Mutation in sodium-calcium exchanger 1 (NCX1) causes cardiac fibrillation in zebrafish

Cardiac fibrillation, a form of cardiac arrhythmia, is the most common cause of embolic stroke and death associated with heart failure. The molecular mechanisms underlying cardiac fibrillation are largely unknown. Here we report a zebrafish model for cardiac fibrillation. The hearts of zebrafish tremblor (tre) mutants exhibit chaotic movements and fail to develop synchronized contractions. Calcium imaging showed that normal calcium transients are absent in tre cardiomyocytes, and molecular cloning of the tre mutation revealed that the tre locus encodes the zebrafish cardiac-specific sodium-calcium exchanger (NCX) 1, NCX1h. Forced expression of NCX1h or other calcium-handling molecules restored synchronized heartbeats in tre mutant embryos in a dosage-dependent manner, demonstrating the critical role of calcium homeostasis in maintaining embryonic cardiac function. By creating mosaic zebrafish embryos, we showed that sporadic NCX1h-null cells were not sufficient to disrupt normal cardiac function, but clustered wild-type cardiomyocytes contract in unison in tre mutant hearts. These data signify the essential role of calcium homeostasis and NCX1h in establishing rhythmic contraction in the embryonic zebrafish heart.     

Heat stress responses modulate calcium regulations and electrophysiological characteristics in atrial myocytes

Heat stress-induced responses change the ionic currents and calcium homeostasis. However, the molecular insights into the heat stress responses on calcium homeostasis remain unclear. The purposes of this study were to examine the mechanisms of heat stress responses on calcium handling and electrophysiological characteristics in atrial myocytes. We used indo-1 fluorimetric ratio technique and whole-cell patch clamp to investigate the intracellular calcium, action potentials, and ionic currents in isolated rabbit single atrial cardiomyocytes with or without (control) exposure to heat stress (43 °C, 15 min) 5 ± 1 h before experiments. The expressions of sarcoplasmic reticulum ATPase (SERCA2a), and Na⁺-Ca²⁺ exchanger (NCX) in the control and heat stress-treated atrial myocytes were evaluated by Western blot and real-time PCR. As compared with control myocytes, the heat stress-treated myocytes had larger sarcoplasmic reticulum calcium content and larger intracellular calcium transient with a shorter decay portion. Heat stress-treated myocytes also had larger L-type calcium currents, transient outward potassium currents, but smaller NCX currents. Heat stress responses increased the protein expressions, SERCA2a, NCX, and heat shock protein. However, heat stress responses did not change the RNA expression of SERCA2a and NCX. In conclusion, heat stress responses change calcium handling through protein but not RNA regulation.     

Analysis of Na+/Ca2+ exchanger knockout mice

The Na(+)/Ca(2+) exchanger (NCX) on the plasma membrane is thought to be the main calcium extrusion system from the cytosol to the extracellular space in many mammalian excitable cells including cardiac myocytes. However, the precise roles of NCX are still unclear because of lack of its specific inhibitors. We generated NCX1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous mutant died at 9.5 days post coitum. Embryonic hearts did not beat and cardiac myocytes showed apoptosis. These results suggest that NCX1 is required for heart beats and survival of cardiac myocytes in embryos. Heterozygous mutant mice were viable and indistinguishable from wild type mice. mRNA and protein levels in the heart of heterozygous mutant were half as much as wild type mice. In response to pressure overload, mutant mice showed better systolic and diastolic relaxation functions than wild type mice. Intracellular Ca(2+) measurement revealed an increase in calcium content of cytoplasm and sarcoplasmic reticulum (SR) and RNA analysis revealed preserved SR Ca(2+) ATPase expression in the ventricle of mutant mice. These results suggest that NCX plays an important role in cardiac performance in these pathological situations.     

The Na+/Ca2+ exchanger/SR Ca2+ ATPase transport capacity regulates the contractility of normal and hypertrophied feline ventricular myocytes

BACKGROUND: Pressure overload leads to cardiac hypertrophy, which is often followed by heart failure. We tested the hypothesis that depressed contractility in this process results from an imbalance in Ca 2+ transport by the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) and the sarcolemmal Na+/Ca2+ exchanger (NCX). METHODS AND RESULTS: Left ventricular (LV) myocytes (n = 79) from 12 normal (N) and 5 hypertrophied (LVH, by aortic banding) feline hearts were studied. Adenoviral gene transfer was used to introduce green fluorescent protein (GFP), SERCA2, and NCX into N and LVH myocytes. Contraction (videomicroscopy) and Ca2+ transients (Fluo-3) were measured in steady state and after rest periods of 2 to 120 seconds (rest decay and potentiation). LVH hearts were significantly larger than N (7.1 +/- 1.4 versus 4.2 +/- 0.2 g/kg). SERCA protein was significantly less abundant in LVH versus N. Steady state contractions and Ca2+ transients of LVH-GFP myocytes decayed more slowly and rest decay of contractility was more pronounced compared with N-GFP. Infection of LVH (and N) myocytes with SERCA increased basal contractility and reduced rest decay. Infection of LVH myocytes with NCX almost abolished contraction and in N myocytes reduced contractility and increased rest decay. CONCLUSION: These findings suggest that an imbalance of Ca2+ transport by SERCA and the NCX produces the characteristic contractile abnormalities of hypertrophied cardiac myocytes.     

