffer containingno STZ Throughout the study animals were housed in pairs(similar weights to minimize dominance) at 22∘C with fixed12 h lightdark cycles and humidity at 30ndash40 Laboratorychow (Harlan Madison WI) and tap water were availablead libitum Experiments were performed 4 weeks after theinjection of STZ or vehicle

22 Exercise Protocol After one week rats in each of controland diabetic groups were randomly divided into two groupsOne subgroup of control and one subgroup of diabetic ratsremained sedentary while the other two subgroups were sub-jected to ExT according to the protocols used by Musch andTerrell [59] with modifications [60] for three weeks after oneweek after diabetes induction The resulting four subgroupsof animals were referred to as nonexercised controls (Sed-control 119899 = 16) diabetic nonexercise (Sed-Dia 119899 = 16)exercise trained controls (ExT-control 119899 = 20) and exercisetrained diabetic (ExT-Dia 119899 = 20) During the trainingperiod rats were exercised between 5 and 15minday at aninitial treadmill speed of 10mmin up a 0 grade for 5 daysIn order to ensure a significant endurance ExT regimen thetreadmill grade and speed were gradually increased to 10and 25mmin respectively and the exercise duration wasincreased to 60mindayAnimals demonstrating the ability torun steadily on the treadmill with very little or no prompting(with electrical stimulation) were used in the study The Sed-control and Sed-Dia rats were treated similarly to the ExT-control and ExT-Dia subgroup and handled daily except forthe treadmill running

23 Sample Collection At the end of the in vivo protocolanimals in all four subgroups (Sed-control Sed-Dia ExT-control and ExT-Dia) were anesthetized using overdose ofpentabarbital (65mgkg ip) Chest cavities were opened andhearts were removed quick-frozen by dropping into liquid

Oxidative Medicine and Cellular Longevity 3

nitrogen and stored at minus80∘C Soleus muscle from hind legswas also removed quick-frozen and stored at minus80∘C

24 Citrate Synthase Assay The efficacy of ExT was assessedby measuring citrate synthase activity in whole musclehomogenate as previously described [51 61] Citrate synthaseactivities were normalized to total protein content andreported as micromoles per milligram protein per minute

25 Semiquantitative RT-PCR Relative levels of p47phox andp67phox in left and right ventricles and atria tissues weredetermined using semiquantitative RT-PCR One hundredmicron (100 120583m) thick coronal sections were cut on a cryostatfrom the left and right ventricles and a 15-gauge needlestub was used to punch atrial samples Total RNA from leftventricle right ventricle and atrial tissue was isolated by TRIReagent (MRC) method as per the manufacturerrsquos instruc-tions as previously described [62 63] Equivalent amountsof total RNA (1 120583g) from each of Sed-control Sed-Dia ExT-control and ExT-Dia rats were then reverse-transcribed for40min at 37∘C in the presence of 15 120583M of random hex-amers and 100U of MMLV-Reverse transcriptase Primersfor p47phox and p67phox (200 nM) were used in polymerasechain reactions to determine the amount of transcripts ineach sample 120573-actin was coamplified as an internal controlAfter 10min of denaturing at 94∘C the amplification wasperformed at 94∘C for 1min at 56∘C for 1min and at 72∘Cfor 1min for 33 cycles with the final extension at 72∘C for10min At the end of the reaction 7 120583L from each PCRreaction wasmixed with BlueOrange loading dye (Promega)and electrophoresed for 45min at 100V using 1 agarosegels containing ethidium bromide The gels were visualizedwith gel doc system (Kodak ID gel Imager) Band intensitieswere then analyzed with the Kodak analysis software andnormalized to that of their respective 120573-actin bands Oligo-primers for PCR were synthesized in-house at Universityof Nebraska Medical Center Sequences of primers usedwere as follows p47phox 51015840-ACCTGTCGGAGAAGGTGG-T (forward) 51015840-TAGGTCTGAAGGATGATGGG (reverse)p67phox 51015840-AGGACTATCTGGGCAAGGC (forward) 51015840-GCTGCGACTGAGGGTGAAT (reverse) 120573-actin 51015840-GGG-AAATCGTGCGTGACATT (forward) 51015840-CGGATGTCA-ACGTCACACTT (reverse)

26 Western Blotting Western blot analyses were used todetermine p47phox and p67phox in the left ventricle right ven-tricle and atria tissues in the four subgroups of rats Proteinwas extracted from the heart after homogenization in RIPAbuffer (cat number BP-115 Boston BioProducts WorcesterMA USA) supplemented with 1mM phenylmethylsulfonylfluoride and protease inhibitor cocktail Collagen III levelswere measured as an index of myocardial stiffness (diabeticcardiomyopathy) in atrial tissue 30ndash40 120583g of each proteinsample was mixed with an equal volume of 4 SDS samplebuffer fractionated on 75 polyacrylamide-sodium dodecylsulfate gel and transferred to a PVDF membrane (Milli-pore) After transfer the membranes were blocked with 5

Table 1 Characteristics of the Sed-control Sed-Dia ExT-controland ExT-Dia groups 119899 = 10 in each group Values represent mean plusmnSE lowast119875 lt 001 versus Sed-control 119875 lt 005 versus Sed-Dia group

Sed-control Sed-Dia ExT-control ExT-Dia(119899 = 10) (119899 = 10) (119899 = 10) (119899 = 10)

Body weight(g) 323 plusmn 7 209 plusmn 7lowast 226 plusmn 2lowast 208 plusmn 12lowast

Blood glucose(mgdL) 72 plusmn 6 362 plusmn 18lowast 78 plusmn 7 315 plusmn 11

Citratesynthase(mmolgmin)

458 plusmn 034 401 plusmn 042 693 plusmn 056lowast 694 plusmn056lowast

nonfat dry milk powder in TBST (20mMTrisHCl pH 74150mMNaCl 01 (vv) Tween 20) at room temperaturefor 1 h Subsequently membranes were probed with a pri-mary anti-p47phox rabbit polyclonal or anti-p67phox rabbitpolyclonal or anti-collagen III goat polyclonal antibodyfrom Santa Cruz Biotechnology overnight at 4∘C followedby incubation with a corresponding peroxidase-conjugatedsecondary antibody for one hourThe signals were visualizedusing an enhanced chemiluminescence (Pierce ChemicalRockford) and detected by a UVP digital imaging systemImageJ-NIH program was used to quantify the signal

27 Statistical Analysis Data was presented as mean plusmn SEMand subjected to Student-Newman-Keuls test119875 values lt 005were considered to indicate statistical significance

