international journal of cardiology · novel insights into the development of chagasic...

International Journal of Cardiology 167 (2013) 3011–3020

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International Journal of Cardiology

j ourna l homepage: www.e lsev ie r .com/ locate / i j ca rd

Novel insights into the development of chagasic cardiomyopathy: Role ofPI3Kinase/NO axis

Danilo Roman-Campos a,⁎, Policarpo Sales-Junior c, Hugo L. Duarte a, Enéas R. Gomes b, Aline Lara b,Paula Campos b, Nazareth N. Rocha d,e, Rodrigo R. Resende f, Anderson Ferreira g, Sílvia Guatimosim b,Ricardo T. Gazzinelli c, Catherine Ropert c, Jader S. Cruz a,⁎a Laboratórios de Membranas Excitáveis e de Biologia Cardiovascular, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais,Belo Horizonte, MG, Brazilb Laboratório de Sinalização Intracelular em Cardiomiócitos, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais,Belo Horizonte, MG, Brazilc Laboratório de Imunopatologia, Instituto René Rachou, FIOCRUZ, Belo Horizonte, MG, Brazild Instituto Biomédico, Universidade Federal Fluminense, Niteroi, Brazile Cenabio, Universidade Federal do Rio de Janeiro, RJ, Brazilf Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazilg Laboratório de Biologia Cardíaca, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

⁎ Corresponding authors. Tel.: +55 31 3409 2668.E-mail addresses: [email protected] (D. Roman-Cam

(J.S. Cruz).

0167-5273/$ – see front matter © 2012 Elsevier Irelandhttp://dx.doi.org/10.1016/j.ijcard.2012.09.020

a b s t r a c t
a r t i c l e i n f o
Article history:
Received 23 November 2011Received in revised form 10 August 2012Accepted 10 September 2012Available online 29 September 2012
Keywords:Chagas' diseaseNitric oxideCardiac electrophysiologyHypertrophyHeart failureIntracellular calcium

Background: Chagas' disease is one of the leading causes of heart failure in Latin American countries. Despite itsgreat social impact, there is nodirect evidence in the literature explaining the development of heart failure inChagas'disease. Therefore, the main objective of the study was to investigate the development of the Chagas' diseasetowards its chronic phase and correlate with modifications in the cellular electrophysiological characteristics ofthe infected heart.Methods and results: Using a murine model of Chagas' disease, we confirmed and extended previous findings ofaltered electrocardiogram and echocardiogram in this cardiomyopathy. The observed changes in the electrocar-diogram were correlated with the prolonged action potential and reduced transient outward potassium currentdensity. Reduced heart function was associated with remodeling of intracellular calcium handling, altered extra-cellular matrix content, and to a set of proteins involved in the control of cellular contractility in ventricularmyocytes. Furthermore, disruption of calcium homeostasis was partially due to activation of the PI3Kinase/nitricoxide signaling pathway. Finally, we propose a causal link between the inflammatorymediators andheart remod-
eling during chagasic cardiomyopathy.Conclusion: Altogether our results demonstrate that heart failure in Chagas' disease may occur due to electricalandmechanical remodeling of cardiac myocytes, and suggest that AKT/PI3K/NO axis could be an important phar-macological target to improve the disease outcome.
© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Chagasic cardiomyopathy is one of the most common causes of heartfailure and sudden death in Latin America [1]. It is caused by infection bythe protozoan parasite Trypanosoma cruzi [2]. There are currently morethan 10 million infected people in the world, and 25 million people livein areas where infectious parasites are present [3]. Chagas' disease is amultifactorial illness that has acute, generally asymptomatic, and chronicphases, in which 20–40% of patients develop cardiomyopathy character-ized by myocarditis, arrhythmia and depressed cardiomyocyte/heart

pos), [email protected]

Ltd. All rights reserved.

function [1]. The mechanism responsible for the cardiomyopathy is con-troversial, and several hypotheses have been raised, including the persis-tence of the parasite or parasite antigens at the inflammatory site and theimportant role of autoimmunehost response [4,5]. In the acute phase, theheart manifestation is accompanied by electrical [6] and mechanicalremodeling [4] along with alterations in the extracellular matrix content[7]. In cardiac chronic phase, it is clear that cardiomyocytes have reducedcontractile reserve [4,8]. Additional studies using electrocardiograficmeasurements [9] have suggested electrical conduction disturbances,however, very little is known about subcellular mechanisms to supportthese previous findings.

