Myofibrillar responsiveness to cAMP,PKA, and caffeine in an animal model of heart failure

We investigated whether an alteration of myofilament calcium responsiveness and contractile activation may in part contribute to heart failure. A control group of Broad Breasted White turkey poults was given regular feed without additive, whereas the experimental group was given the control ration with 700 ppm of furazolidone at 1 week of age for 3 weeks (DCM). At 4 weeks of age, left ventricular trabeculae carneae were isolated from hearts and calcium-force relationships studied. No differences in calcium-activation between fibers from control or failing hearts were noted under standard experimental conditions. Also failing hearts demonstrated no significant shift in the population of troponin T isoforms but we did observe a significant 4-fold decrease in TnT content in failing hearts compared to non-failing hearts. Addition of caffeine, however, resulted in a greater leftward shift on the calcium axis in fibers from failing hearts. At pCa 6, caffeine increased force by 26+/-2.1% in control fibers and 44.5+/-8.7% in myopathic fibers. Cyclic AMP resulted in a greater rightward shift on the calcium axis in failing myocardium. In control muscles, the frequency of minimum stiffness (f(min)) was higher than in muscles from failing hearts. cAMP and caffeine both shifted f(min) to higher frequencies in control fibers whereas in fibers from failing hearts both caused a greater shift. These results lead us to conclude that heart failure exerts differential effects on cAMP and caffeine responsiveness. Our data suggest that changes at the level of the thin myofilaments may alter myofilament calcium responsiveness and contribute to the contractile dysfunction seen in heart failure.     

Restoration of calcium handling properties of adult cardiac myocytes from hypertrophied hearts

Reductions in cardiac sarcoplasmic reticulum calcium-ATPase (Serca2a) levels are thought to underlie the prolonged calcium (Ca(2+)) transients and consequent reduced contractile performance seen in human cardiac hypertrophy and heart failure. In freshly isolated cardiac myocytes from rats with monocrotaline-induced right ventricular hypertrophy we found reduced sarcoplasmic reticulum Serca2a expression and prolonged Ca(2+)transients, characteristic of hypertrophic cardiac disease.Modulation of intracellular Ca(2+)levels, Ca(2+) kinetics or Ca(2+)sensitivity is the focus of many current therapeutic approaches to improve contractile performance in the hypertrophic or failing heart. However, the functional effects of increasing Serca2a expression on Ca(2+) handling properties in myocytes from an animal model of cardiac hypertrophy are largely unknown. Here, we describe enhancement of the deficient Ca(2+) handling properties evident in myocytes from hypertrophied hearts following adenoviral-mediated transfer of the human Serca2a gene to these myocytes.These results highlight the importance of Serca2a deficiencies in the hypertrophic phenotype of cardiac muscle and suggest a simple, effective approach for manipulation of normal cardiac function.     

Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension

Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventricle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca(2+) handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca(2+) transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(endo)plasmic reticular Ca(2+)-ATPase activity, increased sarcoplasmic reticular Ca(2+)-release fraction, and increased Ca(2+) spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca(2+) handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs.     

压力负荷性心衰小鼠左心室跨壁L-型钙电流的变化

为了观察正常和心衰时心内膜下和心外膜下心肌细胞L-型钙电流(ICa-L)的差别,我们采用主动脉弓狭窄的方法建立小鼠压力超负荷性心衰模型,采用全细胞膜片钳技术记录了正常、主动脉狭窄(band)及假手术对照(sham)组动物左心室游离壁内、外膜下心肌细胞的动作电位时程(action potential duration,APD)和ICa-L。结果显示:(1)与sham组同龄的正常小鼠左心室心内膜下细胞动作电位复极达90％的时程(APD90)为(38．2±6．44)ms,较心外膜下细胞的APD90(15.67±5.31)ms明显延长,二者的比值约为2．5:1;内膜下细胞和外膜下细胞ICa-L密度没有差异,峰电流密度分别为(-2.7±0.49)pA/pF和(-2.54±0．53)pA/pF;(2)Band组内、外膜下细胞的动作电位复极达50％的时程(APD50)、APD90均较sham组显著延长,尤以内膜下细胞延长突出,分别较sham组延长了400％和360％,内、外膜下细胞APD90的比值约为4.2:1;(3)与sham组相比, band组内膜下细胞ICa-L密度显著减小,在+10 mV～+40 mV的4个电压下分别降低了20．2％、21．4％、21.6％和25.7％(P< 0．01),但其激活电位、峰电位和翻转电位没有改变;band组外膜下细胞的ICa-L密度与同期sham组相比无明显变化;band组钙通道激活、失活及复活的动力学特征与sham组相比没有改变。以上结果提示,生理状态下小鼠左心室内、外膜下细胞ICa-L密度不存在明显差别,提示ICa-L与APD跨壁异质性的产生无关;心衰时左心室内、外膜下细胞APD明显延长,以内膜下细胞延长尤为突出,内膜下细胞ICa-L密度明显减少,而外膜下细胞ICa-L密度无明显改变,这种ICa-L的非同步变化在心衰时可能起到对抗APD延长、减少复极离散度的有益作用。     