3 Results

31 General Characteristics of Animals Table 1 summarizesthe characteristics of the Sed-control Sed-Dia ExT-controland ExT-Dia animals used in the present study Mean bodyweight of all animals at the start of the study was 186 plusmn 2 gAs shown in Table 1 after 28 days the mean body weight ofSed-control animals increased to 323plusmn7 g whereas the meanbodyweight of ExT-control animals was 226plusmn2 gMean bodyweight of sedentary and exercised trained diabetic animalsincreased modestly to 209 plusmn 7 and 208 plusmn 12 g respectivelyIn the animals injected with citrate buffer only mean bloodglucose levels did not change significantly during the study(720 plusmn 60mgdL at start and 780 plusmn 70mgdL at the timeof sacrifice) Sed-Dia animals had significant high bloodglucose levels at the time of sacrifice 362plusmn18mgdL whereasExT had significantly lowered blood glucose levels 315 plusmn11mgdL We have used citrate synthase activity as a markerof increased aerobic metabolism in the soleus muscle Theenzyme citrate synthase catalyzes the formation of citratefrom acetic acid and oxaloacetic acid in the first step ofKrebs cycle Regular exercise increases the size and number ofskeletal muscle mitochondria to increase respiratory capacityof the muscle Increased mitochondria translate to increasedcitrate synthase activity reflecting the increased activity ofmuscle associated with ExT [51 61] Cardiac muscle does notincrease respiratory capacity in response to ExT as skeletal


lowast

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Figure 1 Expression of left ventricle p47phox and p67phox in Sed-control Sed-Dia ExT-control and ExT-Dia animals (a) RT-PCR of p47phoxtop a representative gel bottom quantification of p47phox transcript normalized to 120573-actin as loading control 119899 = 10 in each group(b)Western blot analysis of left ventricle p47phox in four groups top a representative gel bottom bar graph of summary data of densitometryanalyses of p47phox protein level normalized to actin for loading variations 119899 = 6ndash8 in each group Values represent mean plusmn SE (c) RT-PCR ofleft ventricle p67phox in Sed-control Sed-Dia ExT-control and ExT-Dia animals top a representative gel bottom quantification of p67phoxtranscript normalized to 120573-actin as loading control 119899 = 6 in each group (d) Protein expression of p67phox in four groups top a representativeWestern blot bottom densitometry analysis of p67phox protein normalized to 120573-actin as loading control 119899 = 3-4 in each group Values arerepresented as mean plusmn SE lowast119875 lt 005 versus Sed-control +119875 lt 005 versus Sed-Dia group

muscle does [64] Therefore measuring the citrate synthaseactivity in soleus muscle is extensively used as a metabolicmarker in assessing oxidative and respiratory capacity Inthe current study Sed-control and diabetic animals had alower citrate synthase activity (458 plusmn 034 120583molgmin and401 plusmn 042 120583molgmin resp) than ExT animals (693 plusmn056 120583molgmin for ExT-control and 694plusmn056 120583molgminfor ExT-diabetic)These data also indicate that similar level ofExT was performed in the two groups of animals

32 Increased Expression of p47phox and p67phox in Left Ven-tricle Alleviated with ExT in Diabetic Animals Expressionsof p47phox and p67phox were determined by semiquantita-tive RT-PCR and Western blot using actin as an internalreference in four groups of rats namely Sed-control Sed-Dia ExT-control and ExT-Dia (Figure 1) There were sig-nificantly increased transcripts of p47phox I (458 increase)and protein (23-fold) in the left ventricle of 4-week Sed-Dia versus 4-week Sed-control rats (Figures 1(a) and 1(b))


Sed-Dia animals also demonstrated increased steady-statelevels of mRNA encoding p67phox (increased 781 ofthe 4-week Sed-control) (Figure 1(c)) and protein (16-fold)(Figure 1(d)) in the left ventricleThreeweeks of ExT initiatedafter 1 week of diabetes reduced mRNA and protein levels ofp47phox and p67phox in the left ventricle of the heart to levelssimilar to those of the ExT controls (asymp40ndash50 less than Sed-Dia)

33 Increased Expression of p47phox and p67phox in RightVentricle Alleviated with ExT in Diabetic Animals Similar tothe left ventricle 4 weeks of diabetes also increased p47phoxmRNA and protein expression in the right ventricle albeitnot reaching statistical significance compared to Sed-control(Figure 2(a)) Steady-state levels of mRNA encoding p67phoxin the right ventricle of the heart increased slightly (177)However there were no significant differences in p67phoxprotein expression between the two groups (Figures 2(c) and2(d)) Three weeks of ExT regimen attenuated the increasein steady-state levels of mRNA encoding p47phox to similarlevels in ExT-control and ExT-Dia groups

34 Increased Expression of p47phox and p67phox in the AtriaAlleviated with ExT in Diabetic Animals As shown inFigure 3 steady-state levels of mRNA encoding p47phox andp67phox were increased significantly in atria of the diabeticgroup The increase was 301 for p47phox and 355 forp67phox over sedentary controls (Figures 3(a) and 3(c)) Asexpected threeweeks of ExT initiated after 1week of diabetessignificantly lowered mRNA levels of p47phox and p67phox inthe atria of ExT-Dia heart compared to Sed-Dia group Pro-tein expression of p47phox was increased 74 while p67phoxprotein expression increased but did not reach statisticalsignificance (Figures 3(b) and 3(d)) Interestingly p47phoxmRNA and protein expression in ExT-control and ExT-Diaatria was decreased to almost similar levels suggesting thatExT attenuated the increase in steady-state levels of p47phoxexpression in atria

35 Increased Expression of Arterial Collagen III Is Allevi-ated with ExT in Diabetic Animals As shown in Figure 4expression of the extracellular matrix protein collagen IIIsignificantly increased by 53 in 4 weeks Sed-Dia groupThis increase in steady-state levels of collagen III suggestsincreasing ventricular stiffening a characteristic feature ofdiabetic cardiomyopathy Three weeks of ExT initiated after1 week of STZ-induced diabetes attenuated the increase incollagen III protein expression (38) in the diabetic groupat the same level as control while three weeks of ExT hadno effect on collagen III expression in nondiabetic controlanimals

4 Discussion

The principal finding of the present study is that expressionof the cytosolic subunits of NADPH oxidase p47phox andp67phox is upregulated in hearts of STZ-induced diabetic

rats and the increased expression was attenuated with ExTSince ExT was initiated after 1 week of diabetes it is likelythat it is preventing or minimizing rather than reversing theincrease in expression induced by diabetes Previously wehave reported that Sed-Dia animals demonstrated significantreductions in fractional shortening ejection fraction strokevolume and cardiac output compared with Sed-controlanimals and ExT (for 3weeks) implemented 4weeks after theonset of diabetes significantly increased percent ejection frac-tion attenuated the increase in left ventricular end-systolicdiameter and improved dPdt and isoproterenol inducedincrease in dPdt in the diabetic group [60] It is possible thatthese changes in expression of NADPH oxidase subunits inthe heart are associated with improvement in cardiac func-tion in diabetes

There is increasing information that mitochondria andNADPH oxidase play a fundamental role in ROS produc-tion in the diabetic heart [12] Furthermore ROS-mediatedincrease in peroxynitrite formation induces apoptosis incardiomyocytes in vitro and in the myocardium in vivo asshown by Levrand et al [65] suggesting that oxidative stressis a commonmediator for apoptosis induced cardiac damagein diabetic rats [13] Our data suggest that the increasedoxidative stress levels observed in diabetic hearts may be inpart due to the upregulation of NADPH oxidase subunits andthis change can be alleviated by ExTThese results suggest thatbeneficial effects of ExT may involve improvement in oxida-tive stress commonly observed in diabetic cardiomyopathy