Abnormalities of calcium cycling in the hypertrophied and failing heart

Progressive deterioration of cardiac contractility is a central feature of congestive heart failure (CHF) in humans. In this report we review those studies that have addressed the idea that alterations of intracellular calcium (Ca(2+)) regulation is primarily responsible for the depressed contractility of the failing heart. The review points out that Ca(2+)transients and contraction are similar in non-failing and failing myocytes at very slow frequencies of stimulation (and other low stress environments). Faster pacing rates, high Ca(2+)and beta-adrenergic stimulation reveal large reductions in contractile reserve in failing myocytes. The underlying cellular basis of these defects is then considered. Studies showing changes in the abundance of L-type Ca(2+)channels, Ca(2+)transport proteins [sarcoplasmic reticulum Ca(2+)ATPase (SERCA2), phospholamban (PLB), Na(+)/Ca(2+) exchanger (NCX)] and Ca(2+) release channels (RYR) in excitation-contraction coupling and Ca(2+)release and uptake by the sarcoplasmic reticulum (SR) are reviewed. These observations support our hypotheses that (i) defective Ca(2+)regulation involves multiple molecules and processes, not one molecule, (ii) the initiation and progression of CHF inolves defective Ca(2+)regulation, and (iii) prevention or correction of Ca(2+)regulatory defects in the early stages of cardiac diseases can delay or prevent the onset of CHF.     

Endothelin-1 prolongs intracellular calcium transient decay in neonatal rat cardiac myocytes

Endothelin-1 (ET-1) is involved in the development of cardiac hypertrophy and heart failure. We investigated the effects of ET-1 on intracellular calcium transient and its mechanisms. Neonatal rat cardiomyocytes were prepared and calcium transient was measured using fura-2. Treatment with ET-1 for 48 h prolonged calcium transient decay. In the presence of thapsigargin, ET-1 did not alter calcium transient decay. On the other hand, the prolonged calcium transient decay was maintained even when sodium was removed from the bath solution. These results indicate that ET-1-induced prolongation of calcium transient decay is mainly due to the suppression of calcium uptake by sarcoplasmic reticulum, but not inhibition of the sodium/calcium exchanger. Northern blotting analysis revealed that sarcoplasmic reticulum ATPase (SERCA2) mRNA was decreased in ET-1-treated cardiomyocytes, and that this decrease was inhibited by BQ-123 but not by BQ-788. Moreover, pretreatment with chelerythrine partially restored the ET-1-induced decrease in SERCA2 mRNA, whereas phorbol 12-myristate 13-acetate markedly reduced SERCA2 gene expression. Real-time RT-PCR analysis showed abundant ETA receptor gene expression in cardiomyocytes. ET-1 reduces SERCA2 gene expression through the ETA receptor and PKC pathway, and prolongs intracellular calcium transient decay. Specific inhibition of the ETA receptor may be a possible therapeutic strategy for improving cardiac performance.     

Combined phospholamban ablation and SERCA1a overexpression result in a new hyperdynamic cardiac state

OBJECTIVE: Phospholamban ablation or ectopic expression of SERCA1a in the heart results in significant increases in cardiac contractile parameters. The aim of the present study was to determine whether a combination of these two genetic manipulations may lead to further augmentation of cardiac function. METHODS: Transgenic mice with cardiac specific overexpression of SERCA1a were mated with phospholamban deficient mice to generate a model with SERCA1a overexpression in the phospholamban null background (SERCA1(OE)/PLB(KO)). The cardiac phenotype was characterized using quantitative immunoblotting, sarcoplasmic reticulum calcium uptake and single myocyte mechanics and calcium kinetics. RESULTS: Quantitative immunoblotting revealed an increase of 1.8-fold in total SERCA level, while SERCA2 was decreased to 50% of wild types. Isolated myocytes indicated increases in the maximal rates of contraction by 195 and 125%, the maximal rates of relaxation by 200 and 124%, while the time for 80% decay of the Ca(2+)-transient was decreased to 43 and 75%, in SERCA1(OE)/PLB(KO) hearts, compared to SERCA1a overexpressors and phospholamban knockouts, respectively. These mechanical alterations reflected parallel alterations in V(max) and EC(50) for Ca(2+) of the sarcoplasmic reticulum Ca(2+) transport system. Furthermore, there were no significant cardiac histological or pathological alterations, and the myocyte contractile parameters remained enhanced, up to 12 months of age. CONCLUSIONS: These findings suggest that a combination of SERCA1a overexpression and phospholamban ablation results in further enhancement of myocyte contractility over each individual alteration.     

Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.     

The cardiac sarcoplasmic/endoplasmic reticulum calcium ATPase: a potent target for cardiovascular diseases

The cardiac isoform of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a) is a calcium ion (Ca(2+)) pump powered by ATP hydrolysis. SERCA2a transfers Ca(2+) from the cytosol of the cardiomyocyte to the lumen of the sarcoplasmic reticulum during muscle relaxation. As such, this transporter has a key role in cardiomyocyte Ca(2+) regulation. In both experimental models and human heart failure, SERCA2a expression is significantly decreased, which leads to abnormal Ca(2+) handling and a deficient contractile state. Following a long line of investigations in isolated cardiac myocytes and small and large animal models, a clinical trial is underway that is restoring SERCA2a expression in patients with heart failure by use of adeno-associated virus type 1. Beyond its role in contractile abnormalities in heart failure, SERCA2a overexpression has beneficial effects in a host of other cardiovascular diseases. Here we describe the mechanism of Ca(2+) regulation by SERCA2a, examine the beneficial effects as well as the failures, risks and complexities associated with SERCA2a overexpression, and discuss the potential of SERCA2a as a target for the treatment of cardiovascular disease.     

The role of L-type calcium current in the generation of repolarization-induced contraction in cardiac myocytes

OBJECTIVE: Early experiments into the arrhythmogenic transient inward current frequently showed apparent coupling of this current to repolarization from a depolarizing voltage clamp step. Calcium transients have subsequently been shown to couple to such repolarization and are the result of calcium release from the sarcoplasmic reticulum. We have investigated whether this phenomenon is due to calcium entry via non-inactivated calcium channels or to voltage-activated SR release. METHODS: Voltage clamp steps were imposed on isolated guinea pig and rabbit cardiac myocytes. Calcium release was monitored by tracking cell contraction. L-type calcium current at the moment of repolarization was manipulated by the rapid application of 2 mM cadmium or 10 mM calcium. RESULTS: Repolarization-induced contraction was abolished by the rapid application of 2 mM cadmium immediately prior to repolarization, and was augmented by the rapid change of extracellular calcium concentration from 2 mM to 10 mM immediately prior to repolarization. There is no evidence of coupling of drive train-induced aftercontractions to repolarization from the final action potential of the drive train and 2 mM cadmium does not alter the appearance or timing of these aftercontractions. Simulation of phase 1 repolarization in the mammalian cardiac action potential decreases rather than increases twitch amplitude. CONCLUSION: Repolarization-induced contraction results from calcium entry through non-inactivated calcium channels, not from voltage-activated release. It plays no physiological role in contributing to the stimulated twitch and no pathological role in generating drive train-induced aftercontractions.     

Cardiac-specific overexpression of catalase rescues ventricular myocytes from ethanol-induced cardiac contractile defect

Oxidative stress is intimately involved in alcoholic cardiomyopathy. Catalase is responsible for detoxification of hydrogen peroxide (H(2)O(2)) and may interfere with ethanol-induced cardiac toxicity. To test this hypothesis, a transgenic mouse line was produced to overexpress catalase (~50-fold) in the heart, ranging from sarcoplasm, the nucleus and peroxisomes within myocytes. Mechanical and intracellular Ca(2+) properties were evaluated in ventricular myocytes from catalase transgenic (CAT) and wild-type FVB mice. Protein abundance of sarco (endo) plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), Na(+)/Ca(2+) exchanger (NCX), dihydropyridine Ca(2+) receptor (DHPR), ryanodine receptor (RyR), Akt and phosphorylated Akt (pAkt) were measured by western blot. CAT itself did not alter body and organ weights, as well as myocyte contractile properties. Acute exposure of ethanol elicited a concentration-dependent depression in cell shortening and intracellular Ca(2+) in FVB mice with maximal inhibitions of 65.4% and 35.8%, respectively. The ethanol-induced cardiac depression was significantly attenuated in myocytes from CAT with maximal inhibitions of 42.4% and 27.3%. CAT also abrogated the ethanol-induced inhibition of maximal velocity of shortening/relengthening, prolongation of relengthening duration and intracellular Ca(2+) clearing time. Cell shortening at different extracellular Ca(2+) revealed stronger myocyte-shortening amplitude under lower (0.5 mM) Ca(2+) in CAT mice. Protein expression of NCX, RyR, Akt and pAkt were elevated in myocytes from CAT mice, while those of SERCA, PLB and DHPR were not affected. In conclusion, our data suggest that catalase overexpression may protect cardiac myocytes from ethanol-induced contractile defect, partially through improved intracellular Ca(2+) handling and Akt signaling.     

