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ABSTRACT: Oxidative stress has been implicated in the pathogenesis of heart failure and atrial fibrillation and can result in increased peroxynitrite production in the myocardium. Atrial and ventricular canine cardiac myocytes were superfused with SIN-1 (3-morpholinosydnonimine N-ethylcarbamide), aperoxynitrite donor, to evaluate the acute electrophysiologic effects of peroxynitrite. Perforated whole cell patch clamp techniques were used to record action potentials. SIN-1 (200 µM) increased the action potential duration (APD) in atrial and ventricular myocytes; however, in the atria, APD prolongation was rate-independent, while in the ventricle APD prolongation was rate dependent. In addition to prolongation of the action potential, beat to beat variability of repolarization was significantly increased in ventricular, but not atrial myocytes. We examined the contribution of intracellular calcium cycling to the effects of SIN-1 by treating myocytes with the SERCA blocker, thapsigargin (5-10 µM). Inhibition of calcium cycling prevented APD prolongation in the atrial and ventricular myocytes, and prevented the SIN-1 induced increase in ventricular beat to beat APD variability. Collectively, these data demonstrate that peroxynitrite affects atrial and ventricular electrophysiology differentially. A detailed understanding of oxidative modulation of electrophysiology in specific chambers is critical to optimize therapeutic approaches for cardiac diseases.
Journal of cardiovascular pharmacology 01/2013; · 2.83 Impact Factor
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ABSTRACT: The GTP binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O(2)()). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia/reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O(2)() generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared to WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-hour reperfusion) caused significantly larger MI in TG mice compared to WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91phox, O(2)(), and PAK1. Thus, these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.
AJP Heart and Circulatory Physiology 11/2012; · 3.71 Impact Factor
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Ingrid M Bonilla,
Andriy Belevych,
Arun Sridhar, Yoshinori Nishijima,
Hsiang-Ting Ho,
Quanhua He,
Monica Kukielka,
Dmitry Terentyev,
Radmila Terentyeva,
Bin Liu,
Victor P Long,
Sandor Gyorke,
Cynthia A Carnes,
George E Billman
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ABSTRACT: The risk of sudden cardiac death is increased following myocardial infarction (MI). Exercise training reduces arrhythmia susceptibility but the mechanism is unknown. We used a canine model of sudden cardiac death (healed infarction, with ventricular tachyarrhythmias induced by an exercise plus ischemia test, VF+); we previously reported that endurance exercise training was antiarrhythmic in this model. A total of 41 VF+ animals were studied, after random assignment to 10 weeks of endurance exercise training (EET, n=21) or a matched sedentary period (n=20). Following (> 1 week) the final attempted arrhythmia induction, isolated myocytes were used to test the hypotheses that the endurance exercise-induced antiarrhythmic effects resulted from normalization of cellular electrophysiology and/or normalization of calcium handling. EET prevented VF and shortened in vivo repolarization (P<0.05). EET normalized action potential duration and variability compared to the sedentary group. EET resulted in a further decrement in I(to) compared to the sedentary VF+ group (P<0.05). Sedentary VF+ dogs had a significant reduction in I(Kr), which was restored by exercise training (P<0.05). Compared to controls, myocytes from the sedentary VF+ group displayed calcium alternans, increased calcium spark frequency, and increased phosphorylation of S2814 on RyR2. These abnormalities in intracellular calcium handling were attenuated by exercise training (P<0.05). Exercise training prevented ischemically-induced VF in association with a combination of beneficial effects on cellular electrophysiology and calcium handling.
