Redistribution of connexin45 in gap junctions of connexin43-deficient hearts

OBJECTIVE: Adult ventricular myocytes express two gap junction channel proteins: connexin43 (Cx43) and connexin45 (Cx45). Cx43-deficient mice exhibit slow ventricular epicardial conduction, suggesting that Cx43 plays an important role in intercellular coupling in the ventricle. Cx45 is much less abundant than Cx43 in working ventricular myocytes. Its role in ventricular conduction has not been defined, nor is it known whether expression or distribution of Cx45 is altered in Cx43-deficient mice. The present study was undertaken to determine (1) whether expression of Cx45 is upregulated and (2) whether gap junction structure and distribution are altered in Cx43-deficient mice. METHODS: Ventricular tissue from neonatal Cx43(+/+), Cx43(+/-) and Cx43(-/-) and adult Cx43(+/+) and Cx43(+/-) mice was analyzed by immunoblotting and confocal immunofluorescence microscopy. RESULTS: Total Cx45 protein abundance measured by immunoblotting was not different in Cx43-deficient or null hearts compared to wild-type control hearts. However, the amount and distribution of Cx45 immunoreactive signal measured by quantitative confocal analysis were markedly reduced in both Cx43(+/-) and Cx43(-/-) hearts. CONCLUSION: Although the total content of Cx45 is not upregulated in Cx43-deficient hearts, the localization of Cx45 to cardiac gap junctions depends on the expression level of Cx43 and is dramatically altered in mice that express no Cx43.     

c-Jun N-terminal kinase activation mediates downregulation of connexin43 in cardiomyocytes

Loss of gap junctions and impaired intercellular communication are characteristic features of pathological remodeling in heart failure as a result of stress or injury, yet the underlying regulatory mechanism has not been identified. Here, we report that in cultured myocytes, rapid loss of the gap junction protein connexin43 (Cx43) occurs in conjunction with the activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase, on stress stimulation. To investigate the specific role of JNK activation in the regulation of connexin in cardiomyocytes, an activated mutant of mitogen-activated protein kinase kinase 7 (mutant D), a JNK-specific upstream activator, was expressed in myocytes by adenovirus-mediated gene transfer. JNK activation in infected cardiomyocytes resulted in significant reduction of Cx43 expression at both mRNA and protein levels and impaired cell-cell communication. To evaluate the role of JNK in the regulation of Cx43 expression and gap junction structure in vivo, a Cre-LoxP-mediated gene-switch system was used to establish a transgenic animal model with targeted activation of JNK in ventricular myocardium. The transgenic hearts exhibited significant downregulation of Cx43 expression and loss of gap junctions in myocardium that may contribute to the cardiac dysfunction and premature death phenotype. Our report represents the first evidence, both in vitro and in vivo, implicating JNK as an important mediator of stress-induced Cx43 downregulation and impaired intercellular communication in the failing heart.     

Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1

AIMS: Remodelling of gap junctions, involving reduction of total gap junction quantity and down-regulation of connexin43 (Cx43), contributes to the arrhythmic substrate in congestive heart failure. However, little is known of the underlying mechanisms. Recent studies from in vitro systems suggest that the connexin-interacting protein zonula occludens-1 (ZO-1) is a potential mediator of gap junction remodelling. We therefore examined the hypothesis that ZO-1 contributes to reduced expression of Cx43 gap junctions in congestive heart failure. METHODS AND RESULTS: Left ventricular myocardium from healthy control human hearts (n = 5) was compared with that of explanted hearts from transplant patients with end-stage congestive heart failure due to idiopathic dilated cardiomyopathy (DCM; n = 5) or ischaemic cardiomyopathy (ICM; n = 5). Immunoconfocal and immunoelectron microscopy showed that ZO-1 is specifically localized to the intercalated disc of cardiomyocytes in control and failing ventricles. ZO-1 protein levels were significantly increased in both DCM and ICM (P = 0.0025), showing a significant, negative correlation to Cx43 levels (P = 0.0029). There was, however, no significant alteration of ZO-1 mRNA (P = 0.537). Double immunolabelling demonstrated that a proportion of ZO-1 label is co-localized with Cx43, and that co-localization of Cx43 with ZO-1 is significantly increased in the failing ventricle (P = 0.003). Interaction between the two proteins was confirmed by co-immunoprecipitation. The proportion of Cx43 that co-immunoprecipitates with ZO-1 was significantly increased in the failing heart. CONCLUSION: Our findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.     