Diabetic cardiomyopathy one of the leading causes ofincreasedmorbidity andmortality in the diabetic populationis characterized by the prolongation of action potential dura-tion delayed cytosolic Ca2+ clearance and impaired systolicand diastolic left ventricular function [42 60] Several factorsare believed to contribute to the pathogenesis of diabetic car-diomyopathy including insulin resistance enhanced renin-angiotensin system activation hyperglycemia and damagefrom ROS [66] These pathophysiological factors associatedwith diabetes including hyperglycemia and elevated Ang IIlevels have been shown to increase the expression ofNADPHoxidase subunits and the enzymersquos activity [32 43 67] leadingto myocardial oxidative stress A pivotal role of NADPHoxidase in Ang II-induced cardiac hypertrophy and intersti-tial fibrosis was demonstrated by using mice with targeteddisruption of the NADPH oxidase subunit gp91phox [34]p47phox subunit of NADPH oxidase has also been demon-strated to play a key role in the enzymersquos activation byagonists Using p47phox knockout mice and p47phox cDNAtransfection and antisense techniques Li et al [68] demon-strated the necessity of p47phox subunit for the activationof NADPH oxidase when exposed to the agonists Ang IITNF-120572 or phorbol 12-myristate 13-acetate an activator ofprotein kinase C The phosphorylation of p47phox is believedto induce conformational changes that facilitate its binding tothe other cytosolic subunits including p67phox and their sub-sequent binding to the membrane-bound cytochrome b

558

to allow optimal O2

minus production [69] Precise mechanismsunderlying agonist-induced nonphagocytic NADPH oxidaseactivation are not yet understood but it is clear that both




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(d)

Figure 2 Expression of right ventricle p47phox and p67phox in Sed-control Sed-Dia ExT-control and ExT-Dia animals (a) RT-PCRof p47phoxtop a representative gel bottom bar graph shows quantification of densitometry analysis of p47phox normalized to 120573-actin 119899 = 10 in eachgroup Values represent mean plusmn SE (b) Protein expression of p47phox in four groups top a representative gel bottom densitometry analysesof p47phox protein level normalized to actin 119899 = 3-4 in each group Values represent mean plusmn SE (c) RT-PCR of right ventricle p67phox inSed-control Sed-Dia ExT-control and ExT-Dia animals top a representative gel bottom quantification of p67phox transcript normalized to120573-actin as loading control 119899 = 6 in each group (d) Protein expression of p67phox in four groups top a representative Western blot bottomdensitometry analysis of p67phox protein normalized to 120573-actin as loading control 119899 = 3-4 in each group Values are represented as mean plusmnSE lowast119875 lt 005 versus Sed-control +119875 lt 005 versus Sed-Dia group

acute proteinmodification and chronic changes in expressionlevels may be involved

Our findings indicate that the cardiac tissue expressionsfor p47phox and p67phox are upregulated in the diabetic ratsThis upregulation was greatest in the left ventricle and atriawhile it was slightly elevated in the right ventricle of thediabetic group These findings are consistent with clinicaldata showing an association of DM with an increased riskof systolic diastolic and any left ventricular dysfunction andthe relative sparing of the right ventricle [1 2] In ventricularmyocytes it was reported that hyperglycemic conditions

increase the production of ROS via an enhanced expressionof p47phox protein which could be blocked by an AT

1R

antagonist [42] Cifuentes et al [32] have also shown thatAng II infusion via theAT

1Ractivation induces an enhanced

expression of p67phox and gp91phox proteins in aortic adventi-tia Additionally an increased NADPH oxidase activity and a7-fold upregulation of gp91phox mRNA in aortic tissue havebeen reported in STZ-induced diabetic rats [43] Further-more increased NADPH oxidase activity and collagen 3 weredemonstrated in pathogenesis of the heart failure as seenin hearts of Duchenne muscular dystrophy rat model [70]




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Figure 3 Expression of atrium p47phox and p67phox in Sed-control Sed-Dia ExT-control and ExT-Dia animals (a) RT-PCR of p47phox topa representative gel bottom bar graph shows quantification of densitometry analysis of p47phox normalized to 120573-actin 119899 = 10 in each groupValues represent mean plusmn SE (b) Protein expression of p47phox in four groups top a representative gel bottom densitometry analyses ofp47phox protein level normalized to actin 119899 = 6ndash8 in each group Values represent mean plusmn SE (c) RT-PCR of atrium p67phox in Sed-controlSed-Dia ExT-control and ExT-Dia animals top a representative gel bottom quantification of p67phox transcript normalized to 120573-actin asloading control 119899 = 6 in each group (d) Protein expression of p67phox in four groups top a representativeWestern blot bottom densitometryanalysis of p67phox protein normalized to 120573-actin as loading control 119899 = 3-4 in each group Values are represented as mean plusmn SE lowast119875 lt 005versus Sed-control +119875 lt 005 versus Sed-Dia group

and also a significant increase in collagen III was found inendomyocardial biopsies obtained from patients with DM[71] Ang II has also been shown to stimulate collagen I andcollagen III in cardiac fibroblast and apocynin an inhibitorof NADPH oxidase prevents this initiation suggesting acritical role for ROS in cardiac remodelling [72] Althougha medical treatment with different antioxidant has beenproposed for diabetic cardiomyopathy for decades unfortu-nately such treatments have failed to attenuate cardiac dys-function or improve outcomes [73] This may be due to lackof improving the ROS load in an appropriate measure in themyocardium rather than an overall reduction in ROS

Physical activity is widely accepted as a key element inthe prevention of type 2 diabetes [74] and also has bene-ficial effects in patients with established heart disease [75]although the physiologicalmechanisms explaining howphys-ical activity promotes health remain to be fully elucidatedExT maintains cardiac output by blunting diabetes-inducedbradycardia and the reduction in force ofmyocardial contrac-tility [60] ExT has been shown to elicit a number of beneficialphysiological changes in animals and clinical studies Theseinclude (1) a reduction in blood glucose body fat andinsulin resistance [45 47] (2) improved glycemic control andlipid metabolism [45 76] (3) improved baroreflex sensitivity




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Tubulin

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Figure 4 Collagen III expression in atria top representativeWestern blot showing collagen III protein expression in Sed-controlSed-Dia ExT-control and ExT-Dia animals bottom densitometryanalysis of collagen III expression normalized to 120573-tubulin asloading control 119899 = 6 in each group Values represent mean plusmn SElowast119875 lt 001 versus Sed-control group +119875 lt 005 versus Sed-Diagroup

[46] (4) reduced plasma Ang II levels [49ndash51 54] and (5)reduced renal sympathetic nerve activity and arterial pressureresponses to Ang II [50 52 54 77] Additionally the ability ofExT to upregulate antioxidant enzyme expression and activityin the cardiovascular system has been clearly demonstrated[48 54 55 78 79] Previously our lab showed ExT ini-tiated after the onset of diabetes blunts primarily beta(1)-adrenoceptor expression loss and improves cardiac functionin diabetic rats [60]

The current study adds to this literature by demonstratingfor the first time that ExT significantly reduces p47phoxmRNA expression in the left and right ventricles and atriaof diabetic rats In diabetic rats ExT significantly reducedp67phox mRNA expression of the left ventricle and attenuatedthe increased p67phox message in the atria The increase inNADPH oxidase subunit expression observed in the diabeticcondition and its attenuation by ExT may occur via a varietyof mechanisms Hyperglycemia enhanced renin-angiotensinsystem activity and elevated levels of circulating cytokinesare all clinical manifestations associated with the diabeticcondition and each is known to promote NADPH-derivedO2

minus production TNF-120572 a cytokine that activates transcrip-tion factors which induce the expression of genes involved ininflammation and cell growth has been identified as an ago-nist for NADPH oxidase [68] Levels of TNF-120572 are increasedin response to both hyperglycemic conditions and elevatedAng II levels and the increase is mediated via AT

1R [68 80]

Hyperglycemia and elevated Ang II levels have been clearlyshown to upregulate the expression of NADPH oxidasesubunits and increase NADPH oxidase activity and O