Journal of Applied Physiology 10/2012; · 3.75 Impact Factor
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ABSTRACT: Glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (GLUTs), which includes GLUT4 (the major cardiac isoform) and GLUT12 (a novel, second insulin-sensitive isoform). Potential regional patterns in glucose transport across the cardiac chambers have not been examined; thus, we hypothesized that insulin-responsive GLUT4 and -12 protein and gene expression would be chamber specific in healthy subjects and during chronic heart failure (HF). Using a canine model of tachypacing-induced, progressive, chronic HF, total GLUT protein and messenger RNA in both ventricles and atria (free wall and appendage) were investigated by immunoblotting and real-time PCR. In controls, GLUT4, but not GLUT12, protein content was significantly higher in the atria compared with the ventricles, with the highest content in the right atrium (RA; P < 0.001). GLUT4 and GLUT12 mRNA levels were similar across the cardiac chambers. During chronic HF, GLUT4 and GLUT12 protein content was highest in the left ventricle (LV; by 2.5- and 4.2-fold, respectively, P < 0.01), with a concomitant increase in GLUT4 and GLUT12 mRNA (P < 0.001). GLUT4, but not GLUT12, protein content was decreased in RA during chronic HF (P = 0.001). In conclusion, GLUT4 protein was differentially expressed across the chambers in the healthy heart, and this regional pattern was reversed during HF. Our data suggest that LV was the primary site dependent on both GLUT4 and GLUT12 during chronic HF. In addition, the paradoxical decrease in GLUT4 content in RA may induce perturbations in atrial energy production during chronic HF.
AJP Regulatory Integrative and Comparative Physiology 08/2011; 301(5):R1300-6. · 3.34 Impact Factor
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Cardiovascular research 06/2011; · 5.80 Impact Factor
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Yoshinori Nishijima,
Arun Sridhar,
Ingrid Bonilla,
Murugesan Velayutham,
Mahmood Khan,
Radmila Terentyeva,
Chun Li,
Periannan Kuppusamy,
Terry S Elton,
Dmitry Terentyev,
Sandor Györke,
Jay L Zweier,
Arturo J Cardounel,
Cynthia A Carnes
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ABSTRACT: Heart failure is a common antecedent to atrial fibrillation; both heart failure and atrial fibrillation are associated with increased myocardial oxidative stress. Chronic canine heart failure reduces atrial action potential duration and atrial refractoriness. We hypothesized that inducible nitric oxide synthase 2 (NOS2) contributes to atrial oxidative stress and electrophysiologic alterations.
A 16-week canine tachypacing model of heart failure was used (n= 21). At 10 weeks, dogs were randomized to either placebo (n = 12) or active treatment (n = 9) with NOS cofactor, tetrahydrobiopterin (BH(4), 50 mg), and NOS substrate (L-arginine, 3 g) twice daily for 6 weeks. A group of matched controls (n = 7) was used for comparison. Heart failure increased atrial NOS2 and reduced atrial BH(4), while L-arginine was unchanged. Treatment reduced inducible atrial fibrillation and normalized the heart failure-induced shortening of the left atrial myocyte action potential duration. Treatment increased atrial [BH(4)] while [L-arginine] was unchanged. Treatment did not improve left ventricular function or dimensions. Heart failure-induced reductions in atrial [BH(4)] resulted in NOS uncoupling, as measured by NO and superoxide anion (O(2)(·-)) production, while BH(4) and L-arginine treatment normalized NO and O(2)(·-). Heart failure resulted in left atrial oxidative stress, which was attenuated by BH(4) and L-arginine treatment.
Chronic non-ischaemic heart failure results in atrial oxidative stress and electrophysiologic abnormalities by depletion of BH(4) and uncoupling of NOS2. Modulation of NOS2 activity by repletion of BH(4) may be a safe and effective approach to reduce the frequency of atrial arrhythmias during heart failure.
