Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts

Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na+/Ca2+ exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafish tremblor (tre) embryos. The tre ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects in tre mutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown of SERCA2 activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast to tre mutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygous tre embryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development.     

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.     

心脏钠钙交换--心力衰竭治疗的一个新目标

心力衰竭是一个非常复杂的过程,其发生与细胞内钙调节的异常有关,Na /Ca2 交换体(NCX)是心肌细胞内钙稳态的重要调节机制之一,也是心脏收缩功能决定性因素.目前对肥厚衰竭心肌Ca2 交换活性改变及其与心功能障碍以及心律失常产生的关系已有较多研究,本文就其研究现状予以综述.     

Adenovirally delivered shRNA strongly inhibits Na+-Ca2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes

The cardiac Na(+)-Ca(2+) exchanger (NCX1) is the main mechanism for Ca(2+) efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na(+)-Ca(2+) exchange activity. Exchanger expression was inhibited by up to approximately 94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca(2+) transient amplitude, a depressed rate of Ca(2+) rise and decline, elevated diastolic [Ca(2+)], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca(2+) pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.     

Calcium and the heart: an essential partnership

Calcium ions are important in many aspects of normal cardiac function as well as in the response to certain pathologic states. The contribution that myocardial calcium influx makes to the cardiac action potential and the pharmacologic efficacy of compounds designated as calcium channel blockers is examined with respect to current knowledge regarding the structure and characteristics of cardiac sarcolemmal calcium channels. Once intracellular, calcium provides the link between cardiac electrical activity and actual mechanical shortening of cardiomyocytes through a complex interaction of regulatory and structural contractile proteins. This is followed by calcium clearance from the cytosol; the mechanisms by which this occurs are manipulated by drugs such as the digitalis glycosides to enhance myocardial contractility. The importance of intracellular 'second messengers' (eg, cyclic AMP) in constituting a final common pathway for the effects of certain cardiotonic agents is defined. The significance of abnormal calcium homeostasis under conditions of heart failure, myocardial infarction, ventricular fibrillation and cardiomyopathy is examined. The role of calcium in the mediation of myocardial damage under conditions of ischemia and secondary to a phenomenon known as 'the calcium paradox' is discussed. The finding that neonatal hearts are more vulnerable to ischemic contracture than adult hearts may be partially explained by differences between neonatal and adult myocardial calcium handling. Understanding of the interactions that exist between the calcium ion and the cardiomyocyte requires a sound knowledge of this essential partnership by both the physiologist and the practising physician.     

The small heart mutation reveals novel roles of Na+/K+-ATPase in maintaining ventricular cardiomyocyte morphology and viability in zebrafish

Forward genetic screens in zebrafish have been used to identify mutations in genes with important roles in organogenesis. One of these mutants, small heart, develops a diminutive and severely malformed heart and multiple developmental defects of the brain, ears, eyes, and kidneys. Using a positional cloning approach, we identify that the mutant gene encodes the zebrafish Na+/K+-ATPase alpha1B1 protein. Disruption of Na+/K+-ATPase alpha1B1 function via morpholino "knockdown" or pharmacological inhibition with ouabain phenocopies the mutant phenotype, in a dose-dependent manner. Heterozygosity for the mutation sensitizes embryos to ouabain treatment. Our findings present novel genetic and morphological details on the function of the Na+/K+-ATPase alpha1B1 in early cardiac morphogenesis and the pathogenesis of the small heart malformation. We demonstrate that the reduced size of the mutant heart is caused by dysmorphic ventricular cardiomyocytes and an increase in ventricular cardiomyocyte apoptosis. This study provides a new insight that Na+/K+-ATPase alpha1B1 is required for maintaining ventricular cardiomyocyte morphology and viability.     

Tropomodulin1 is required in the heart but not the yolk sac for mouse embryonic development

Tropomodulin (Tmod)1 caps the pointed ends of actin filaments in sarcomeres of striated muscle myofibrils and in the erythrocyte membrane skeleton. Targeted deletion of mouse Tmod1 leads to defects in cardiac development, fragility of primitive erythroid cells, and an absence of yolk sac vasculogenesis, followed by embryonic lethality at embryonic day 9.5. The Tmod1-null embryonic hearts do not undergo looping morphogenesis and the cardiomyocytes fail to assemble striated myofibrils with regulated F-actin lengths. To test whether embryonic lethality of Tmod1 nulls results from defects in cardiac myofibrillogenesis and development or from erythroid cell fragility and subsequent defects in yolk sac vasculogenesis, we expressed Tmod1 specifically in the myocardium of the Tmod1-null mice under the control of the alpha-myosin heavy chain promoter Tg(alphaMHC-Tmod1). In contrast to Tmod1-null embryos, which fail to undergo cardiac looping and have defective yolk sac vasculogenesis, both cardiac and yolk sac morphology of Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos are normal at embryonic day 9.5. Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos develop into viable and fertile mice, indicating that expression of Tmod1 in the heart is sufficient to rescue the Tmod1-null embryonic defects. Thus, although loss of Tmod1 results in myriad defects and embryonic lethality, the Tmod1(-/-) primary defect is in the myocardium. Moreover, Tmod1 is not required in erythrocytes for viability, nor do the Tmod1(-/-) fragile primitive erythroid cells affect cardiac development, yolk sac vasculogenesis, or viability in the mouse.     