Furthermore, there was no consensus regarding whether Chagas'disease was able to disrupt intracellular calcium handling during T. cruziinfection [10].

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3012 D. Roman-Campos et al. / International Journal of Cardiology 167 (2013) 3011–3020

In this study we provide evidence that Chagas' disease presents me-chanical, morphological and electrical heart remodeling in acute andchronic phases. Most importantly, these alterations are correlated withthe presence of inflammatory mediators during the establishment ofchagasic cardiomyopathy. Taken together, our results offer a reasonableexplanation in a multilayered study that demonstrates the molecularbasis of the main events observed in the acute and chronic phases ofChagas' disease.

2. Materials and methods

2.1. Animals

Animals were maintained at the Federal University of Minas Gerais (UFMG), Brazilin accordance with NIH guidelines for the care and use of animals. Experiments wereperformed according to approved protocols from the Institutional Animal Care andUse Committee at UFMG.

2.2. Mice and parasites

Eight-week-old male C57BL/6 and iNOS knockout mice were infected intraperito-neally with 50 bloodstream trypomastigote forms of Colombian T. cruzi strain [11],which was maintained by serial passages in mice at the Centro de Pesquisas RenéRachou/FIOCRUZ (Belo Horizonte, Brazil).

2.3. Echocardiography

Echocardiographywas donewith a Vevo 7700 (Visual Sonics, Toronto, Canada) usinga 30 MHz transducer placed on the thorax of mice anesthetized with 1.5% isoflurane.Continuous monitoring of electrocardiography and respiratory frequency was done. Thediastolic LV posterior wall and interventricular septum thicknesses and LV mass wereevaluated in the M mode and B mode, respectively, at the level of the papillary muscles.Stroke volume was calculated in the bidimensional mode using Simpson's method. Allthe parameters analyzed followed the guidelines set by the American Society of Echocar-diography [12].

2.4. Electrocardiography

Mice were anesthetized with injections of xylazine (2%) and ketamine (3.5 μg/μL/g).Electrodes in DII derivation and a dorsal electrode used as the ground electrode werethen implanted. Twenty-four hours after surgery, one-hour EKG analysis was performedin awake mice.

2.5. Quantitative real time PCR

Isolated left ventricular myocytes were processed for RNA purification. Cells wereground with a mortar and pestle with liquid nitrogen, and total RNA was extracted usingTRIzol. For quantitative PCR (qPCR), total RNA was treated with DNase I (Ambion, Austin,TX, USA) and first strand cDNAwas synthesized using a High Capacity cDNATranscriptionKit (Applied Biosystems, CA, USA). After reverse transcription, the cDNAwas subjected toqPCR on a 7500 Real Time PCR System (Applied Biosystems, CA, USA) using Power SYBRGreen PCR Master Mix (Applied Biosystems, CA, USA). Relative quantification of geneexpression was done with the 2-ΔΔCt method using β-actin expression to normalizethe data. The sequences of primers used are available upon request.

2.6. Western blot

Isolated left ventricular myocytes were processed, and 30–60 μg of protein wasseparated by SDS‐PAGE. Antibodies and their sources were as follows: anti-TnI andanti-phospho‐TnI (Cell Signaling), anti-eNOS (AbCam), anti-nNOS (AbCam) andanti-α-tubulin (Sigma Chemical Co). Immunodetection was done using enhancedchemiluminescence (Amersham Biosciences). Protein levels are expressed as a ratioof optical densities. The control used was α-tubulin, to detect any variations in proteinloading.

2.7. Histomorphometric analyses

Themicewere sacrificed, and heartswere carefully removed,weighed andfixed in for-malin (10% w/v in isotonic saline). Sections (5 μm) were stained and processed for lightmicroscopic studies and morphometric analysis. Hematoxylin and eosin (HE) was usedto count the number of parasite nests and to quantify inflammatory infiltrates. Picrosiriusstaining followedbypolarized-lightmicroscopywas used to visualize and analyze collagen[13]. All staining was performed in paraffin-embedded heart sections mounted on glassslides. The images were digitized through a JVC TK-1270/JGB microcamera to performthe analysis.