mRNA Expression Levels in Failing Human Hearts Predict Cellular Electrophysiological Remodeling: A Population-Based Simulation Study

Differences in mRNA expression levels have been observed in failing versus non-failing human hearts for several membrane channel proteins and accessory subunits. These differences may play a causal role in electrophysiological changes observed in human heart failure and atrial fibrillation, such as action potential (AP) prolongation, increased AP triangulation, decreased intracellular calcium transient (CaT) magnitude and decreased CaT triangulation. Our goal is to investigate whether the information contained in mRNA measurements can be used to predict cardiac electrophysiological remodeling in heart failure using computational modeling. Using mRNA data recently obtained from failing and non-failing human hearts, we construct failing and non-failing cell populations incorporating natural variability and up/down regulation of channel conductivities. Six biomarkers are calculated for each cell in each population, at cycle lengths between 1500 ms and 300 ms. Regression analysis is performed to determine which ion channels drive biomarker variability in failing versus non-failing cardiomyocytes. Our models suggest that reported mRNA expression changes are consistent with AP prolongation, increased AP triangulation, increased CaT duration, decreased CaT triangulation and amplitude, and increased delay between AP and CaT upstrokes in the failing population. Regression analysis reveals that changes in AP biomarkers are driven primarily by reduction in I, and changes in CaT biomarkers are driven predominantly by reduction in I and SERCA. In particular, the role of I is pacing rate dependent. Additionally, alternans developed at fast pacing rates for both failing and non-failing cardiomyocytes, but the underlying mechanisms are different in control and heart failure.     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.     

Mg-ATPase and CA2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts – effects of calcium sensitizing agents MCI-154, DPI 201–106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca²⁺ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca²⁺ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10^–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca²⁺ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca²⁺ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca²⁺ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.     

Modeling the cellular basis of altered excitation-contraction coupling in heart failure

Ca transients measured in failing human ventricular myocytes exhibit reduced amplitude and slowed relaxation [Beuckelmann, D.J., Nabauer, M., Erdmann, E., 1992. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055; Gwathmey, J.K., Copelas, L., MacKinnon, R., Schoen, F.J., Feldman, M.D., Grossman, W., Morgan, J.P., 1987. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. 61, 70-76; Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.H., Kass, D.A., Marban, E., Tomaselli, G.F., 1996. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. 78(2); Li, H.G., Jones, D.L., Yee, R., Klein, G.J., 1992. Electrophysiologic substrate associated with pacing-induced hert failure in dogs: potential value of programmed stimulation in predicting sudden death. J. Am. Coll. Cardiol. 19(2), 444-449; Vermeulen, J.T., McGuire, M.A., Opthof, T., Colonel, R., Bakker, J.M.T.d., Klopping, C., Janse, M.J., 1994. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.] and blunted frequency dependence [Davies, C.H., Davia, K., Bennett, J.G., Pepper, J.R., Poole-Wilson, P.A., Harding, S.E., 1995. Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure. Circulation, 92, 2540-2549; Hasenfuss, G., Reinecke, H., Studer, R., Meyer, M., Pieske, B., Holtz, J., Holubarsch, C., Posival, H., Just, H., Drexler, H., 1994. Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442; Hasenfuss, G., Reinecke, H., Studer, R., Pieske, B., Meyer, M., Drexler, H., Just, H., 1996. Calcium cycling proteins and force-frequency relationships in heart failure. Basic Res. Cardiol. 91, 17-22; Monte, F.D., O'Gara, P., Poole-Wilson, P.A., Yacoub, M., Harding, S.E., 1995. Cell geometry and contractile abnormalities of myocytes from failing human left ventricle. Cardiovasc. Res. 30, 281-290; Philips, P.J., Gwathmey, J.K., Feldman, M.D., Schoen, F.J., Grossman, W., Morgan, J.P., 1990. Post-extrasystolic potentiation and the force-frequency relationships: differential augmentation of myocardial contractility in working myocardium from patients with end-stage heart failure. J. Mol. Cell. Cardiol. 22, 99-110; Pieske, B., Hasenfuss, G., Holubarsch, C., Schwinger, R., Bohm, M., Just, H., 1992. Alterations of the force-frequency relationship in the failing human heart depend on the underlying cardiac disease. Basic Res. Cardiol. 87, 213-221.]. Analyses of protein levels in these failing hearts reveal that the SR Ca-ATPase is down-regulated on average by 50% and that the Na/Ca exchanger is up-regulated on average by a factor of two. In this paper, we test the hypothesis that this altered pattern of expression of Ca handling proteins is sufficient to account for changes in excitation-contraction coupling properties measured experimentally at the cellular level. To do this, we present an integrated model of excitation-contraction coupling in the guinea pig ventricular cell. The model is used to determine the effects of SR Ca-ATPase down-regulation and Na/Ca exchanger up-regulation on action potential duration, Ca transient shape and amplitude, and isometric force. Model analyses demonstrate that changes in Ca handling proteins play a direct and critical role in prolongation of action potential duration, and in reduction of contractile force in heart failure.     

Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43+/-; 66% mean reduction in Cx43) mice for 6 h at 10-15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43+/- myocytes had significantly shorter action potential durations (APD) and increased steady-state (Iss) and inward rectifier (I(K1)) potassium currents compared with those of wild-type littermate cells. In Cx43+/- hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of Iss compared with unpaced Cx43+/- hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.     

Simulation and mechanistic investigation of the arrhythmogenic role of the late sodium current in human heart failure

Heart failure constitutes a major public health problem worldwide. The electrophysiological remodeling of failing hearts sets the stage for malignant arrhythmias, in which the role of the late Na(+) current (I(NaL)) is relevant and is currently under investigation. In this study we examined the role of I(NaL) in the electrophysiological phenotype of ventricular myocytes, and its proarrhythmic effects in the failing heart. A model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of I(NaL). A sensitivity analysis of the model was performed and simulations of the pathological electrical activity of the cell were conducted. The proposed model for the human I(NaL) and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. The sensitivity analysis of the modulation of electrophysiological parameters of myocytes from failing hearts due to ion channels remodeling, revealed a role for I(NaL) in the prolongation of action potential duration (APD), triangulation of the shape of the AP, and changes in Ca(2+) transient. A mechanistic investigation of intracellular Na(+) accumulation and APD shortening with increasing frequency of stimulation of failing myocytes revealed a role for the Na(+)/K(+) pump, the Na(+)/Ca(2+) exchanger and I(NaL). The results of the simulations also showed that in failing myocytes, the enhancement of I(NaL) increased the reverse rate-dependent APD prolongation and the probability of initiating early afterdepolarizations. The electrophysiological remodeling of failing hearts and especially the enhancement of the I(NaL) prolong APD and alter Ca(2+) transient facilitating the development of early afterdepolarizations. An enhanced I(NaL) appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of [Ca(2+)](i) homeostasis of failing myocytes.     

Changes in ionic currents and beta-adrenergic receptor signaling in hypertrophied myocytes overexpressing G alpha(q)

Transgenic overexpression of G alpha(q) causes cardiac hypertrophy and depressed contractile responses to beta-adrenergic receptor agonists. The electrophysiological basis of the altered myocardial function was examined in left ventricular myocytes isolated from transgenic (G alpha(q)) mice. Action potential duration was significantly prolonged in G alpha(q) compared with nontransgenic (NTG) myocytes. The densities of inward rectifier K(+) currents, transient outward K(+) currents (I(to)), and Na(+)/Ca(2+) exchange currents were reduced in G alpha(q) myocytes. Consistent with functional measurements, Na(+)/Ca(2+) exchanger gene expression was reduced in G alpha(q) hearts. Kinetics or sensitivity of I(to) to 4-aminopyridine was unchanged, but 4-aminopyridine prolonged the action potential more in G alpha(q) myocytes. Isoproterenol increased L-type Ca(2+) currents (I(Ca)) in both groups, with a similar EC(50), but the maximal response in G alpha(q) myocytes was approximately 24% of that in NTG myocytes. In NTG myocytes, the maximal increase of I(Ca) with isoproterenol or forskolin was similar. In G alpha(q) myocytes, forskolin was more effective and enhanced I(Ca) up to approximately 55% of that in NTG myocytes. These results indicate that the changes in ionic currents and multiple defects in the beta-adrenergic receptor/Ca(2+) channel signaling pathway contribute to altered ventricular function in this model of cardiac hypertrophy.     