Zebrafish model for human long QT syndrome

Long QT syndrome (LQTS) is a disorder of ventricular repolarization that predisposes affected individuals to lethal cardiac arrhythmias. To date, an appropriate animal model of inherited LQTS does not exist. The zebrafish is a powerful vertebrate model used to dissect molecular pathways of cardiovascular development and disease. Because fundamental electrical properties of the zebrafish heart are remarkably similar to those of the human heart, the zebrafish may be an appropriate model for studying human inherited arrhythmias. Here we describe the molecular, cellular, and electrophysiological basis of a zebrafish mutant characterized by ventricular asystole. Genetic mapping and direct sequencing identify the affected gene as kcnh2, which encodes the channel responsible for the rapidly activating delayed rectifier K(+) current (I(Kr)). We show that complete loss of functional I(Kr) in embryonic hearts leads to ventricular cell membrane depolarization, inability to generate action potentials (APs), and disrupted calcium release. A small hyperpolarizing current restores spontaneous APs, implying wild-type function of other ionic currents critical for AP generation. Heterozygous fish manifest overt cellular and electrocardiographic evidence for delayed ventricular repolarization. Our findings provide insight into the pathogenesis of homozygous kcnh2 mutations and expand the use of zebrafish mutants as a model system to study human arrhythmias.     

Reversal of slow growth and heartbeat through the restoration of mitochondrial function in clk-1-deficient mouse embryos by exogenous administration of coenzyme Q10

The longevity gene clk-1/coq7 encodes an enzyme that is essential for the biosynthesis of coenzyme Q (CoQ) in mitochondria and regulates the lifespan and behavioral timing in Caenorhabditis elegans and the chronological lifespan in fission yeast. However, whether the mammalian clk-1/coq7 ortholog (clk-1) regulates these phenotypes in mammals remains to be fully evaluated due to the embryonic lethality of clk-1-deficient (clk-1(-/-)) mice. To investigate whether clk-1 regulates biological functions, such as growth and heartbeat, through CoQ in mouse embryos, we cultivated the cells and hearts of clk-1(-/-) mouse embryos at embryonic day 10.5 (E10.5) for at least 10 days in the presence of fetal bovine serum. In embryonic cells, cardiomyocytes, and hearts, the growth and heart rates were significantly slowed in clk-1(-/-) compared with wild-type or heterozygous mouse tissues. Moreover, frequent apoptosis and a significant reduction in mitochondrial functions, including membrane potential and ATP production, were observed in the clk-1(-/-) cells and hearts. The slowed growth and heart rates and the reduced mitochondrial function of clk-1(-/-) embryonic cells and hearts in culture were almost completely rescued by the administration of exogenous CoQ(10). The results indicate that clk-1 regulates growth and heart rates through CoQ-mediated mitochondrial functions in mouse embryos.     

胚胎干细胞移植到大鼠梗死心脏后的分化研究

目的研究胚胎下细胞在梗死心脏微环境下向心肌细胞、成纤维细胞的分化情况。方法将大鼠分为两组,梗死组为正常大鼠通过结扎左前降支(LAD)制备,对照组为正常大鼠。将4,6-二氨基(DAPI)标记的具有伞能分化能力的鼠胚胎干细胞(mESCs)注射人急性心肌梗死大鼠(18只)或对照绀大鼠(16只)的心脏,观察胚胎干细胞在休内的分化情况。结果 DAPI标记的移植mESCs在对照和梗死心脏均能成活并形成稳定的移植岛,同时在移植区有巨噬细胞浸润。mESCs移植2～4周后,心脏特异性肌钙蛋白T(cTnT)阳性的移植mESCs比例在正常心脏较梗死心脏高(2.67%±0.79%比1.06%±0.52%,P0.01),但4周后cTnT阳性的DAP1标记细胞在正常和梗死心脏的比例差异无统计学意义(1.17%±0.98%比1.07±1,02%,P0.05)。mESCs在埘照组和梗死组心脏都能分化为成纤维细胞。结论移植mESCs不仪能仔活,还可分化进入大鼠梗死心肌细胞。但是,梗死心脏的微环境不能选择性促进mESCs分化进入心肌细胞。     