2.8. Cardiomyocyte isolation

Adult left and right ventricular myocytes were enzymatically isolated using the colla-genase (1 mg/ml)/calcium gradient method as previously described [14]. Myocytes werefreshly isolated and stored in DMEM media (Sigma Chemical Co., MO, USA) until theywere used for experiments (within 6–8 h). Only calcium-tolerant, quiescent, rod-shapedmyocytes showing clear cross striations were studied.

2.9. Electrophysiological recordings

The whole-cell patch-clamp method was used in the current-clamp configurationto record APs, and voltage-clamp configuration to record IK, IK1 and ICa,L. The protocolsand solutions used were as previously described [14].

Briefly, we recorded 30–50 AP per cell (at 1 Hz), until a stable recording conditionwas achieved, and we used the last recorded AP to perform the complete analysis. Weevaluated the overshoot amplitude, maximal rate of depolarization and duration at 10,30, 50, 70 and 90% of AP repolarization. IK1 was recorded from a holding potential of−40 mV, and then stepped to a series of membrane test potentials (1.5 s duration) every15 s between −130 and −40 mV with increments of 10 mV. To measure total out-ward potassium current we applied depolarization steps from −40 to 70 mV (3 s du-ration) from a holding potential of −80 mV every 15 s. We used a 50 ms pre-pulse to−40 mV from−80 mV to inactivate Na+ current (INa). L-type Ca2+ current (ICa,L) wasblocked with 100 μM of Cd2+. The peak outward current is mainly composed by tran-sient outward potassium current (Ito), which was the one used for analysis. ICa,L wasrecorded in the presence of 1.8 mM extracellular Ca2+. Membrane potential was firststepped from a holding potential of −80 mV to −40 mV for 50 ms (to inactivateNa+ channels), and then stepped to different membrane voltages between −40 and50 mV (300 ms duration).

In the experiments to evaluate the participation of the NO and PI3K signaling path-ways, we loaded isolated cardiac myocytes at least 30 min prior to experiments with6 μM L-NMMA (L-NG-monomethyl arginine citrate — a nitric oxide synthase inhibitor)or 20 μM LY294002 (a phosphoinositide 3-kinase inhibitor). The extracellular solutionused for these experiments was supplemented with 100 μM L-arginine.

2.10. Calcium imaging

Ca2+ imaging experiments were performed in Fluo-4 AM loaded-cardiomyocytes(10 μM; Invitrogen, Eugene, OR, USA) field stimulated at 1 Hz [14]. For calcium tran-sient, the confocal line-scan imaging was performed by a Zeiss LSM 510META confocalmicroscope equipped with an argon laser (488 nm) and a 63× oil immersion objective.Fluorescence line-scan images were acquired at a sampling rate of 1.54 ms/line. The in-tracellular Ca2+ level was reported as F/F0, where F0 is the resting Ca2+ fluorescence.The images obtained were used to evaluate calcium release asynchronicity [15]. Theamplitude of the intracellular [Ca2+]i transient evoked by the application of a Ca2+-and Na+-free solution containing 10 mM caffeine was used as an indicator of SRCa2+ load, as previously shown [16]. Ca2+ spark frequency was recorded in restingventricular myocytes and analyzed using SparkMaster software [17].

2.11. Intracellular NO measurements

Isolated cardiac myocytes were loadedwith 5 μMDAF-2 FM (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate;Molecular Probes, Eugene, OR, USA) [13] for 30 min at37 °C, as previously reported by us [14]. ImageJ software was used for image processingand analysis.

2.12. Statistical analysis

All experiments using isolated cardiomyocytes used 3–8 differenthearts. All results areexpressed asmean±standard error of themean (SEM), and the number of cells is given asn. Differences between groups were determined using Student's t-test, one-way ortwo-way ANOVA, followed by Bonferroni post-hoc test, depending on the experimentevaluated. Statistical methods used are described in the figure legends. Differences wereconsidered significant when Pb0.05.