Cardiovascular research 04/2011; 91(1):71-9. · 5.80 Impact Factor
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ABSTRACT: There is emerging evidence that treatment with thyroid hormone (TH) can improve postischemic cardiac function. 3,5-Diiodothyropropionic acid (DITPA), a TH analog, has been proposed to be a safer therapeutic agent than TH because of its negligible effects on cardiac metabolism and heart rate. However, conflicting results have been reported for the cardiac effects of DITPA. Importantly, recent clinical trials demonstrated no symptomatic benefit in patients with DITPA despite some improved hemodynamic and metabolic parameters. To address these issues, dose-dependent effects of DITPA were investigated in mice for baseline cardiovascular effects and postischemic myocardial function and/or salvage. Mice were treated with subcutaneous DITPA at 0.937, 1.875, 3.75, or 7.5 mg·kg(-1)·day(-1) for 7 days, and the results were compared with untreated mice for ex vivo and/or in vivo myocardial ischemia-reperfusion (I/R). DITPA had no effects on baseline body temperature, body weight, or heart rate; however, it mildly increased blood pressure. In isolated hearts, baseline contractile function was significantly impaired in DITPA-pretreated mice; however, postischemic recovery was comparable between untreated and DITPA-treated groups. In vivo baseline cardiac parameters were significantly affected by DITPA, with increased ventricular dimensions and decreased contractile function. Importantly, DITPA-treated mice demonstrated high prevalence of fatal cardiac rhythm abnormalities during in vivo ischemia and/or reperfusion. There were no improvements in myocardial infarction and postischemic fractional shortening with DITPA. Myocardial sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and heat shock protein (HSP) levels remained unchanged with DITPA treatment. Thus DITPA administration impairs baseline cardiac parameters in mice and can be fatal during in vivo acute myocardial I/R.
AJP Heart and Circulatory Physiology 02/2011; 300(2):H702-11. · 3.71 Impact Factor
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ABSTRACT: In heart failure (HF), abnormal myocyte Ca(2+) handling has been implicated in cardiac arrhythmias and contractile dysfunction. In the present study, we investigated the relationships between Ca(2+) handling, reduced myocyte contractility, and enhanced arrhythmogenesis during HF progression in a canine model of non-ischaemic HF.
Key Ca(2+) handling parameters were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) [Ca(2+)] in isolated ventricular myocytes at different stages of HF. The progression of HF was associated with an early and continuous increase in ryanodine receptor (RyR2)-mediated SR Ca(2+) leak. The increase in RyR2 activity was paralleled by an increase in the frequency of diastolic spontaneous Ca(2+) waves (SCWs) in HF myocytes under conditions of β-adrenergic stimulation. In addition to causing arrhythmogenic-delayed afterdepolarizations, SCWs decreased the amplitude of subsequent electrically evoked Ca(2+) transients by depleting SR Ca(2+). At late stages of HF, Ca(2+) release oscillated essentially independent of electrical pacing. The increased propensity for the generation of SCWs in HF myocytes was attributable to reduced ability of the RyR2 channels to become refractory following Ca(2+) release. The progressive alterations in RyR2 function and Ca(2+) cycling in HF myocytes were associated with sequential modifications of RyR2 by CaMKII-dependent phosphorylation and thiol oxidation.
These findings suggest that destabilized RyR2 activity due to excessive CaMKII phopshorylation and oxidation resulting in impaired post-release refractoriness is a common mechanism involved in arrhythmogenesis and contractile dysfunction in the failing heart.
Cardiovascular research 02/2011; 90(3):493-502. · 5.80 Impact Factor
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Andriy E Belevych,
Sarah E Sansom,
Radmila Terentyeva,
Hsiang-Ting Ho, Yoshinori Nishijima,
Mickey M Martin,
Hitesh K Jindal,
Jennifer A Rochira,
Yukiko Kunitomo,
Maha Abdellatif,
Cynthia A Carnes,
Terry S Elton,
Sandor Györke,
Dmitry Terentyev
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ABSTRACT: In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.