2

minus

production [32 39 67 81] Based on these findings it appearsthat hyperglycemia and elevated Ang II associated with dia-betes work via the AT

1receptor to increase TNF-120572 levels and

Diabetes(hyperglycemia)

uarr expression of p47phox and p67phox

Reactive oxygen species(ROS)

Increased collagen III

ExT

Cardiomyopathy(heart failure)

Figure 5 Amelioration of cardiomyopathy by exercise trainingHyperglycemia induces overexpression of cytoplasmic subunits ofNADH oxidase (p47phox and p67phox) in left ventricle Overexpres-sion of these subunits exhibits high reactive oxygen species andleads to increased collagen III and hence cardiomyopathy Exercisetraining (ExT) mitigates the expression of p47phox and p67phoxconsequently ameliorates heart dysfunctions

NADPH oxidase activity while ExT suppresses expressionof TNF-alpha and thus offers a potential protection againstTNF-alpha-induced insulin resistance [82] and increasedROS production [83 84]

Our study provides evidence that increase in oxidativestress specifically in left ventricle may be due to an enhancedtranscription as well as translation of p47phox and p67phox

subunits of NADPH oxidase Expression of p67phox wasincreased at transcription level but not at the protein level inatria of diabetic rats suggesting that increased transcriptiondid not contribute to changes in protein expression in thistissue Translocation of cytosolic subunits (p47phox p67phoxp40phox and rac1) and their association with membrane-bound cytochrome b

558(consisting of one p22phox and one

gp91phox subunit) follows during acute oxidase activation[85] In present studies perhaps there was only translocationof p67phox in the atria of diabetic heart rather than overallexpression of synthesis a possibility that remains to beexplored

The numerous physiological benefits of ExT offer sev-eral mechanisms via which the expression and activity ofNADPH oxidase may be attenuated in diabetes ExT hasbeen shown to normalize diabetes-related elevations in bloodglucose [45ndash47] plasma Ang II [49 50 53] and TNF-120572[83 84] Normalizing these factors could lower levels of ROS-inducing agonists thus dampening NADPH oxidase activityAdditionally ExT has repeatedly been shown to upregulateantioxidant expression and activity including SOD catalaseand glutathione in aortic and cardiac tissues [48 49 5456 78 79] However while the literature provides someinsight the specific mechanism by which ExT lowered themRNAexpression of p47phox and p67phox in our current studyremains to be elucidated


5 Conclusions

In summary our data show that themRNA expressions of theNADPH oxidase subunits p47phox and p67phox are upregu-lated in cardiac tissue in the diabetic condition Furthermoreour data show that ExT attenuates the upregulated expres-sion of these NADPH oxidase subunits and normalizes theincreased collagen III levels (Figure 5) and provides supportwith a potentialmechanistic link for exercise training as beingan effective nonpharmacological tool in regulating oxidativestress levels in the diabetic heart Future areas of researchwill need to focus on understanding some of the mechanismsinvolved in diabetic cardiomyopathy and the therapeuticstrategies such as ExT to halt or slow its progression

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Funding fromNational Institutes of Health Heart Lung andBlood Institute R56HL124104 andAmericanHeart Associa-tion 14SDG19980007 grant supported theworkThe technicalassistance of Dr Xuefei Liu is greatly appreciated

References

[1] S Dandamudi J Slusser D W Mahoney M M Redfield RJ Rodeheffer and H H Chen ldquoThe prevalence of diabeticcardiomyopathy a population-based study in Olmsted CountyMinnesotardquo Journal of Cardiac Failure vol 20 no 5 pp 304ndash309 2014

[2] R B Devereux M J Roman M Paranicas et al ldquoImpact ofdiabetes on cardiac structure and function the Strong Heartstudyrdquo Circulation vol 101 no 19 pp 2271ndash2276 2000

[3] A Ilercil R B Devereux M J Roman et al ldquoRelationship ofimpaired glucose tolerance to left ventricular structure andfunction the Strong Heart studyrdquo American Heart Journal vol141 no 6 pp 992ndash998 2001

[4] S Rubler J Dlugash Y Z Yuceoglu T Kumral A W Bran-wood and A Grishman ldquoNew type of cardiomyopathy asso-ciated with diabetic glomerulosclerosisrdquo The American Journalof Cardiology vol 30 no 6 pp 595ndash602 1972

[5] S Boudina and E D Abel ldquoDiabetic cardiomyopathy revisitedrdquoCirculation vol 115 no 25 pp 3213ndash3223 2007

[6] A Aneja W H W Tang S Bansilal M J Garcia and M EFarkouh ldquoDiabetic cardiomyopathy insights into pathogenesisdiagnostic challenges and therapeutic optionsrdquo American Jour-nal of Medicine vol 121 no 9 pp 748ndash757 2008

[7] I Falcao-Pires and A F Leite-Moreira ldquoDiabetic cardiomyopa-thy understanding the molecular and cellular basis to progressin diagnosis and treatmentrdquo Heart Failure Reviews vol 17 no3 pp 325ndash344 2012

[8] J W Baynes and S R Thorpe ldquoRole of oxidative stress indiabetic complications a new perspective on an old paradigmrdquoDiabetes vol 48 no 1 pp 1ndash9 1999

[9] D Giugliano A Ceriello and G Paolisso ldquoOxidative stress anddiabetic vascular complicationsrdquo Diabetes Care vol 19 no 3pp 257ndash267 1996

[10] T Yu S-S Sheu J L Robotham and Y Yoon ldquoMitochondrialfission mediates high glucose-induced cell death through ele-vated production of reactive oxygen speciesrdquo CardiovascularResearch vol 79 no 2 pp 341ndash351 2008

[11] S Boudina H Bugger S Sena et al ldquoContribution of impairedmyocardial insulin signaling to mitochondrial dysfunction andoxidative stress in the heartrdquoCirculation vol 119 no 9 pp 1272ndash1283 2009

[12] Y Teshima N Takahashi S Nishio et al ldquoProduction of reac-tive oxygen species in the diabetic heart roles of mitochondriaand NADPH oxidaserdquo Circulation Journal vol 78 no 2 pp300ndash306 2014

[13] MM Dallak D P Mikhailidis M A Haidara et al ldquoOxidativestress as a common mediator for apoptosis induced-cardiacdamage in diabetic ratsrdquoOpenCardiovascularMedicine Journalvol 2 no 1 pp 70ndash78 2008

[14] N Takahashi O Kume O Wakisaka et al ldquoNovel strategy toprevent atrial fibrosis and fibrillationrdquo Circulation Journal vol76 no 10 pp 2318ndash2326 2012

[15] R J Gryglewski R M J Palmer and S Moncada ldquoSuperoxideanion is involved in the breakdown of endothelium-derivedvascular relaxing factorrdquoNature vol 320 no 6061 pp 454ndash4561986

[16] A Mugge J H Elwell T E Peterson and D G HarrisonldquoRelease of intact endothelium-derived relaxing factor dependson endothelial superoxide dismutase activityrdquo The AmericanJournal of PhysiologymdashCell Physiology vol 260 no 2 part 1 ppC219ndashC225 1991

[17] B Tesfamariam and R A Cohen ldquoEnhanced adrenergic neu-rotransmission in diabetic rabbit carotid arteryrdquoCardiovascularResearch vol 29 no 4 pp 549ndash554 1995