3. Results

Echocardiographic investigations into the nature and extent ofcardiac dysfunction associated with chagasic cardiomyopathy in miceare shown in Fig. 1. From Fig. 1, it is clear that the infected mice wentfrom left ventricle (LV) hypertrophy in the acute phase (Fig. 1B) toright ventricular chamber dilatation in the chronic phase (Fig. 1C). In ad-dition, we observed that hypertrophy markers were present at 30 and90 dpi (Fig. 1D). The composite results are indicative of a hypertrophicresponse. To ensure that ourmodel was precisely reproducing the alter-ations seen during Chagas' disease, histological examination ofnon-infected, 30, and 90 dpi hearts was done. Infected hearts exhibitedincreased collagen production (Supplemental Fig. 1A) and inflammatory

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Fig. 1. Echocardiographic parameters during T. cruzi infection. Ai) Representative echocardiographic images in control; Aii) 30 days post-infection (30 dpi) and Aiii) 90 dpi mouse.Up and down white arrows indicate right and left ventricles. B, Left ventricular mass is increased at 30 dpi. C) Dilated right ventricle at 90 dpi. D) Increased relative mRNA expres-sion of hypertrophic markers during T. cruzi infection. *Control×30 dpi and #control×90 dpi, Pb0.05.

3013D. Roman-Campos et al. / International Journal of Cardiology 167 (2013) 3011–3020

infiltrate in LV (Supplementary Fig. 1B).mRNA levels of TNF-α and TGF-βwere higher at 30 and 90 dpi (Supplementary Fig. 1C). At 90 dpi, it wasnot possible to find any T. cruzi nests in the LV (Supplementary Fig. 1D),indicating that the infection was controlled, which is in agreement withprevious studies [4]. EKG of control and 30 and 90 dpi were compared,and the results indicated various degrees of electrical disturbances, suchas tachycardia, P-wave alternans (Supplementary Fig. 2B), QRSwaveformdeformation (Supplementary Fig. 2D and E), and atrial overload (Supple-mentary Fig. 2F). Taken together, our data show significant electricalalterations during transition from acute to chronic phase of CD. AlthoughEKG is valuable in measuring cardiac electrical state in vivo, it is limitedin the assessment of specific changes in cardiac myocyte electrophysio-logical activity. Measurements of action potentials from the LVcardiomyocytes revealed marked AP prolongation in 30 dpi (Fig. 2B)and 90 dpi (Fig. 2C) compared to controls (Fig. 2A). Averaged data aredepicted in Fig. 2D. Fig. 2E summarizes AP parameters obtained in con-trol (n=13), 30 dpi (n=11), and 90 dpi (n=13). To investigatewhether these changes in cardiac function have a cellular basis in T.cruzi-infectedmice, we decided to test the possibility that ionic currentsmaybe altered. Fig. 3 shows a reduction in ICa,L inmyocytes isolated frominfected mice, which is demonstrated by the representative families ofICa,L (30 and 90 dpi, Fig. 3B and C, respectively) compared to controls(Fig. 3A and dashed line in Fig. 3E and F). Sequential comparisonsshowed a decrease in ICa,L density in the −20 to 30 mV range for

30 dpi (Fig. 3E) and in the −20 to 20 mV range for 90 dpi (Fig. 3F).Peak LV ICa,L density (pA/pF) was −7.3±0.5 (n=16), −4.6±0.3(n=11) and −4.6±0.5 (n=9) (at 0 mV, Pb0.05) in the control, 30and 90 dpi, respectively. Also, we observed a rightward shift in the po-tential that activates 50% of the L-type Ca2+ channels (Control,V0.5=−11.8±0.85 mV, n=16; 30 dpi, V0.5=−8.9±0.61 mV, n=25; and 90 dpi, V0.5=−6.92±0.82 mV, n=9). The reduction of ICa,Lwould be expected to shorten rather than prolong the AP duration,and is thus not likely to underlie the delayed repolarization.

A major determinant of AP repolarization is the magnitude ofvoltage-dependent K+ currents. Representative transient outward K+

current (Ito) traces for control (Fig. 3G), 30 dpi (Fig. 3H), and 90 dpi(Fig. 3I) are shown. Fig. 3J, K, and L depict representative inwardly recti-fying K+ current (IK1) recordings for control, 30 dpi, and 90 dpi, respec-tively. In infectedmice, total Itowas significantly reduced (Fig. 3M). In allcases, the measured IK1was not different between groups (Fig. 3N). Be-cause decreased Ito would be expected to lengthen AP duration, wecan conclude that the AP prolongation observed in T. cruzi-infectedcardiomyocytes could result from decreased total Ito density.