PLoS ONE 01/2011; 6(12):e28324. · 4.09 Impact Factor
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ABSTRACT: QT prolongation may increase the risk of torsades de pointes (TdP). Early afterdepolarizations (EADs) and transmural dispersion of repolarization have been known to serve as physiological substrates and predictors for TdP. Abnormal Ca(2+) cycling is the proximate cause of EADs, and Ca(2+) cycling is abnormal in heart failure (HF). However, the mechanisms for drug-induced TdP in HF are poorly understood. The purpose of this study was to search for torsadogenic-modifying effects of verapamil, ryanodine, KB-R7943, W-7, KN-93, and H-8 on ventricular premature depolarizations (VPD) and TdP in rabbits with HF. Rabbits with HF were pretreated with propranolol followed by test articles before continuous infusion of dofetilide to induce TdP. In the control hearts, VPD and TdP were induced in all rabbits and the onsets of VPD and TdP were 3.6 +/- 1.3 minutes and 10.3 +/- 1.4 minutes, respectively. Dofetilide lengthened RR, QT and QTc. Verapamil, ryanodine and H-8 significantly delayed onset of VPD (P < 0.05) and suppressed TdP (P < 0.01). KB-R7943, W-7, and KN-93 accelerated onset of TdP. Blockades of L-type Ca(2+) channel, ryanodine channel, and protein kinase A prevent dofetilide-induced TdP, suggesting roles for intracellular Ca(2+) overload and Ca(2+) signaling pathways in drug-induced TdP.
International Heart Journal 07/2010; 51(4):285-90. · 1.16 Impact Factor
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George E Billman, Yoshinori Nishijima,
Andriy E Belevych,
Dmitry Terentyev,
Ying Xu,
Kaylan M Haizlip,
Michelle M Monasky,
Nitisha Hiranandani,
William S Harris,
Sandor Gyorke,
Cynthia A Carnes,
Paul M L Janssen
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ABSTRACT: Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant (P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 +/- 0.04; 1 g/day, 3.02 +/- 0.23; 2 g/day, 3.63 +/- 0.17; and 4 g/day, 6.97 +/- 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 +/- 1.6 vs. post, 40.1 +/- 1.7%; placebo: pre, 39.2 +/- 1.3 vs. post, 38.4 +/- 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to beta-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.
AJP Heart and Circulatory Physiology 04/2010; 298(4):H1219-28. · 3.71 Impact Factor
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ABSTRACT: Treating cancer patients with chemotherapeutics, such as doxorubicin (Dox), cause dilated cardiomyopathy and congestive heart failure because of oxidative stress. On the other hand, heat shock factor-1 (HSF-1), a transcription factor for heat shock proteins (Hsps), is also known to be activated in response to oxidative stress. However, the possible role of HSF-1 activation and the resultant Hsp25 in chemotherapeutic-induced heart failure has not been investigated. Using HSF-1 wild-type (HSF-1(+/+)) and knock-out (HSF-1(-/-)) mice, we tested the hypothesis that activation of HSF-1 plays a role in the development of Dox-induced heart failure. Higher levels of Hsp25 and its phosphorylated forms were found in the failing hearts of Dox-treated HSF-1(+/+) mice. More than twofold increase in Hsp25 mRNA level was found in Dox-treated hearts. Proteomic analysis showed that there is accumulation and aggregation of Hsp25 in Dox-treated failing hearts. Additionally, Hsp25 was found to coimmunoprecipitate with p53 and vice versa. Further studies indicated that the Dox-induced higher levels of Hsp25 transactivated p53 leading to higher levels of the pro-apoptotic protein Bax, but other p53-related proteins remained unaltered. Moreover, HSF-1(-/-) mice showed significantly reduced Dox-induced heart failure and higher survival rate, and there was no change in Bax upon treating with Dox in HSF-1(-/-) mice. From these results we propose a novel mechanism for Dox-induced heart failure: increased expression of Hsp25 because of oxidant-induced activation of HSF-1 transactivates p53 to increase Bax levels, which leads to heart failure.