Cardiac myosin light chain-2: a novel essential component of thick-myofilament assembly and contractility of the heart

Although it is well known that mutations in the cardiac regulatory myosin light chain-2 (mlc-2) gene cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of MLC-2 in the heart are only poorly understood. We have isolated a mutation in zebrafish, tell tale heart (tel(m225)), which selectively perturbs contractility of the embryonic heart. By positional cloning, we identified tel to encode the zebrafish mlc-2 gene. In contrast to mammals, zebrafish have only 1 cardiac-specific mlc-2 gene, which we find to be expressed in atrial and ventricular cardiomyocytes during early embryonic development, but also in the adult heart. Accordingly, loss of zMLC-2 function cannot be compensated for by upregulation of another mlc-2 gene. Surprisingly, ultrastructural analysis of tel cardiomyocytes reveals complete absence of organized thick myofilaments. Thus, our findings provide the first in vivo evidence that cardiac MLC-2 is required for thick-filament stabilization and contractility in the vertebrate heart.     

Restricted inactivation of serum response factor to the cardiovascular system

Serum response factor (SRF) directs programs of gene expression linked to growth and muscle differentiation. To investigate the role of SRF in cardiovascular development, we generated mice in which SRF is knocked out in >80% of cardiomyocytes and >50% of vascular smooth muscle cells (SMC) through SM22alpha-Cre-mediated excision of SRF's promoter and first exon. Mutant mice display vascular patterning, cardiac looping, and SRF-dependent gene expression through embryonic day (e)9.5. At e10.5, attenuation in cardiac trabeculation and compact layer expansion is noted, with an attendant decrease in vascular SMC recruitment to the dorsal aorta. Ultrastructurally, cardiac sarcomeres and Z disks are highly disorganized in mutant embryos. Moreover, SRF mutant mice exhibit vascular SMC lacking organizing actin/intermediate filament bundles. These structural defects in the heart and vasculature coincide with decreases in SRF-dependent gene expression, such that by e11.5, when mutant embryos succumb to death, no SRF-dependent mRNA expression is evident. These results suggest a vital role for SRF in contractile/cytoskeletal architecture necessary for the proper assembly and function of cardiomyocytes and vascular SMC.     

Cardiac conduction is required to preserve cardiac chamber morphology

Electrical cardiac forces have been previously hypothesized to play a significant role in cardiac morphogenesis and remodeling. In response to electrical forces, cultured cardiomyocytes rearrange their cytoskeletal structure and modify their gene expression profile. To translate such in vitro data to the intact heart, we used a collection of zebrafish cardiac mutants and transgenics to investigate whether cardiac conduction could influence in vivo cardiac morphogenesis independent of contractile forces. We show that the cardiac mutant dco^s226 develops heart failure and interrupted cardiac morphogenesis following uncoordinated ventricular contraction. Using in vivo optical mapping/calcium imaging, we determined that the dco cardiac phenotype was primarily due to aberrant ventricular conduction. Because cardiac contraction and intracardiac hemodynamic forces can also influence cardiac development, we further analyzed the dco phenotype in noncontractile hearts and observed that disorganized ventricular conduction could affect cardiomyocyte morphology and subsequent heart morphogenesis in the absence of contraction or flow. By positional cloning, we found that dco encodes Gja3/Cx46, a gap junction protein not previously implicated in heart formation or function. Detailed analysis of the mouse Cx46 mutant revealed the presence of cardiac conduction defects frequently associated with human heart failure. Overall, these in vivo studies indicate that cardiac electrical forces are required to preserve cardiac chamber morphology and may act as a key epigenetic factor in cardiac remodeling.     