Modulation of cardiac gap junction expression and arrhythmic susceptibility

Connexin43 (Cx43), the predominant ventricular gap junction protein, is critical for maintaining normal cardiac electrical conduction, and its absence in the mouse heart results in sudden arrhythmic death. The mechanisms linking reduced Cx43 abundance in the heart and inducibility of malignant ventricular arrhythmias have yet to be established. In this report, we investigate arrhythmic susceptibility in a murine model genetically engineered to express progressively decreasing levels of Cx43. Progressively older cardiac-restricted Cx43 conditional knockout (CKO) mice were selectively bred to produce a heart-specific Cx43-deficient subline ("O-CKO" mice) in which the loss of Cx43 in the heart occurs more gradually. O-CKO mice lived significantly longer than the initial series of CKO mice but still died suddenly and prematurely. At 25 days of age, cardiac Cx43 protein levels decreased to 59% of control values (P<0.01), but conduction velocity was not significantly decreased and no O-CKO mice were inducible into sustained ventricular tachyarrhythmias. By 45 days of age, cardiac Cx43 abundance had decreased in a heterogeneous fashion to 18% of control levels, conduction velocity had slowed to half of that observed in control hearts, and 80% of O-CKO mice were inducible into lethal tachyarrhythmias. Enhanced susceptibility to induced arrhythmias was not associated with altered invasive hemodynamic measurements or changes in ventricular effective refractory period. Thus, moderately severe reductions in Cx43 abundance are associated with slowing of impulse propagation and a dramatic increase in the susceptibility to inducible ventricular arrhythmias.     

Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia

Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca(2+) and H(+) and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.     

Gap Junction Protein Phenotypes of the Human Heart and Conduction System

Connexin Phenotypes in the Human Heart. Introduction: Gap junction channels are major determinants of intercellular resistance to current flow between cardiac myocytes. Alterations in gap junctions may contribute to development of arrhythmia substrates in patients. However, there is significant interspecies variation in the types and amounts of gap junction subunit proteins (connexins) expressed in disparate regions of mammalian hearts. To elucidate determinants of conduction properties in the human heart, we characterized connexin phenotypes of specific human cardiac tissues with different conduction properties. Methods and Results: The distribution and relative abundance of Cx37, Cx40, Cx43, Cx45, and Cx46 were studied immunohistochemically using monospecific antibodies and frozen sections of the sinoatrial node and adjacent atria, the AV node and His bundle, the bundle branches, and the left and right ventricular walls. Patterns of expression of these connexins in the human heart differed from those in previous animal studies. Sinus node gap junctions were small and sparse and contained Cx45 and apparently smaller amounts of Cx40 but no Cx43. AV node gap junctions were also small and contained mainly Cx45 and Cx40 hut, unlike the sinus node, also expressed Cx43. Atrial gap junctions were larger than nodal junctions and contained moderate amounts of Cx40, Cx43, and Cx45. Junctions in the bundle branches were the largest in size and contained abundant amounts of Cx40, Cx43, and Cx45. Gap junctions in ventricular myocardium contained mainly Cx43 and Cx45; only a very small amount of ventricular Cx40 was detected in subendocardial myocyte junctions and endothelial cells of small to medium sized intramural coronary arteries. Minimal Cx37 and Cx46 immunoreactivity was detected between occasional atrial or ventricular myocytes. Conclusions: The relative amounts of individual connexins and the number and size of gap junctions vary greatly in specific regions of the human heart with different conduction properties. These differences likely play a role in regulating cardiac conduction velocity. Differences in the connexin phenotypes of specific regions of the human heart and experimental animal hearts must he considered in future experimental or modeling studies of cardiac conduction.     

Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stress-activated protein kinase

Decreases in the expression of connexin 43 and the integrity of gap junctions in cardiac muscle, induced by the constitutive activation of the c-Jun N-terminal kinase (JNK) signaling pathway, have been linked to conduction defects and sudden cardiac failure in mice [Petrich BG, Gong X , Lerner DL , Wang X , Brown JH , Saffitz JE , Wang Y. c-Jun N-terminal kinase activation mediates downregulation of connexin 43 in cardiomyocytes. Circ Res. 91 (2002) 640-647; B.G. Petrich, B.C. Eloff, D.L. Lerner, A. Kovacs, J.E. Saffitz, D.S. Rosenbaum, Y. Wang, Targeted activation of c-Jun N-terminal kinase in vivo induces restrictive cardiomyopathy and conduction defects. J. Biol. Chem. 2004;279: 15330-15338]. We examined the membrane cytoskeletal protein, alphaII-spectrin, which associates with connexin 43, to learn if changes in its association with connexin 43 are linked to the instability of gap junctions. Several forms of alphaII-spectrin are expressed in the heart, including one, termed alphaII-SH3i, which contains a 20-amino-acid sequence next to the SH3 domain of repeat 10. In adult mouse heart, antibodies to all forms of alphaII-spectrin labeled the sarcolemma, transverse ("t-") tubules and intercalated disks of cardiomyocytes. In contrast, antibodies specific for alphaII-SH3i labeled only gap junctions and transverse tubules. In transgenic hearts, in which the JNK pathway was constitutively activated, alphaII-SH3i was lost specifically from gap junctions but not from t-tubules while other isoforms of alphaII-spectrin were retained at intercalated disks. Immunoprecipitations confirmed the decreased association of alphaII-SH3i with connexin 43 in transgenic hearts compared to controls. Furthermore, activation of JNK in neonatal myocytes blocked the formation of gap junctions by exogenously expressed Cx43-GFP fusion protein. Similarly, overexpression of the SH3i fragment in the context of repeats 9-11 of alphaII-spectrin specifically caused the accumulation of Cx43-GFP in the perinuclear region and inhibited its accumulation at gap junctions. These results support a critical role for the alphaII-SH3i isoform of spectrin in intracellular targeting of Cx43 to gap junctions and implicates alphaII-SH3i as a potential target for stress signaling pathways that modulate intercellular communication.     

心力衰竭大鼠心脏缝隙连接蛋白43的变化及厄贝沙坦的干预作用

目的观察压力超负荷所致心力衰竭大鼠心室肌中缝隙连接蛋白43表达的变化以及厄贝沙坦对心力衰竭时缝隙连接重构的干预作用。方法采用腹主动脉缩窄法建立大鼠心力衰竭模型,随机分为厄贝沙坦组和心力衰竭组,分别用厄贝沙坦(50 mg/kg)和安慰剂治疗,另设假手术对照组。术后16周用颈总动脉插管法测定心功能,用免疫印迹法检测心肌细胞缝隙连接蛋白43蛋白表达的变化,并以透射电镜观察缝隙连接空间重构的变化。结果心力衰竭大鼠心肌中缝隙连接蛋白43表达明显下调并出现空间重构,厄贝沙坦可明显上调缝隙连接蛋白43蛋白的表达并改善缝隙连接的空间重构。结论压力超负荷所致心力衰竭大鼠心室肌存在明显缝隙连接重构,这可能是心力衰竭时心肌电生理重构的机制之一,而此过程与血管紧张素Ⅱ的作用有关,血管紧张素受体拮抗剂厄贝沙坦可以明显改善心力衰竭时出现的缝隙连接重构。     

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes

Objectives. To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes.Background. Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response.Methods. Cultures were exposed to 0.1 μmol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy.Results. Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n = 4, p < 0.05). This response was inhibited by the presence of 1.0 μmol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [³⁵S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point).Conclusions. Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.     