AJP Heart and Circulatory Physiology 04/2010; 298(6):H1832-41. · 3.71 Impact Factor
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Andriy E Belevych,
Dmitry Terentyev,
Serge Viatchenko-Karpinski,
Radmila Terentyeva,
Arun Sridhar, Yoshinori Nishijima,
Lance D Wilson,
Arturo J Cardounel,
Kenneth R Laurita,
Cynthia A Carnes,
George E Billman,
Sandor Gyorke
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ABSTRACT: Although cardiac alternans is a known predictor of lethal arrhythmias, its underlying causes remain largely undefined in disease settings. The potential role of, and mechanisms responsible for, beat-to-beat alternations in the amplitude of systolic Ca(2+) transients (Ca(2+) alternans) was investigated in a canine post-myocardial infarction (MI) model of sudden cardiac death (SCD).
Post-MI dogs had preserved left ventricular (LV) function and susceptibility to ventricular fibrillation (VF) during exercise. LV wedge preparations from VF dogs were more susceptible to action potential (AP) alternans and the frequency-dependence of Ca(2+) alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca(2+) transients ([Ca(2+)](c)) alternated in phase with changes in diastolic Ca(2+) in sarcoplasmic reticulum ([Ca(2+)](SR)), but the dependence of [Ca(2+)](c) amplitude on [Ca(2+)](SR) was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca(2+) release for a given amplitude of Ca(2+) current and elevated diastolic RyR-mediated SR Ca(2+) leak. SR Ca(2+) uptake activity did not differ between VF and control cells. VF myocytes had an increased rate of reactive oxygen species production and increased RyR oxidation. Treatment of VF myocytes with reducing agents normalized parameters of Ca(2+) handling and shifted the threshold of Ca(2+) alternans to higher frequencies.
Redox modulation of RyRs promotes generation of Ca(2+) alternans by enhancing the steepness of the Ca(2+) release-load relationship and thereby providing a substrate for post-MI arrhythmias.
Cardiovascular research 08/2009; 84(3):387-95. · 5.80 Impact Factor
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ABSTRACT: We sought to define the underlying mechanisms for atrial fibrillation (AF) during chronic heart failure (HF).
Preliminary studies showed that 4 months of HF resulted in irreversible systolic dysfunction (n = 9) and a substrate for sustained inducible AF (>3 months, n = 3). We used a chronic (4-month) canine model of tachypacing-induced HF (n = 10) to assess atrial electrophysiological remodelling, relative to controls (n = 5). Left ventricular fractional shortening was reduced from 37.2 +/- 0.83 to 13.44 +/- 2.63% (P < 0.05). Left atrial (LA) contractility (fractional area change) was reduced from 34.9 +/- 7.9 to 27.9 +/- 4.23% (P < 0.05). Action potential durations (APDs) at 50 and 90% repolarization were shortened by approximately 60 and 40%, respectively, during HF (P < 0.05). HF-induced atrial remodelling included increased fibrosis, increased I(to), and decreased I(K1), I(Kur), and I(Ks) (P < 0.05). HF induced increases in LA Kv channel interacting protein 2 (P < 0.05), no change in Kv4.3, Kv1.5, or Kir2.3, and reduced Kir2.1 (P < 0.05). When I(Ca-L) was elicited by action potential (AP) clamp, HF APs reduced the integral of I(Ca) in control myocytes, with a larger reduction in HF myocytes (P < 0.05). I(CaL) measured with standard voltage clamp was unchanged by HF. Incubation of myocytes with N-acetylcysteine (a glutathione precursor) attenuated HF-induced electrophysiological alterations. LA angiotensin-1 receptor expression was increased in HF.
Chronic HF causes alterations in ion channel expression and ion currents, resulting in attenuation of the APD and atrial contractility and a substrate for persistent AF.
Cardiovascular research 07/2009; 84(2):227-36. · 5.80 Impact Factor
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Dmitry Terentyev,
Inna Györke,
Andriy E Belevych,
Radmila Terentyeva,
Arun Sridhar, Yoshinori Nishijima,
Esperanza Carcache de Blanco,
Savita Khanna,
Chandan K Sen,
Arturo J Cardounel,
Cynthia A Carnes,
Sandor Györke
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ABSTRACT: Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.