[18] Z S Katusic and P M Vanhoutte ldquoSuperoxide anion is anendothelium-derived contracting factorrdquoTheAmerican Journalof PhysiologymdashHeart and Circulatory Physiology vol 257 no 1part 2 pp H33ndashH37 1989

[19] P H Sugden and A Clerk ldquoCellular mechanisms of cardiachypertrophyrdquo Journal of Molecular Medicine vol 76 no 11 pp725ndash746 1998

[20] A Akki M Zhang C Murdoch A Brewer and A M ShahldquoNADPH oxidase signaling and cardiac myocyte functionrdquoJournal of Molecular and Cellular Cardiology vol 47 no 1 pp15ndash22 2009

[21] K K Griendling D Sorescu and M Ushio-Fukai ldquoNAD(P)Hoxidase role in cardiovascular biology and diseaserdquo CirculationResearch vol 86 no 5 pp 494ndash501 2000

[22] H D Wang S Xu D G Johns et al ldquoRole of NADPH oxidasein the vascular hypertrophic and oxidative stress response toangiotensin II in micerdquo Circulation Research vol 88 no 9 pp947ndash953 2001

[23] J-M Li and A M Shah ldquoDifferential NADPH- versus NADH-dependent superoxide production by phagocyte-type endothe-lial cell NADPH oxidaserdquo Cardiovascular Research vol 52 no3 pp 477ndash486 2001

[24] A Cave D Grieve S Johar M Zhang and A M ShahldquoNADPH oxidase-derived reactive oxygen species in cardiacpathophysiologyrdquoPhilosophical Transactions of the Royal SocietyB Biological Sciences vol 360 no 1464 pp 2327ndash2334 2005

[25] C Patterson J Ruef N R Madamanchi et al ldquoStimulation ofa vascular smooth muscle cell NAD(P)H oxidase by thrombinevidence that p47119901ℎ119900119909may participate in forming this oxidase invitro and in vivordquo Journal of Biological Chemistry vol 274 no28 pp 19814ndash19822 1999


[26] K P Patel W G Mayhan K R Bidasee and H ZhengldquoEnhanced angiotensin II-mediated central sympathoexcita-tion in streptozotocin-induced diabetes role of superoxideanionrdquo American Journal of PhysiologymdashRegulatory IntegrativeandComparative Physiology vol 300 no 2 pp R311ndashR320 2011

[27] H M Siragy ldquoAT(1) and AT(2) receptors in the kidney rolein disease and treatmentrdquo American Journal of Kidney Diseasesvol 36 no 3 supplement 1 pp S4ndashS9 2000

[28] J R Sowers M Epstein and E D Frohlich ldquoDiabetes hyper-tension and cardiovascular diseasemdashan updaterdquo Hypertensionvol 37 no 4 pp 1053ndash1059 2001

[29] V Koshkin O Lotan and E Pick ldquoThe cytosolic componentp47(phox) is not a sine qua non participant in the activationof NADPH oxidase but is required for optimal superoxideproductionrdquo The Journal of Biological Chemistry vol 271 no48 pp 30326ndash30329 1996

[30] HM Siragy A Awad P Abadir andRWebb ldquoThe angiotensinII type 1 receptor mediates renal interstitial content of tumornecrosis factor-alpha in diabetic ratsrdquo Endocrinology vol 144no 6 pp 2229ndash2233 2003

[31] K K Griendling C A Minieri J D Ollerenshaw and RW Alexander ldquoAngiotensin II stimulates NADH and NADPHoxidase activity in cultured vascular smooth muscle cellsrdquoCirculation Research vol 74 no 6 pp 1141ndash1148 1994

[32] M E Cifuentes F E Rey O A Carretero and P J PaganoldquoUpregulation of p67(phox) and gp91(phox) in aortas fromangiotensin II-infused micerdquo The American Journal of Physi-ologymdashHeart and Circulatory Physiology vol 279 no 5 ppH2234ndashH2240 2000

[33] D Lang S IMosfer A Shakesby FDonaldson andM J LewisldquoCoronary microvascular endothelial cell redox state in leftventricular hypertrophy the role of angiotensin IIrdquo CirculationResearch vol 86 no 4 pp 463ndash469 2000

[34] J K Bendall A C Cave C Heymes N Gall and A MShah ldquoPivotal role of a gp91phox-containing NADPH oxidase inangiotensin II-induced cardiac hypertrophy in micerdquo Circula-tion vol 105 no 3 pp 293ndash296 2002

[35] K K Griendling T JMurphy and RWAlexander ldquoMolecularbiology of the renin-angiotensin systemrdquoCirculation vol 87 no6 pp 1816ndash1828 1993

[36] K K Griendling D Sorescu B Lassegue and M Ushio-FukaildquoModulation of protein kinase activity and gene expression byreactive oxygen species and their role in vascular physiologyand pathophysiologyrdquo Arteriosclerosis Thrombosis and Vascu-lar Biology vol 20 no 10 pp 2175ndash2183 2000

[37] K K Griendling andM Ushio-Fukai ldquoReactive oxygen speciesas mediators of angiotensin II signalingrdquo Regulatory Peptidesvol 91 no 1ndash3 pp 21ndash27 2000

[38] P J Pagano J K Clark M E Cifuentes-Pagano S M ClarkG M Callis and M T Quinn ldquoLocalization of a constitutivelyactive phagocyte-like NADPH oxidase in rabbit aortic adventi-tia enhancement by angiotensin IIrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14483ndash14488 1997

[39] H Zhang A Schmeisser C D Garlichs et al ldquoAngiotensinII-induced superoxide anion generation in human vascularendothelial cells role of membrane-bound NADH-NADPH-oxidasesrdquo Cardiovascular Research vol 44 no 1 pp 215ndash2221999

[40] M Zhang A L Kho N Anilkumar et al ldquoGlycated pro-teins stimulate reactive oxygen species production in car-diac myocytes involvement of Nox2 (gp91phox)-containing

NADPH oxidaserdquo Circulation vol 113 no 9 pp 1235ndash12432006

[41] S Nishio Y TeshimaN Takahashi et al ldquoActivation of CaMKIIas a key regulator of reactive oxygen species production indiabetic rat heartrdquo Journal of Molecular and Cellular Cardiologyvol 52 no 5 pp 1103ndash1111 2012

[42] J R Privratsky L EWold J R SowersM T Quinn and J RenldquoAT1blockade prevents glucose-induced cardiac dysfunction

in ventricular myocytes role of the AT1receptor and NADPH

oxidaserdquo Hypertension vol 42 no 2 pp 206ndash212 2003[43] U Hink H Li H Mollnau et al ldquoMechanisms underly-

ing endothelial dysfunction in diabetes mellitusrdquo CirculationResearch vol 88 no 2 pp E14ndashE22 2001

[44] D H J Thijssen A J Maiorana G OrsquoDriscoll N T CableM T E Hopman and D J Green ldquoImpact of inactivity andexercise on the vasculature in humansrdquo European Journal ofApplied Physiology vol 108 no 5 pp 845ndash875 2010

[45] S Li B Culver and J Ren ldquoBenefit and risk of exercise onmyocardial function in diabetesrdquoPharmacological Research vol48 no 2 pp 127ndash132 2003

[46] A Loimaala H V Huikuri T Koobi M Rinne A Nenonenand I Vuori ldquoExercise training improves baroreflex sensitivityin type 2 diabetesrdquo Diabetes vol 52 no 7 pp 1837ndash1842 2003