Protein kinase Cε mediates salutary effects on electrical coupling induced by ischemic preconditioning

BACKGROUND: Ischemic preconditioning delays the onset of electrical uncoupling and prevents loss of the primary ventricular gap junction protein connexin 43 (Cx43) from gap junctions during subsequent ischemia. OBJECTIVE: To test the hypothesis that these effects are mediated by protein kinase C epsilon (PKCepsilon), we studied isolated Langendorff-perfused hearts from mice with homozygous germline deletion of PKCepsilon (PKCepsilon-KO). METHODS: Cx43 phosphorylation and distribution were measured by quantitative immunoblotting and confocal microscopy. Changes in electrical coupling were monitored using the 4-electrode technique to measure whole-tissue resistivity. RESULTS: The amount of Cx43 located in gap junctions, measured by confocal microscopy under basal conditions, was significantly greater in PKCepsilon-KO hearts compared with wild-type, but total Cx43 content measured by immunoblotting was not different. These unanticipated results indicate that PKCepsilon regulates subcellular distribution of Cx43 under normal conditions. Preconditioning prevented loss of Cx43 from gap junctions during ischemia in wild-type but not PKCepsilon-KO hearts. Specific activation of PKCepsilon, but not PKCdelta, also prevented ischemia-induced loss of Cx43 from gap junctions. Preconditioning delayed the onset of uncoupling in wild-type but hastened uncoupling in PKCepsilon-KO hearts. Cx43 phosphorylation at the PKC site Ser368 increased 5-fold after ischemia in wild-type hearts, and surprisingly, by nearly 10-fold in PKCepsilon-KO hearts. Preconditioning prevented phosphorylation of Cx43 in gap junction plaques at Ser368 in wild-type but not PKCepsilon-KO hearts. CONCLUSION: Taken together, these results indicate that PKCepsilon plays a critical role in preconditioning to preserve Cx43 signal in gap junctions and delay electrical uncoupling during ischemia.     

Remodeling of gap junctions and slow conduction in a mouse model of desmin-related cardiomyopathy

OBJECTIVE: We studied a transgenic mouse model of human desmin-related cardiomyopathy with cardiac-specific expression of a 7-amino acid deletion mutation in desmin (D7-des) to test the hypothesis that impaired linkage between desmin and desmosomes alters expression and function of the electrical coupling protein, connexin43 (Cx43). METHODS: Expression of Cx43 and selected mechanical junctions proteins was characterized in left ventrices of D7-des and control mice by quantitative confocal microscopy and immunoblotting. Remodeling of gap junctions was also analyzed by electron microscopic morphometry. The electrophysiological phentoype of D7-des mice was characterized by electrocardiography and optical mapping of transmembrane voltage. RESULTS: Cx43 signal at intercalated disks was decreased by approximately 3-fold in D7-des ventricular tissue due to reductions in both gap junction number and size. Immunoreactive signal at cell-cell junctions was also reduced significantly for adhesion molecules and linker proteins of desmosomes and fascia adherens junctions. Electron microscopy showed decreased gap junction remodeling. However, immunoblotting showed that the total tissue content of Cx43 and mechanical junction proteins was not reduced, suggesting that diminished signal at cell-cell junctions was not due to insufficient protein expression, but to failure of these proteins to assemble properly within electrical and mechanical junctions. Remodeling of gap junctions in D7-des mice led to slowing of ventricular conduction as demonstrated by optical electrophysiological mapping. CONCLUSIONS: These results illustrate how a defect in a protein conventionally thought to fulfill a mechanical function in the heart can also lead to electrophysiological alterations that may contribute to arrhythmogenesis.     

N-cadherin haploinsufficiency affects cardiac gap junctions and arrhythmic susceptibility