Circulation Research 12/2008; 103(12):1466-72. · 9.49 Impact Factor
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ABSTRACT: The goal of the present study was to assess how reduced SERCA2a expression affects in vivo myocardial ischemia/reperfusion (I/R) injury. We specifically wanted to determine to what extent hearts with reduced SERCA2a levels are susceptible to in vivo I/R injury. Therefore, we examined the effects of different ischemic periods on post-ischemic myocardial injury in wild-type (WT) and SERCA2a heterozygous knockout (SERCA2a(+/-)) mice expressing lower levels of SERCA2a pump in vivo. Following 20-min ischemia and 48-hour reperfusion, SERCA2a(+/-) mice developed significant myocardial infarction (MI) compared to negligible infarction in WT mice (14+/-3% vs. 3+/-1%, P<0.01); whereas following 30-min ischemia, the infarction was significantly larger in SERCA2a(+/-) mice compared to WT mice (49+/-5% vs. 37+/-3%, P<0.05). Further, echocardiographic analysis revealed worsened postischemic contractile function in SERCA2a(+/-) mice compared to WT mice. Thus, these findings demonstrate that maintaining optimal SERCA2a function is critical for myocardial protection from I/R injury and postischemic functional recovery.
Journal of Molecular and Cellular Cardiology 12/2008; 46(2):285-7. · 5.17 Impact Factor
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Arun Sridhar, Yoshinori Nishijima,
Dmitry Terentyev,
Radmila Terentyeva,
Rebecca Uelmen,
Monica Kukielka,
Ingrid M Bonilla,
Gail A Robertson,
Sandor Györke,
George E Billman,
Cynthia A Carnes
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ABSTRACT: Ventricular tachyarrhythmias are the most common cause of sudden cardiac death (SCD); a healed myocardial infarction increases the risk of SCD. We determined the contribution of specific repolarization abnormalities to ventricular tachyarrhythmias in a postinfarction model of SCD. For our methods, we used a postinfarction canine model of SCD, where an exercise and ischemia test was used to stratify animals as either susceptible (VF(+)) or resistant (VF(-)) to sustained ventricular tachyarrhythmias. Our results show no changes in global left ventricular contractility or volumes occurred after infarction. At 8-10 wk postmyocardial infarction, myocytes were isolated from the left ventricular midmyocardial wall and studied. In the VF(+) animals, myocyte action potential (AP) prolongation occurred at 50 and 90% repolarization (P < 0.05) and was associated with increased variability of AP duration and afterdepolarizations. Multiple repolarizing K(+) currents (I(Kr), I(to)) and inward I(K1) were also reduced (P < 0.05) in myocytes from VF(+) animals compared with control, noninfarcted dogs. In contrast, only I(to) was reduced in VF(-) myocytes compared with controls (P < 0.05). While afterdepolarizations were not elicited at baseline in myocytes from VF(-) animals, afterdepolarizations were consistently elicited after the addition of an I(Kr) blocker. In conclusion, the loss of repolarization reserve via reductions in multiple repolarizing currents in the VF(+) myocytes leads to AP prolongation, repolarization instability, and afterdepolarizations in myocytes from animals susceptible to SCD. These abnormalities may provide a substrate for initiation of postmyocardial infarction ventricular tachyarrhythmias.