[47] S Rousseau-Migneron L Turcotte G Tancrede and ANadeau ldquoTransient increase in basal insulin levels in severelydiabetic rats submitted to physical trainingrdquo Diabetes Researchvol 9 no 2 pp 97ndash100 1988

[48] M E Davis H Cai L McCann T Fukai and D G HarrisonldquoRole of c-Src in regulation of endothelial nitric oxide synthaseexpression during exercise trainingrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 284 no 4pp H1449ndashH1453 2003

[49] H Kohno S Furukawa H Naito K Minamitani D Ohmoriand F Yamakura ldquoContribution of nitric oxide angiotensin IIand superoxide dismutase to exercise-induced attenuation ofblood pressure elevation in spontaneously hypertensive ratsrdquoJapanese Heart Journal vol 43 no 1 pp 25ndash34 2002

[50] R W Braith and D G Edwards ldquoNeurohormonal abnormal-ities in heart failure impact of exercise trainingrdquo CongestiveHeart Failure vol 9 no 2 pp 70ndash76 2003

[51] T L Llewellyn NM SharmaH Zheng andK P Patel ldquoEffectsof exercise training on SFO-mediated sympathoexcitation dur-ing chronic heart failurerdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 306 no 1 pp H121ndashH1312014

[52] C E Negrao M C Irigoyen E D Moreira P C Brum PM Freire and E M Krieger ldquoEffect of exercise training onRSNA baroreflex control and blood pressure responsivenessrdquoThe American Journal of Physiology vol 265 no 2 part 2 ppR365ndashR370 1993

[53] I H Zucker and R U Pliquett ldquoNovel mechanisms ofsympatho-excitation in chronic heart failurerdquo Heart FailureMonitor vol 3 no 1 pp 2ndash7 2002

[54] K Husain ldquoInteraction of physical training and chronic nitro-glycerin treatment on blood pressure nitric oxide and oxi-dantsantioxidants in the rat heartrdquo Pharmacological Researchvol 48 no 3 pp 253ndash261 2003

[55] T Fukai M R Siegfried M Ushio-Fukai Y Cheng G Kojdaand D G Harrison ldquoRegulation of the vascular extracellularsuperoxide dismutase by nitric oxide and exercise trainingrdquoJournal of Clinical Investigation vol 105 no 11 pp 1631ndash16392000


[56] S K Powers S L Lennon J Quindry and J LMehta ldquoExerciseand cardioprotectionrdquo Current Opinion in Cardiology vol 17no 5 pp 495ndash502 2002

[57] I Fridovich ldquoThe biology of oxygen radicalsrdquo Science vol 201no 4359 pp 875ndash880 1978

[58] R Balzan D R Agius and W H Bannister ldquoCloned prokary-otic iron superoxide dismutase protects yeast cells againstoxidative stress depending on mitochondrial locationrdquo Bio-chemical and Biophysical Research Communications vol 256no 1 pp 63ndash67 1999

[59] T I Musch and J A Terrell ldquoSkeletal muscle blood flow abnor-malities in rats with a chronic myocardial infarction rest andexerciserdquo The American Journal of PhysiologymdashHeart and Cir-culatory Physiology vol 262 no 2 pp H411ndashH419 1992

[60] K R Bidasee H Zheng C-H Shao S K Parbhu G J Rozan-ski and K P Patel ldquoExercise training initiated after the onset ofdiabetes preserves myocardial function effects on expression ofbeta-adrenoceptorsrdquo Journal of Applied Physiology vol 105 no3 pp 907ndash914 2008

[61] P A Srere ldquo[1] Citrate synthase [EC 4137 Citrate oxaloace-tate-lyase (CoA-acetylating)]rdquo Methods in Enzymology vol 13pp 3ndash11 1969

[62] N M Sharma H Zheng P P Mehta Y-F Li and K P PatelldquoDecreased nNOS in the PVN leads to increased sympathoex-citation in chronic heart failure role for CAPON and Ang IIrdquoCardiovascular Research vol 92 no 2 pp 348ndash357 2011

[63] N M Sharma H Zheng Y-F Li and K P Patel ldquoNitric oxideinhibits the expression of AT

1receptors in neuronsrdquo American

Journal of Physiology Cell Physiology vol 302 no 8 pp C1162ndashC1173 2012

[64] P M Siu D A Donley R W Bryner and S E Alway ldquoCitratesynthase expression and enzyme activity after endurance train-ing in cardiac and skeletal musclesrdquo Journal of Applied Physiol-ogy vol 94 no 2 pp 555ndash560 2003

[65] S Levrand C Vannay-Bouchiche B Pesse et al ldquoPeroxynitriteis a major trigger of cardiomyocyte apoptosis in vitro and invivordquo Free Radical Biology and Medicine vol 41 no 6 pp 886ndash895 2006

[66] V J Dzau ldquoTissue angiotensin and pathobiology of vasculardisease a unifying hypothesisrdquo Hypertension vol 37 no 4 pp1047ndash1052 2001

[67] T Fukui N Ishizaka S Rajagopalan et al ldquop22phox mRNAexpression and NADPH oxidase activity are increased in aortasfrom hypertensive ratsrdquo Circulation Research vol 80 no 1 pp45ndash51 1997

[68] J-M Li A M Mullen S Yun et al ldquoEssential role of theNADPH oxidase subunit p47119901ℎ119900119909 in endothelial cell superoxideproduction in response to phorbol ester and tumor necrosisfactor-120572rdquo Circulation Research vol 90 no 2 pp 143ndash150 2002

[69] B M Babior ldquoNADPH oxidase an updaterdquo Blood vol 93 no5 pp 1464ndash1476 1999

[70] I A Williams and D G Allen ldquoThe role of reactive oxygenspecies in the hearts of dystrophin-deficient mdx micerdquo TheAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 293 no 3 pp H1969ndashH1977 2007

[71] M Shimizu K Umeda N Sugihara et al ldquoCollagen remod-elling inmyocardia of patients with diabetesrdquo Journal of ClinicalPathology vol 46 no 1 pp 32ndash36 1993

[72] P J Lijnen J F van Pelt and R H Fagard ldquoStimulation of reac-tive oxygen species and collagen synthesis by angiotensin II incardiac fibroblastsrdquo Cardiovascular Therapeutics vol 30 no 1pp e1ndashe8 2012

[73] J Kasznicki and J Drzewoski ldquoHeart failure in the diabeticpopulationmdashpathophysiology diagnosis and managementrdquoArchives of Medical Science vol 10 no 3 pp 546ndash556 2014

[74] P De Feo and P Schwarz ldquoIs physical exercise a core therapeu-tical element for most patients with type 2 diabetesrdquo DiabetesCare vol 36 supplement 2 pp S149ndashS154 2013

[75] J Myers M Prakash V Froelicher D Do S Partington andJ Edwin Atwood ldquoExercise capacity and mortality amongmen referred for exercise testingrdquo The New England Journal ofMedicine vol 346 no 11 pp 793ndash801 2002

[76] J M Goguen and L A Leiter ldquoLipids and diabetes mellitusa review of therapeutic optionsrdquo Current Medical Research andOpinion vol 18 supplement 1 pp s58ndashs74 2002

[77] H Zheng N M Sharma X Liu and K P Patel ldquoExercisetraining normalizes enhanced sympathetic activation fromthe paraventricular nucleus in chronic heart failure role ofangiotensin IIrdquo American Journal of Physiology RegulatoryIntegrative and Comparative Physiology vol 303 no 4 ppR387ndashR394 2012