To determine whether T. cruzi infection is associated with defects inintracellular Ca2+ homeostasis, [Ca2+]i transients were measured infield-stimulated cardiac cells. Representative [Ca2+]i transients from acontrol and infected mice (30 and 90 dpi) stimulated at 1 Hz areshown in Fig. 4A, B and C. At 30 dpi, the amplitude of [Ca2+]i showed

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Fig. 2. Action potential lengthening in isolated left ventricular myocytes of infected mice. A) Representative action potential measured in isolated cardiac myocytes from control; B)30 and C) 90 days post-infection mouse. D) Averaged time to action potential repolarization. E) Average values for resting membrane potential (R.M.P.), overshoot and dV/dt. Datarepresent mean±s.e.m. of control, 30 dpi and 90 dpi. *Control×30 dpi and #control×90 dpi, Pb0.05.


no significant difference, while in 90 dpi, this value was increased by56% (Fig. 4C and D). To determine whether an increase of SR loadwould contribute to changes in [Ca2+]i, we evaluated SR Ca2+ load bycaffeine exposure (Fig. 4E). The amplitude of [Ca2+]i transients elicitedby caffeine in infected cells (90 dpi) was 71% higher than that of control,while the value at 30 dpiwas not different. [Ca2+]i declinewas prolongedby 20% in 30 dpi and became faster at 90 dpi (~21%, Fig. 4F).

SR Ca2+ load is an important factor determining Ca2+ release andshould have an impact on Ca2+ spark properties in cardiomyocytes.We next used confocal line scan imaging to visualize Ca2+ sparks incardiomyocytes (Fig. 5A, B and C). Cells infected with T. cruzi for90 days show a significant increase in the amplitude of Ca2+ sparks(Fig. 5D) without any effect on Ca2+ spark frequency (Fig. 5E). At30 dpi, Ca2+ sparks were not affected (Fig. 5D and E).

To generate a more detailed analysis of the [Ca2+]i, we decided toevaluate the dyssynchrony of Ca2+ release [14]. Fig. 5F, G andH are rep-resentative line scan images taken from control, 30 dpi and 90 dpi.Dyssynchronicity in SR Ca2+ release, indicating uncoupling betweenL-type Ca2+ channels and ryanodine receptors, is shown in Fig. 5I.

Because we observed profound alterations in excitation–contractioncoupling only in cardiomyocytes from 90 dpi, we decided to evaluate asubset of proteins involved in the control of calciumhandling and cellularcontraction. In Fig. 6, it can be observed that at 90 dpi, there is a reductionin the expression of troponin-I and there was no difference in its phos-phorylation (Fig. 6A and B). However, we observed a decrease in the ex-pression of phospholamban, as shown in Fig. 6C. Interestingly, there wasa trend to increase the phosphorylation degree of phospholamban but itdid not reach statistical significance (Fig. 6D). Additionally, we observedthat SERCA-2A expression was reduced at 90 dpi (Fig. 6E).

It has been shown that T. cruzi infection provokes sustained NO bio-synthesis in cardiomyocytes [18], which raises the possibility that NO is

responsible for the observed alterations during Chagas' disease. To con-firm this hypothesis we assessed NO levels in infected LV myocytesloaded with a NO-sensitive probe DAF-FM (Fig. 7Aa–Ab). We foundthat baseline NO levels were significantly increased in isolated LVmyocytes at 30 dpi (Fig. 7Ac). In agreement with the increased NO,mRNA levels of iNOS, nNOS and eNOS were elevated at 30 and 90 dpi(Fig. 7B) and protein levels of nNOS and eNOS were higher at 90 dpi(Fig. 7C–D).

Finally, to directly confirm the involvement of NO, we pre-incubatedLV myocytes from mice at 30 dpi with L-NMMA (competitive inhibitorof NOS), which resulted in the recovery of ICa,L density (Fig. 8A, middlepanel), when compared tomyocytes frommice at 30 dpi (Fig. 8A, upperpanel). Additionally, infected knockout mice for iNOS demonstrated at30 dpi increased NO production (Supplementary Fig. 3A) and reducedICa,L (Supplementary Fig. 3B), indicating that eNOS and/or nNOSisoforms are likely involved in ICa,L attenuation during T. cruzi infection.In addition, we evaluated an upstream step in the NO signaling pathwayand determined that pre-incubation of isolated LV myocytes at 30 dpiwith LY294002 (PI3K inhibitor) resulted in partial recovery of ICa,Ldensity (Fig. 8A, bottom panel), indicating that PI3K/NO axis may beinvolved in the downregulation of ICa,L during T. cruzi infection. Fig. 8Bsummarizes these findings.