Proteomic analysis reveals significant elevation of heat shock protein 70 in patients with chronic heart failure due to arrhythmogenic right ventricular cardiomyopathy

As proteins are the ultimate biological determinants of phenotype of disease, we screened altered proteins associated with heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC) to identify biomarkers potential for rapid diagnosis of heart failure. By 2-dimensional gel electrophoresis and mass spectrometry, we identified five commonly altered proteins with more than 1.5 fold changes in eight ARVC failing hearts using eight non-failing hearts as reference. Noticeably, one of the altered proteins, heat shock protein 70 (HSP70), was increased by 1.64 fold in ARVC failing hearts compared with non-failing hearts. The increase of cardiac HSP70 was further validated by Western blot, immunochemistry, and enzyme-linked immunosorbent assay (ELISA) in failing hearts due to not only ARVC, but also dilated (DCM, n = 18) and ischemic cardiomyopathy (ICM, n = 8). Serum HSP70 was also observed to be significantly increased in heart failure patients derived from the three forms of cardiomyopathies. In addition, we observed hypoxia/serum depletion stimulation induced significantly elevation of intracellular and extracellular HSP70 in cultured neonatal rat cardiomyocytes. For the first time to our knowledge, we revealed and clearly demonstrated significant up-regulation of cardiac and serum HSP70 in ARVC heart failure patients. Our results indicate that elevated HSP70 is the common feature of heart failure due to ARVC, DCM, and ICM, which suggests that HSP70 may be used as a biomarker for the presence of heart failure due to cardiomyopathies of different etiologies and may hold diagnostic/prognostic potential in clinical practice.     

Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury

The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (K_ATP) channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane K_ATP channels ensures a rapid energy-sparing reduction in action potential duration (APD) in response to metabolic challenges, while cellular signaling that reduces surface K_ATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kir6.2 K_ATP channel subunit initiating a cascade responsible for K_ATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB) model of heart failure is coupled with a 35–40% reduction in surface expression of K_ATP channels compared to hearts from sham-operated mice. Linkage between K_ATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of K_ATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial K_ATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by K_ATP channels. Recognition of CaMKII-dependent downregulation of K_ATP channel expression as a mechanism for vulnerability to injury in failing hearts points to strategies targeting this interaction for potential preventives or treatments.     

8-Bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes

Superfusion with 8-bromo-cyclic GMP or intracellular injection of cyclic GMP inhibits calcium-dependent slow action potentials in embryonic chick or guinea pig ventricular cells, suggesting that cyclic GMP inhibits calcium currents. Recently, cyclic GMP has been shown to reduce cyclic AMP-stimulated calcium currents in voltage-clamped ventricular myocytes. Since earlier results in intact cells had suggested that cyclic GMP might inhibit basal (i.e., unstimulated by cyclic AMP) calcium currents, we directly investigated the effect of 8-bromo-cyclic GMP on basal calcium channel currents (using barium as the charge carrier) in voltage-clamped ventricular myocytes isolated from embryonic chick hearts. Superfusion with 1 mM 8-bromo-cyclic GMP (without prior cyclic AMP elevation) progressively decreased peak calcium channel currents (-68% at 15 min after the onset of drug exposure). In contrast, the currents were unchanged during 15 min superfusion with control solution, or 1 mM 8-bromo-GMP (the noncyclic inactive analog of 8-bromo-cyclic GMP). The present results in voltage-clamped embryonic chick heart cells indicate that cyclic GMP can inhibit basal calcium channel currents, apparently through a cyclic AMP-independent mechanism.     

Histidine button engineered into cardiac troponin I protects the ischemic and failing heart

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.     