[78] J W E Rush J R Turk and M H Laughlin ldquoExercise trainingregulates SOD-1 and oxidative stress in porcine aortic endothe-liumrdquoThe American Journal of PhysiologymdashHeart and Circula-tory Physiology vol 284 no 4 pp H1378ndashH1387 2003

[79] K Husain and S R Hazelrigg ldquoOxidative injury due to chronicnitric oxide synthase inhibition in rat effect of regular exerciseon the heartrdquo Biochimica et Biophysica Acta (BBA)mdashMolecularBasis of Disease vol 1587 no 1 pp 75ndash82 2002

[80] N ShanmugamMA ReddyMGuha andRNatarajan ldquoHighglucose-induced expression of proinflammatory cytokine andchemokine genes in monocytic cellsrdquo Diabetes vol 52 no 5pp 1256ndash1264 2003

[81] S Rajagopalan S Kurz T Munzel et al ldquoAngiotensin II-mediated hypertension in the rat increases vascular superoxideproduction via membrane NADHNADPH oxidase activationContribution to alterations of vasomotor tonerdquo The Journal ofClinical Investigation vol 97 no 8 pp 1916ndash1923 1996

[82] A M W Petersen and B K Pedersen ldquoThe anti-inflammatoryeffect of exerciserdquo Journal of Applied Physiology vol 98 no 4pp 1154ndash1162 2005

[83] W Aldhahi and O Hamdy ldquoAdipokines inflammation and theendothelium in diabetesrdquo Current Diabetes Reports vol 3 no4 pp 293ndash298 2003

[84] M Straczkowski I Kowalska S Dzienis-Straczkowska et alldquoChanges in tumor necrosis factor-alpha system and insulinsensitivity during an exercise training program in obese womenwith normal and impaired glucose tolerancerdquo European Journalof Endocrinology vol 145 no 3 pp 273ndash280 2001

[85] Z Guo Z Xia J Jiang and J H McNeill ldquoDownregulation ofNADPH oxidase antioxidant enzymes and inflammatorymarkers in the heart of streptozotocin-induced diabetic rats byN-acetyl-L-cysteinerdquoAmerican Journal of Physiology Heart andCirculatory Physiology vol 292 no 4 pp H1728ndashH1736 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



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BioMed Research International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


fibrosis [12ndash14] Furthermore O2

minus has been shown to impairnitric oxide (NO) dependent vasodilation [15 16] and thusenhance endothelium-dependent vasoconstriction [17 18]and precipitate cardiomyocyte hypertrophy [19]

ROS is generated by all cell types within the heartincluding cardiomyocytes endothelial cells vascular smoothmuscle cells (VSMCs) fibroblasts and infiltrating inflam-matory cells [20] Nicotinamide adenine dinucleotide phos-phate (NADPH) oxidases (NOXs) are membrane-associatedenzymes which are the primary physiological producersof O2

minus [21] and ROS [20 22] NOXs consist of 4 majorsubunits a plasma membrane-spanning cytochrome b

558

composed of a large gp91phox subunit and a smaller p22phox

subunit whereas 2 cytosolic subunits are p47phox and p67phoxEndothelial cells (ECs) andmyocytes express all four compo-nents of NADPHoxidase [23 24] while VSMCs express all ofthe subunits with the exception of p67phox subunit [25] Thelowmolecular weight G protein rac2 (in some cells rac1) par-ticipates in the assembly of the active complex and a secondG protein rap1A is thought to be involved in the deactivationof NADPH oxidase enzyme activity

DM is associated with increased levels of Ang II [26ndash30] and numerous studies have shown that Ang II upsurgesproduction of O

2

minus by activating NADPH oxidases [31ndash34]in VSMCs [35ndash37] adventitial fibroblasts [32 38] ECs [3339] and cardiomyocytes [34] Additionally hyperglycemiaa key clinical manifestation of diabetes also produces ROSvia NADPH oxidase activation by glycation end products[40] while blocking of ROS formation is known to preventhyperglycemic damage [41] In rat ventricularmyocytes highglucose conditions induced cardiac contractile dysfunctionand cardiomyopathy occurs via Ang II type 1 receptor (AT

1R)

mediated activation of NADPH oxidase [27 28 30] Highglucose conditions may also increase the activity of NADPHoxidase by increasing the expression of its various subunitsresulting in increased production of ROS [8 42] Infusionof Ang II has also been shown to increase NADPH oxidaseactivity and O

2

minus production by increasing expression ofp67phox and gp91phox in aortic adventitial cells [32] Exposureof ventricularmyocytes to high glucose also increases expres-sion of p47phox and production of ROS and these increaseswere blocked by AT

1R antagonist L-158809 [42] Addition-

ally Hink et al [43] found increasedNADPH oxidase activityand a 7-fold increase in gp91phox mRNA in aortic tissue fromrats with streptozotocin- (STZ-) induced diabetes

Aerobic exercise of moderate intensity and frequencyis one of a number of cardiac rehabilitation programs pre-scribed to patients with chronic heart failure [44] Exercisetraining (ExT) reduces blood glucose body fat and insulinresistance and improves glycemic control lipid metabolismand baroreflex sensitivity in diabetes [45ndash48] AdditionallyExT lowers plasma Ang II levels [49ndash51] and reduces renalsympathetic nerve activity and arterial pressure responses toAng II [50ndash53] Previous studies have also shown that ExTincreases expression of superoxide dismutase (SOD) cata-lase and glutathione in cardiac tissues [49 54ndash56]The abilityof these antioxidant enzymes to reduce ROS has also beenwell documented [57 58] However the impact of ExT on

NADPHoxidase activity and expression of its subunits in dia-betes is not entirely clear These findings have led us to inves-tigate whether diabetes-induced cardiac dysfunction may bedue in part to increased expression of p47phox and p67phox andfurther whether ExT could improveattenuate the increasedexpression of these subunits induced by diabetes

2 Materials and Methods

21 Induction andVerification of Experimental Diabetes Ani-mals used for this study were approved by the Animal Careand Use Committee of the University of Nebraska MedicalCenter and all experiments were conducted according to theAPSrsquos Guiding Principles for Research Involving Animals andHuman Beings and the National Institutes of Health Guidefor the Care and Use of Laboratory Animals Male Sprague-Dawley rats weighing between 180 and 190 g were purchasedfrom Sasco Breeding Laboratories (Omaha NE) After 1week of acclimatization in housing facilities diabetes wasinduced by a single intraperitoneal injection (65mgkg) ofstreptozotocin (STZ Sigma) in a 2 solution of cold 01Mcitrate buffer (pH 45) Onset of diabetes was identified bypolydipsia polyuria and blood glucose levels gt250mgdLControl animals were injected with citrate buffer containingno STZ Throughout the study animals were housed in pairs(similar weights to minimize dominance) at 22∘C with fixed12 h lightdark cycles and humidity at 30ndash40 Laboratorychow (Harlan Madison WI) and tap water were availablead libitum Experiments were performed 4 weeks after theinjection of STZ or vehicle