4. Discussion

Heart dysfunction is a common finding in Chagas' disease (CD) [8,9].Heart malfunctioning during acute and chronic phases of the diseasehas been reported. However, many of these studies did not explorewhich signaling pathways were actually involved.

We found different degrees of electrical conduction disturbances inthe acute and chronic phases. These alterations were followed by

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tachycardia, which was first attributed to sympathetic and parasympa-thetic denervation due to the production of auto-antibodies againstmuscarinic (M2) [19] and adrenergic (β1) [20] receptors during CD.The re-entry phenomenon is a recurrent finding during T. cruzi infectionwhich contributes to the occurrence of sudden death [9]. The hypertro-phy observed in our study, using the Colombian strain of T. cruzi, couldbe attributed to diverse signaling pathways triggered by ANP, BNPand/or TNF-α increased levels [8,21]. TNF-α is able to trigger NF-kB,leading to activation of transcription factors inducing cardiomyocyte hy-pertrophy [22]. The hypertrophy in the acute phasewas not followed byan increased ejection fraction, further confirming our initial findings ofreduced cardiomyocyte contractile dysfunction [4]. It is interesting to

note that in the early chronic phase we observed RV dilation without,as one would expect a clear reduction in LV ejection fraction. It couldbe that the preserved ejection fraction is due to compensatory mecha-nisms caused by increased calcium release and SR calcium content inthis disease stage [23].

In our study we observed reduced TnI expression at 90 dpi and noalteration in its phosphorylated form. Thus, it is possible that the re-duction in the cellular contractile performance in the early chronicphase, as we previously reported [4] could be related to the decreasein TnI levels. One potential drawback would be the possibility of TnItruncation which could decrease the actual amount of TnI present inthe analyzed samples. In the early chronic phase we observed an

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Fig. 4. SR calcium release is altered in isolated left ventricular myocytes of infected mice. A) Representative confocal images (top) and averaged fluorescence intensity (bottom) inline scan mode in control, B) 30 days post-infection, and C) 90 days post-infection (dpi). D) Averaged fluorescence intensity. E) SR calcium content elucidated by applying 10 mMcaffeine. F) Calcium transient decay time constant. *Control×30 dpi and #control×90 dpi, Pb0.05.


increased calcium release that was probably triggered as a compensatorymechanism.

The QT interval in electrocardiogram is composed of ventricular de-polarization and repolarization [24]. Here we demonstrate that duringCD there is a significant prolongation of the action potential duration.Thus, longer QT-interval would be expected creating, as a consequence,

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Fig. 5. Calcium sparks and asynchronicity during T. cruzi infection. A) Representative calciumfrequency of calcium sparks. F,G,H) Representative images showing asynchronicity in SR*Control×30 dpi and #control×90 dpi, Pb0.05. The vertical calibration bar on panels A, B,

a substrate for lethal arrhythmias. There is only one report in the litera-ture demonstrating action potential prolongationwith reduced Ito in theacute phase of CD [6]. In the same study, however, the authors showedthat there was no alteration in either the action potential or Ito in thechronic phase. This apparent discrepancy between our findings andthose of Pacioretty and colleagues is probably due to different parasite

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Fig. 6. Altered contractility proteins expression at 90 dpi. In panels A to E the top image is a representative Western blot and the bottom shows an average densitometric analysis (n,number of heart samples analyzed. A) TnI expression and B) TnI-P/TnI ratio. C) PLN expression and D) PLN-P/PLN ratio. E) SERCA-2A expression. α-Tubulin expression levels wereused as a loading control. *Pb0.05 when compared to controls.


strains and animal models used. It is postulated that cardiac phenotypeof CD depends on the host genetic background as well as the parasitestrain. Both would contribute to a distinct immunological response,which would determine the final outcome observed in CD.