Regulation of the Na+/Ca2+ exchanger (NCX) in the murine embryonic heart

OBJECTIVE: The Na+/Ca2+ exchanger (NCX) is involved in embryonic heart development and function demonstrated by the abnormal myofibrillar organization, defects in heartbeat, and early embryonic death of NCX-null embryos. It was therefore the aim of our study to identify key functional regulators of the embryonic NCX. METHODS: NCX current (I(NCX)) density was measured as the Ni2+ (5 mM)-sensitive current applying the whole-cell patch-clamp technique in early (EDS, E10.5V) and late developmental stage (LDS, E16.5V) mouse ventricular cardiomyocytes. RESULTS: Compared to LDS, cardiomyocytes derived from EDS showed a significantly higher basal I(NCX) density for the I(NCX) forward (-120 mV: -4.1+/-1 pA/pF, n=15 versus -1.7+/-0.4, n=11, p<0.05) and reverse modes (+60 mV: 4.0+/-0.9 pA/pF, n=15 versus 1.8+/-0.4, n=11, p<0.05). There was 2-3-fold elevation of forward and reverse current in LDS on application of ATP-gamma-S (2 mM) together with forskolin (1 microM) as well as intracellular application of the catalytic subunit of cAMP-dependent protein kinase (cPKA, 200 U/mL), cAMP (200 microM), phorbol 12-myristate-13-acetate (PMA), a direct activator of protein kinase C (PKC), and 8-Br-cGMP, a membrane permeable analog of cGMP. The specific PKC inhibitor Ro 31-8220 significantly reduced I(NCX) by 70%. Co-application of 20 microM PKA inhibitor Fragment 14-22 (PKI), a specific inhibitor of PKA, and cAMP significantly reduced the exchanger activity by approx 60%. Despite these obvious effects in LDS we could not detect a significant impact of these compounds on I(NCX) in EDS-derived cardiomyocytes. Application of the alkaline phosphatase to test for constitutive phosphorylation of NCX did not affect I(NCX) density in LDS but led to an approx 80% reduction of I(NCX) in EDS. CONCLUSION: In EDS cardiomyocytes I(NCX) density is upregulated, at least in part by the high phosphorylation of the exchanger protein. At LDS, embryonic cardiomyocytes showed a strong increase of I(NCX) density upon stimulation by PKC- and PKA-dependent signalling pathways.     

Cardiomyocyte-restricted deletion of connexin43 during mouse development

Although the gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development, mice with a global inactivation of Cx43 survive until birth but die perinatally due to an obstruction of the right ventricular outflow tract of the heart. To analyze the functional role of Cx43 gap junction channels in cardiomyocytes of the developing and early postnatal heart, we used alphaMyHC-Cre mice to ablate Cx43 expression selectively in cardiomyocytes during development. We found efficient ablation of Cx43 in cardiomyocytes during embryonic development starting at embryonic day (ED) 9.5 in the ventricular wall. Analyses of cardiac Cx43 protein at birth indicated complete loss of Cx43 expression in cardiomyocytes. All mice homozygously deficient for Cx43 in cardiomyocytes died until postnatal day (PD) 16. Heterozygous inactivation of Cx43 in cardiomyocytes neither altered atrial nor ventricular activation, but homozygous ablation led to changes in ventricular activation, i.e. significant decrease of the QRS-amplitude and prolonged QRS-duration already at PD 4. Cardiac morphology was similar to controls until PD 1, but subtle morphological changes were found in a subgroup of mutant mice at later stages. Besides narrowing of the ventricular outlet region at PD 6, hypertrophy of ventricular myocardium was found at PD 12. Our data indicate that complete inactivation of cardiac Cx43 during development predisposes hearts to develop postnatal morphological alterations, which differ from outflow tract obstructions described for Cx43 null mice. In addition, complete loss of cardiac Cx43 protein during development correlates with slowed ventricular activation at PD 4, impairs viability during development, and leads to death of all mutant mice until PD 16.     

Cardiomyocyte-restricted deletion of connexin43 during mouse development

Although the gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development, mice with a global inactivation of Cx43 survive until birth but die perinatally due to an obstruction of the right ventricular outflow tract of the heart. To analyze the functional role of Cx43 gap junction channels in cardiomyocytes of the developing and early postnatal heart, we used αMyHC-Cre mice to ablate Cx43 expression selectively in cardiomyocytes during development. We found efficient ablation of Cx43 in cardiomyocytes during embryonic development starting at embryonic day (ED) 9.5 in the ventricular wall. Analyses of cardiac Cx43 protein at birth indicated complete loss of Cx43 expression in cardiomyocytes. All mice homozygously deficient for Cx43 in cardiomyocytes died until postnatal day (PD) 16. Heterozygous inactivation of Cx43 in cardiomyocytes neither altered atrial nor ventricular activation, but homozygous ablation led to changes in ventricular activation, i.e. significant decrease of the QRS-amplitude and prolonged QRS-duration already at PD 4. Cardiac morphology was similar to controls until PD 1, but subtle morphological changes were found in a subgroup of mutant mice at later stages. Besides narrowing of the ventricular outlet region at PD 6, hypertrophy of ventricular myocardium was found at PD 12. Our data indicate that complete inactivation of cardiac Cx43 during development predisposes hearts to develop postnatal morphological alterations, which differ from outflow tract obstructions described for Cx43 null mice. In addition, complete loss of cardiac Cx43 protein during development correlates with slowed ventricular activation at PD 4, impairs viability during development, and leads to death of all mutant mice until PD 16.