Sarcoplasmic reticulum Ca(2+)ATPase and cell contraction in developing rabbit heart

The purpose of the present study was to determine whether age-related changes in the expression and function of the cardiac isoform of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) play a role in SR Ca(2+)release and cell contraction. SERCA2a protein levels and subcellular localization were compared between fetal, neonatal, juvenile and adult New Zealand White rabbits. Studies of SERCA function in isolated myocytes were performed in situ by examining the rate of reloading of the SR Ca(2+)stores following caffeine-induced depletion. We found that significant quantities of SERCA2a were present early in immature heart and that SERCA2a expression reached adult levels within 15-30 days after birth. Furthermore, SERCA2a protein is present as a series of transverse striations within the cell as early as 1 day of age. In contrast to previous studies of SERCA in vitro, the SERCA protein function in situ was found to be comparable between neonatal and adult myocytes in maintaining SR Ca(2+)stores. These results indicate that the paucity of SR Ca(2+)release in immature ventricular cardiac myocytes is not the result of immaturity in SERCA2a expression.     

Sarcolipin and phospholamban as regulators of cardiac sarcoplasmic reticulum Ca2+ ATPase

The cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a) plays a critical role in maintaining the intracellular calcium homeostasis during cardiac contraction and relaxation. It has been well documented over the years that altered expression and activity of SERCA2a can lead to systolic and diastolic dysfunction. The activity of SERCA2a is regulated by two structurally similar proteins, phospholamban (PLB) and sarcolipin (SLN). Although, the relevance of PLB has been extensively studied over the years, the role SLN in cardiac physiology is an emerging field of study. This review focuses on the advances in the understanding of the regulation of SERCA2a by SLN and PLB. In particular, it highlights the similarities and differences between the two proteins and their roles in cardiac patho-physiology.     

Hypoxia and HIF-1 suppress SERCA2a expression in embryonic cardiac myocytes through two interdependent hypoxia response elements

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA2a) is an essential component of cardiomyocyte excitation–contraction (EC)-coupling. Suppression of SERCA2a expression induces contractile dysfunction and has been reported in various forms of ischemic cardiac disease as well as in hypobaric hypoxia. The present study investigated whether SERCA2a expression is regulated by hypoxia in embryonic mouse cardiomyocytes and explored the underlying mechanism. We show that in cultured embryonic cardiomyocytes hypoxia (1% O₂) induce time-dependent downregulation of SERCA2a expression. This mechanism manifested as specific changes in cardiac myocyte calcium signals induced by reduced expression and activity of SERCA2a. Chemical activation of hypoxia-inducible factor-1 (HIF-1) by DFO or overexpression of normoxia-stabile HIF-1α (HIF-1α/VP16) suppressed endogenous SERCA2a expression as well as the activity of the SERCA2a-promoter-luciferase reporter. Analysis of the SERCA2a promoter found two putative HIF-1 binding HRE-sites. Site-specific promoter mutagenesis revealed that co-operative HIF-1 binding to both of these hypoxia response elements on the SERCA2a promoter is required for expressional suppression. This mechanism establishes a link between oxygen supply and calcium activity in embryonic cardiac myocytes that is exploited in cardiac development, and further may offer a possible explanation for the functional depression of SERCA2a seen in ischemic and hypoxic myocardium.     

Junctophilins: molecular components contributing junctional membrane complexes between the cell-surface membrane and endoplasmic/sarcoplasmic reticulum

The junctional membrane complex between the plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ER/SR) is an important structural foundation for functional crosstalk between ionic channels. In cardiac myocytes, functional coupling between cell-surface and intracellular Ca(2+) channels produces Ca(2+) transients for contraction. Junctophilins, a novel family of junctional membrane complex proteins, contribute to the stabilization of the junctional membrane complex by anchoring the ER/SR and interacting with the PM. Mutant mice lacking the cardiac-type junctophilin exhibited embryonic lethality due to heart failure, and the mutant cardiac myocytes showed deficiency of the junctional membrane complexes and abnormal Ca(2+) signaling.     

Enhanced Ca(2+)-activated Na(+)-Ca(2+) exchange activity in canine pacing-induced heart failure

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.     

Ryanodine受体在心力衰竭和心律失常中的作用

Ryanodine受体（ryanodine receptor,RyR）是肌浆网膜上的钙释放通道。钙释放失调在心力衰竭发展过程中具有关键作用。此外,RyR基因突变会导致肌浆网钙渗漏触发心律失常。因此,针对RyR已成为心力衰竭和心律失常治疗的一个新的策略。