There are many mechanisms that could contribute to the attenua-tion of Ito. In our study we propose that TNF-α plays an importantrole. We observed that hearts isolated at different points of infectionhad a sustained TNF-α concentration, even in the chronic phase.TNF-α is able to stimulate both NO and superoxide production in car-diac myocytes [18,25], and both are well-known molecules involvedin the regulation of Ito. There are evidences that NO is able to reduceIto. It has been demonstrated [26] that exposing CHO cells expressingKv4.3 channels to NO leads to Kv4.3 current attenuation, and theauthors postulated that the mechanism involved in this process isdependent on the activation of PKA and/or PP2. However, the authorsdid not confirm this result in ventricular myocytes. Interestingly, weobserved reduced Ito density that recovered partially incubating isolatedcardiomyocytes with L-NMMA, indicating a possible modulation of Itoby NO [14]. Another interesting study demonstrated that after incubationof cardiomyocytes with TNF-α for 24 h, there was an attenuation of Itodue to activation of iNOS and superoxide production [25]. In our studywe provide compelling evidence of increased NO production throughincreased expression of iNOS, nNOS and eNOS. Additionally, as TNF-α,IFN-γ and IL1-β can induce iNOS expression in cardiac myocytes [18],the chronic inflammationobserved in our study sets an important scenar-io to allow sustained iNOS expression. Considering superoxide produc-tion it was demonstrated that isolated cardiomyocytes incubated withangiotensin II had increased superoxide production through activationof gp91phox, leading to reduced expression of Kv4.2 channels resulting

in reduced Ito [27]. Many studies indicate that during T. cruzi infection su-peroxide production is increased in cardiomyocytes [25,26]. Additionally,it was demonstrated that a reduction of Ito in the acute phase of CD couldbe due to the activation of PKC [28]. There aremany pathways thatwouldlead to reduction of Ito during CD. However, the exact molecular mecha-nism is still an openquestion that deserves future attention. Despite alter-ations observed in Itoduring CD,wedid notfindany significant changes inthe IK1 current.

In a previous study, our group demonstrated that both LV and RVmyocytes had reduced contractile ability in the acute and chronicphases of CD [4]. It is known that ICa,L is involved in the activation ofcardiomyocyte contraction [30]. In our study, we observed reducedICa,L in LV myocytes. Reduction in ICa,L is not a consistent finding indifferent models of heart disease [31,32]. Previous studies have dem-onstrated that ICa,L is modulated by various intracellular signalingpathways. Here we demonstrate that activation of the AKT/PI3K/NOpathway is likely involved in ICa,L reduction observed in CD. DuringT. cruzi infection, internalization of the parasite is dependent on theactivation of TGF-β receptors [33]. It is known that in cardiacmyocytes,TGF-β leads to the AKT/PI3K/NO signaling pathway activation [34].During T. cruzi infection, AKT/PI3K/NO axis activation is able to preventinfected cells from dying by repressing apoptotic machinery such ascaspase-9, BCL2 and the transcription factor FOXO-16 [35]. In ourstudy, we demonstrated increased TGF-β production in acute andchronic phases of CD, which could account for the constant activationof the AKT/PI3K/NO signaling pathway, leading to increased andsustained production of NO. In fact, we demonstrated that there issustained NO production during CD. The ability of NO to modulate ICa,Lhas been extensively studied by others. It is known that increased

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Fig. 7. Nitric oxide production is increased during T. cruzi infection in isolated cardiomyocytes. A) Representative images showing increased basal nitric oxide levels in isolatedcardiomyocytes: control (Aa) versus 30 days-post infection (dpi) myocytes (Ab). Ac) Averaged baseline nitric oxide levels. B) eNOS, nNOS and iNOS gene expression are alteredin acute and chronic phases of Chagas' disease. C) nNOS and D) eNOS protein expression are increased in myocytes isolated at 90 dpi. α-Tubulin expression levels were used asa loading control. *Control×30 dpi and #control×90 dpi, Pb0.05.