AJP Regulatory Integrative and Comparative Physiology 09/2008; 295(5):R1463-72. · 3.34 Impact Factor
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ABSTRACT: Recent studies have demonstrated that increased expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a improves myocardial contractility and Ca2+ handling at baseline and in disease conditions, including myocardial ischemia-reperfusion (I/R). Conversely, it has also been reported that pharmacological inhibition of SERCA might improve postischemic function in stunned hearts or in isolated myocardium following I/R. The goal of this study was to test how decreases in SERCA pump level/activity affect cardiac function following I/R. To address this question, we used a heterozygous SERCA2a knockout (SERCA2a+/-) mouse model with decreased SERCA pump levels and studied the effect of myocardial stunning (20-min ischemia followed by reperfusion) and infarction (30-min ischemia followed by reperfusion) following 60-min reperfusion. Our results demonstrate that postischemic myocardial relaxation was significantly impaired in SERCA2a+/- hearts with both stunning and infarction protocols. Interestingly, postischemic recovery of contractile function was comparable in SERCA2a+/- and wild-type hearts subjected to stunning. In contrast, following 30-min ischemia, postischemic contractile function was reduced in SERCA2a+/- hearts with significantly larger infarction. Rhod-2 spectrofluorometry revealed significantly higher diastolic intracellular Ca2+ in SERCA2a+/- hearts compared with wild-type hearts. Both at 30-min ischemia and 2-min reperfusion, intracellular Ca2+ levels were significantly higher in SERCA2a+/- hearts. Electron paramagnetic resonance spin trapping showed a similar extent of postischemic free-radical generation in both strains. These data provide direct evidence that functional SERCA2a level, independent of oxidative stress, is crucial for postischemic myocardial function and salvage during I/R.
AJP Heart and Circulatory Physiology 04/2008; 294(3):H1426-34. · 3.71 Impact Factor
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ABSTRACT: Previous studies demonstrated an enhanced beta(2)-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on beta(1)-AR and beta(2)-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue beta-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because beta(2)-ARs are located in caveolae, caveolin-3 was also quantified. beta(1)-AR gene expression decreased ( approximately 5-fold), beta(2)-AR gene expression was not changed, and the ratio of beta(2)-AR to beta(1)-AR gene expression was significantly increased in susceptible compared with resistant dogs. beta(1)-AR protein decreased ( approximately 50%) and beta(2)-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned beta(1)-AR gene expression to levels seen in resistant animals but did not alter beta(2)-AR protein levels in susceptible dogs. These data suggest that beta(1)-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves beta(1)-AR gene expression, thereby restoring a more normal beta-AR balance.
AJP Heart and Circulatory Physiology 12/2007; 293(5):H2702-9. · 3.71 Impact Factor
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Yoshinori Nishijima,
Arun Sridhar,
Serge Viatchenko-Karpinski,
Courtney Shaw,
John D Bonagura,
William T Abraham,
Mandar S Joshi,
John Anthony Bauer,
Robert L Hamlin,
Sandor Györke,
David S Feldman,
Cynthia A Carnes
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ABSTRACT: While cardiac resynchronization therapy (CRT) has been shown to reduce morbidity and mortality in heart failure (HF) patients, the fundamental mechanisms for the efficacy of CRT are poorly understood. The lack of understanding of these basic mechanisms represents a significant barrier to our understanding of the pathogenesis of HF and potential recovery mechanisms. Our purpose was to determine cellular mechanisms for the observed improvement in chronic HF after CRT. We used a canine model of chronic nonischemic cardiomyopathy. After 15 months, dogs were randomized to continued RV tachypacing (untreated HF) or CRT for an additional 9 months. Six minute walk tests, echocardiograms, and electrocardiograms were done to assess the functional response to therapy. Left ventricular (LV) midmyocardial myocytes were isolated to study electrophysiology and intracellular calcium regulation. Compared to untreated HF, CRT improved HF-induced increases in LV volumes, diameters and mass (p<0.05). CRT reversed HF-induced prolongations in LV myocyte repolarization (p<0.05) and normalized HF-induced depolarization (p<0.03) of the resting membrane potential. CRT improved HF-induced reductions in calcium (p<0.05). CRT did not attenuate the HF-induced increases in LV interstitial fibrosis. Using a translational approach in a chronic HF model, CRT significantly improved LV structure; this was accompanied by improved LV myocyte electrophysiology and calcium regulation. The beneficial effects of CRT may be attributable, in part, to improved LV myocyte function.
Life Sciences 10/2007; 81(14):1152-9. · 2.53 Impact Factor