Electrical remodeling of cardiac myocytes from mice with heart failure due to the overexpression of tumor necrosis factor-alpha

Mice that overexpress the inflammatory cytokine tumor necrosis factor-alpha in the heart (TNF mice) develop heart failure characterized by atrial and ventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias, and increased mortality (males > females). Abnormalities in Ca2+ handling, prolonged action potential duration (APD), calcium alternans, and reentrant atrial and ventricular arrhythmias were previously observed with the use of optical mapping of perfused hearts from TNF mice. We therefore tested whether altered voltage-gated outward K+ and/or inward Ca2+ currents contribute to the altered action potential characteristics and the increased vulnerability to arrhythmias. Whole cell voltage-clamp recordings of K+ currents from left ventricular myocytes of TNF mice revealed an approximately 50% decrease in the rapidly activating, rapidly inactivating transient outward K+ current Ito and in the rapidly activating, slowly inactivating delayed rectifier current IK,slow1, an approximately 25% decrease in the rapidly activating, slowly inactivating delayed rectifier current IK,slow2, and no significant change in the steady-state current Iss compared with controls. Peak amplitudes and inactivation kinetics of the L-type Ca2+ current ICa,L were not altered. Western blot analyses revealed a reduction in the proteins underlying Kv4.2, Kv4.3, and Kv1.5. Thus decreased K+ channel expression is largely responsible for the prolonged APD in the TNF mice and may, along with abnormalities in Ca2+ handling, contribute to arrhythmias.     

Altered spatial calcium regulation enhances electrical heterogeneity in the failing canine left ventricle: implications for electrical instability

Myocytes across the left ventricular (LV) wall of the mammalian heart are known to exhibit heterogeneity of electrophysiological properties; however, the transmural variation of cellular electrophysiology and Ca(2+) homeostasis in the failing LV is incompletely understood. We studied action potentials (APs), the L-type calcium (Ca(2+)) current (I(Ca,L)), and intracellular Ca(2+) transients ([Ca(2+)](i)) of subendocardial (Endo), midmyocardial (Mid), and subepicardial (Epi) tissue layers in the canine normal and tachycardia pacing-induced failing left ventricles. Heart failure (HF) was associated with significant prolongation of the AP duration in Mid myocytes. There were no differences in I(Ca,L) density in normal Endo, Mid, and Epi myocytes, whereas in the failing heart, I(Ca,L) density was downregulated by 45% and 26% (at +10 mV) in Endo and Mid myocytes, respectively. The rates of sarcoplasmic reticulum (SR) Ca(2+) release and decay of the [Ca(2+)](i) were slowed, and the amplitude of the [Ca(2+)](i) was depressed in Endo and Epi myocytes isolated from failing, compared with normal, hearts. Experiments in sodium (Na(+))-free solutions showed that Epi and Mid myocytes of the failing ventricle exhibit a greater reliance on the Na(+)-Ca(2+) exchanger to remove cytosolic Ca(2+) than myocytes isolated from normal hearts. Simulation studies in Endo, Mid, and Epi canine myocytes demonstrate the importance of L-type current density and SR Ca(2+) uptake in modulating the potentially arrhythmogenic repolarization in HF. In conclusion, these results demonstrate that spatially heterogeneous decreases in I(Ca,L) and defective cytosolic Ca(2+) removal contribute to the altered [Ca(2+)](i) and AP profiles across the canine failing LV. These distinct electrophysiological features in myocytes from a failing heart contribute to a characteristic electrogram arising from increased dispersion of refractoriness across the LV, which may result in significant arrhythmogenic sequellae.     

The Antiarrhythmic and Anticonvulsant Effects of Dietary N-3 Fatty Acids

It has been shown in animals and probably in humans, that n-3 polyunsaturated fatty acids (PUFAs) are antiarrhythmic. We report recent studies on the antiarrhythmic actions of PUFAs. The PUFAs stabilize the electrical activity of isolated cardiac myocytes by modulating sarcolemmal ion channels, so that a stronger electrical stimulus is required to elicit an action potential and the refractory period is markedly prolonged. Inhibition of voltage-dependent sodium currents, which initiate action potentials in excitable tissues, and of the L-type calcium currents, which initiate release of sarcoplasmic calcium stores that increase cytosolic free calcium concentrations and activate the contractile proteins in myocytes, appear at present to be the probable major antiarrhythmic mechanism of the PUFAs. Received: 27 May 1999/Revised: 20 July 1999