NO production activates PKG causing phosphorylation of the α1-Csubunit of the Ca2+ channel. In fact, it was recently demonstrated inisolated cardiomyocytes that PKG phosphorylates the α-1C subunit,

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Fig. 8. Nitric oxide modulates ICa,L in the acute phase of Chagas' disease. A) Representative I30 dpi, 30 dpi+L-NMMA and 30 dpi+LY294002 (right panel traces). B) Current–voltagecontrol+LY294002, 30 dpi and 30 dpi+LY294002 (bottom). *30 dpi×30 dpi+L-NMMA o

leading to a reduction in ICa,L [36]. Transgenic mice overexpressingnNOS have a reduction in ICa,L that can be recovered by applying specif-ic nNOS inhibitors [37]. Moreover, it has been proposed that eNOS

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Ca,L traces in control, control+L-NMMA and control+LY294002 (left panel traces) andrelationships comparing control+L-NMMA, 30 dpi and 30 dpi+L-NMMA (top) andr LY294002, 30 dpi×30 dpi+LY294002, *Pb0.05.


overexpression also leads to attenuation of ICa,L, because it was ob-served reduced cell shortening. However, the authors did not measureICa,L [38]. In our present studywedetermined that iNOS is not responsiblefor ICa,L attenuation during acute phase of Chagas' disease, which stronglysuggest that nNOS and/or eNOS are likely involved. Additionally, it is im-portant to point out that there are probably additional mechanisms in-volved in the control of ICa,L during CD.

The main function of L-type Ca2+ channel activation is to elicit SRCa2+ release [29,30]. It is thought that reduction in ICa,L leads to reducedSR Ca2+release. However, this was not observed in our study. In thechronic phase, the increased SR Ca2+ release could be explained by in-creased SR Ca2+ load, which could be understood as a compensatorymechanism.We did analyze decay time of the caffeine-evoked transientsto estimate the Na+/Ca2+ exchange activity and did not observe any dif-ference between the experimental groups, indicating that the Na+/Ca2+

exchanger is not responsible for the difference in SR Ca2+ content ob-served at 90 dpi. Although SERCA-2A levels are decreased in infectedmice, indicating a smaller Ca2+ reuptake to the SR,we also observed a de-crease in PLNexpression levels in cardiac samples from90 dpi, suggestingthat in this group there will be less PLN inhibition of SERCA-2A. Corrobo-rating this assumption, we found a faster Ca2+ decay in cardiomyocytesfrom 90 dpi mice, indicating that in this group SERCA-2A activity wouldbe higher than in control. Moreover, we know that AP is prolonged incardiomyocytes from 90 dpi cells.

Together, decreased PLN levels and increased AP duration incardiomyocytes from 90 dpi mice can account for the higher Ca2+

transient amplitude found in these cells.Since our measurements of L-type Ca2+ whole-cell current were

made using voltage square pulses, by using this experimental condi-tion we were probably underestimating the influx of Ca2+ in 90 dpicardiomyocytes. In contrast, SR Ca2+ load measurements wereperformed in field stimulated cells. Considering the prolonged actionpotential observed in 90 dpi cells, we argue that during these exper-iments Ca2+ influx through L-Type Ca2+ channels was probablyhigher than the one obtained using square pulses.

Additionally, the asynchronicity observed in the present study in-dicates uncoupling between L-type Ca2+ channels and the ryanodinereceptors (RyR) [15].

In this study we demonstrated that there is increased expressionof nNOS. RyR have many sites that can be S-nitrosylated (~12 sites),increasing RyR open probability [39] mainly through the activationof nNOS. Thus, we decided to evaluate spark frequency as an indirectindicator of RyR open probability [39]. Surprisingly, we did not ob-serve any significant increase in spark frequency in the acute phaseof CD. This result suggests that there are other possible mechanismsinvolved in the putative gain-of-function of RyR in the acute phaseof CD. Another interesting possibility is that increased action potentialduration leads to increased stimulation of RyR, which could explainthe sustained Ca2+ release in acute phase of CD.

Overall, our results point to the AKT/PI3K/NO signaling pathway asa major molecular target to ameliorate chagasic cardiomyopathy.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ijcard.2012.09.020.

Funding sources

This work was supported by grants from Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparoà Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação deAperfeiçoamento de Pessoal de Nível Superior (CAPES) and Programade Apoio a Núcleos de Excelência (PRONEX/FAPEMIG/CNPq).

Acknowledgments

The authors of this manuscript have certified that they comply withthe Principles of Ethical Publishing in the International Journal of

Cardiology.Wewould like to thank CEMEL (Biological Sciences Institute,Federal University of Minas Gerais, Confocal Microscopy Facility). Theauthors would like to thank Cristália Produtos Químicos e FarmacêuticosLTDA for donation of the HEMOFOL (Heparin-sodium salt).

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