22 Exercise Protocol After one week rats in each of controland diabetic groups were randomly divided into two groupsOne subgroup of control and one subgroup of diabetic ratsremained sedentary while the other two subgroups were sub-jected to ExT according to the protocols used by Musch andTerrell [59] with modifications [60] for three weeks after oneweek after diabetes induction The resulting four subgroupsof animals were referred to as nonexercised controls (Sed-control 119899 = 16) diabetic nonexercise (Sed-Dia 119899 = 16)exercise trained controls (ExT-control 119899 = 20) and exercisetrained diabetic (ExT-Dia 119899 = 20) During the trainingperiod rats were exercised between 5 and 15minday at aninitial treadmill speed of 10mmin up a 0 grade for 5 daysIn order to ensure a significant endurance ExT regimen thetreadmill grade and speed were gradually increased to 10and 25mmin respectively and the exercise duration wasincreased to 60mindayAnimals demonstrating the ability torun steadily on the treadmill with very little or no prompting(with electrical stimulation) were used in the study The Sed-control and Sed-Dia rats were treated similarly to the ExT-control and ExT-Dia subgroup and handled daily except forthe treadmill running

23 Sample Collection At the end of the in vivo protocolanimals in all four subgroups (Sed-control Sed-Dia ExT-control and ExT-Dia) were anesthetized using overdose ofpentabarbital (65mgkg ip) Chest cavities were opened andhearts were removed quick-frozen by dropping into liquid














3 Results



lowast

+



p47

phox

120573-a

ctin

p47phox

120573-actin

00

03

05

08

10

(a)

lowast

+



p47phox

120573-actin

0

02

04

06

08

1

12

p47

phox

120573-a

ctin

(b)

lowast

+



p67phox

120573-actin

00

05

10

15

20

p67

phox

120573-a

ctin

(c)

lowast

+



p67phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p67

phox

120573-a

ctin

(d)









4 Discussion





558

to allow optimal O2





p47

phox

120573-a

ctin

lowast+

p47phox

120573-actin

00

02

04

06

08

(a)



p47phox

120573-actin

01

02

03

04

05

06

07

08

09

p47

phox

120573-a

ctin

(b)



lowast

+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

01

02

03

04

05

06

07

p67

phox

120573-a

ctin

(d)





1R







p47

phox

120573-a

ctin

lowast

lowast+

p47phox

120573-actin

00

04

08

12

16

(a)



lowast

lowast +

p47phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p47

phox

120573-a

ctin

(b)



lowast+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

02

04

06

08

1

12

p67

phox

120573-a

ctin

(d)







lowast

+

Collagen III

Tubulin

00

04

08

12

16

20

Col

lage

n II

Itub

ulin





1R [68 80]


2

minus







ExT










5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results



lowast

+



p47

phox

120573-a

ctin

p47phox

120573-actin

00

03

05

08

10

(a)

lowast

+



p47phox

120573-actin

0

02

04

06

08

1

12

p47

phox

120573-a

ctin

(b)

lowast

+



p67phox

120573-actin

00

05

10

15

20

p67

phox

120573-a

ctin

(c)

lowast

+



p67phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p67

phox

120573-a

ctin

(d)









4 Discussion





558

to allow optimal O2





p47

phox

120573-a

ctin

lowast+

p47phox

120573-actin

00

02

04

06

08

(a)



p47phox

120573-actin

01

02

03

04

05

06

07

08

09

p47

phox

120573-a

ctin

(b)



lowast

+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

01

02

03

04

05

06

07

p67

phox

120573-a

ctin

(d)





1R







p47

phox

120573-a

ctin

lowast

lowast+

p47phox

120573-actin

00

04

08

12

16

(a)



lowast

lowast +

p47phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p47

phox

120573-a

ctin

(b)



lowast+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

02

04

06

08

1

12

p67

phox

120573-a

ctin

(d)







lowast

+

Collagen III

Tubulin

00

04

08

12

16

20

Col

lage

n II

Itub

ulin





1R [68 80]


2

minus







ExT










5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

+



p47

phox

120573-a

ctin

p47phox

120573-actin

00

03

05

08

10

(a)

lowast

+



p47phox

120573-actin

0

02

04

06

08

1

12

p47

phox

120573-a

ctin

(b)

lowast

+



p67phox

120573-actin

00

05

10

15

20

p67

phox

120573-a

ctin

(c)

lowast

+



p67phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p67

phox

120573-a

ctin

(d)









4 Discussion





558

to allow optimal O2





p47

phox

120573-a

ctin

lowast+

p47phox

120573-actin

00

02

04

06

08

(a)



p47phox

120573-actin

01

02

03

04

05

06

07

08

09

p47

phox

120573-a

ctin

(b)



lowast

+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

01

02

03

04

05

06

07

p67

phox

120573-a

ctin

(d)





1R







p47

phox

120573-a

ctin

lowast

lowast+

p47phox

120573-actin

00

04

08

12

16

(a)



lowast

lowast +

p47phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p47

phox

120573-a

ctin

(b)



lowast+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

02

04

06

08

1

12

p67

phox

120573-a

ctin

(d)







lowast

+

Collagen III

Tubulin

00

04

08

12

16

20

Col

lage

n II

Itub

ulin





1R [68 80]


2

minus







ExT










5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















4 Discussion





558

to allow optimal O2





p47

phox

120573-a

ctin

lowast+

p47phox

120573-actin

00

02

04

06

08

(a)



p47phox

120573-actin

01

02

03

04

05

06

07

08

09

p47

phox

120573-a

ctin

(b)



lowast

+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

01

02

03

04

05

06

07

p67

phox

120573-a

ctin

(d)





1R







p47

phox

120573-a

ctin

lowast

lowast+

p47phox

120573-actin

00

04

08

12

16

(a)



lowast

lowast +

p47phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p47

phox

120573-a

ctin

(b)



lowast+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

02

04

06

08

1

12

p67

phox

120573-a

ctin

(d)







lowast

+

Collagen III

Tubulin

00

04

08

12

16

20

Col

lage

n II

Itub

ulin





1R [68 80]


2

minus







ExT










5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















p47

phox

120573-a

ctin

lowast+

p47phox

120573-actin

00

02

04

06

08

(a)



p47phox

120573-actin

01

02

03

04

05

06

07

08

09

p47

phox

120573-a

ctin

(b)



lowast

+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

01

02

03

04

05

06

07

p67

phox

120573-a

ctin

(d)





1R







p47

phox

120573-a

ctin

lowast

lowast+

p47phox

120573-actin

00

04

08

12

16

(a)



lowast

lowast +

p47phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p47

phox

120573-a

ctin

(b)



lowast+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

02

04

06

08

1

12

p67

phox

120573-a

ctin

(d)







lowast

+

Collagen III

Tubulin

00

04

08

12

16

20

Col

lage

n II

Itub

ulin





1R [68 80]


2

minus







ExT










5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















p47

phox

120573-a

ctin

lowast

lowast+

p47phox

120573-actin

00

04

08

12

16

(a)



lowast

lowast +

p47phox

120573-actin

0

01

02

03

04

05

06

07

08

09

1

p47

phox

120573-a

ctin

(b)



lowast+

p67phox

120573-actin

00

03

05

08

10

p67

phox

120573-a

ctin

(c)



p67phox

120573-actin

0

02

04

06

08

1

12

p67

phox

120573-a

ctin

(d)







lowast

+

Collagen III

Tubulin

00

04

08

12

16

20

Col

lage

n II

Itub

ulin





1R [68 80]


2

minus







ExT










5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















lowast

+

Collagen III

Tubulin

00

04

08

12

16

20

Col

lage

n II

Itub

ulin





1R [68 80]


2

minus







ExT










5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 Conclusions




Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















exercise training attenuates upregulation of p47 phox in...

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