Cardiac-specific deletion of the murine gene (Cdh2) encoding the cell adhesion molecule, N-cadherin, results in disassembly of the intercalated disc (ICD) structure and sudden arrhythmic death. Connexin 43 (Cx43)-containing gap junctions are significantly reduced in the heart after depleting N-cadherin, therefore we hypothesized that animals expressing half the normal levels of N-cadherin would exhibit an intermediate phenotype. We examined the effect of N-cadherin haploinsufficiency on Cx43 expression and susceptibility to induced arrhythmias in mice either wild-type or heterozygous for the Cx43 (Gja1)-null allele. An increase in hypophosphorylated Cx43 accompanied by a modest decrease in total Cx43 protein levels was observed in the N-cadherin heterozygous mice. Consistent with these findings N-cadherin heterozygotes exhibited increased susceptibility to ventricular arrhythmias compared to wild-type mice. Quantitative immunofluorescence microscopy revealed a reduction in size of large Cx43-containing plaques in the N-cadherin heterozygous animals compared to wild-type. Gap junctions were further decreased in number and size in the N-cad/Cx43 compound heterozygous mice with increased arrhythmic susceptibility compared to the single mutants. The scaffold protein, ZO-1, was reduced at the ICD in N-cadherin heterozygous cardiomyocytes providing a possible explanation for the reduction in Cx43 plaque size. These data provide further support for the intimate relationship between N-cadherin and Cx43 in the heart, and suggest that germline mutations in the human N-cadherin (Cdh2) gene may predispose patients to increased risk of cardiac arrhythmias.     

Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease).

OBJECTIVES: We tested the hypothesis that defective interactions between adhesion junctions and the cytoskeleton caused by the plakoglobin mutation in Naxos disease lead to remodeling of gap junctions and altered expression of the major gap junction protein, connexin43. BACKGROUND: Naxos disease, a recessive form of arrhythmogenic right ventricular cardiomyopathy, is associated with a high incidence of arrhythmias and sudden cardiac death. Naxos disease is caused by a mutation in plakoglobin, a protein that links cell-cell adhesion molecules to the cytoskeleton. METHODS: Myocardial expression of connexin43 and other intercellular junction proteins was characterized in 4 patients with Naxos disease. Immunohistochemistry was performed in all 4 patients, and immunoblotting and electron microscopy were performed in 1 patient who died in childhood before overt arrhythmogenic right ventricular cardiomyopathy had developed. RESULTS: Connexin43 expression at intercellular junctions was reduced significantly in both right and left ventricles in all patients with Naxos disease. Electron microscopy revealed smaller and fewer gap junctions interconnecting ventricular myocytes. Mutant plakoglobin was expressed but failed to localize normally at intercellular junctions. Localization of N-cadherin, alpha- and beta-catenins, plakophilin-2, desmoplakin-1, and desmocollin-2 at intercalated disks appeared normal. CONCLUSIONS: Remodeling of gap junctions occurs early in Naxos disease, presumably because of abnormal linkage between mechanical junctions and the cytoskeleton. Gap junction remodeling may produce a coupling defect which, combined with the subsequent development of pathologic changes in myocardium, could contribute to a highly arrhythmogenic substrate and enhance the risk of sudden death in Naxos disease.     

Remodelling of gap junctions and connexin expression in diseased myocardium

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease.     

Voltage-Gated Na+ Channel Activity and Connexin Expression in C×43-Deficient Cardiac Myocytes

INTRODUCTION: Dynamic interplay between active and passive electrical properties of cardiac myocytes is based on interrelationships between various channels responsible for depolarizing and repolarizing ionic currents and intercellular conductances. Mice with targeted disruption of the connexin43 (Cx43) gene have hearts completely devoid of Cx43, the principal gap junctional protein expressed in mammalian hearts. METHODS AND RESULTS: To determine whether cardiac myocytes that develop in an abnormal environment of reduced intercellular coupling have altered active membrane properties, we studied whole cell action potentials, Na+ channel currents, and Na+ channel expression and distribution via immunoblotting and confocal immunofluorescence in neonatal ventricular myocytes isolated from Cx43 wild-type, heterozygous, and homozygous null hearts. Action potential morphology, peak Na+ current, activation and inactivation kinetics, and Na+ channel protein expression and distribution were not different among myocytes isolated from wild-type, heterozygous, or null hearts. Active membrane properties and Na+ channel activity were completely normal in Cx43-deficient myocytes isolated from hearts that have been shown to exhibit markedly reduced Cx43 expression, gap junction number, and epicardial conduction delay. CONCLUSION: Despite a genetic inability to produce Cx43 and a developmental history that culminates in marked gross cardiac morphologic abnormalities, premature death, and myocardial inexcitability ex vivo, cardiac Na+ channel distribution and function appear to be normal in Cx43 null hearts. Although intimate structural and functional interrelationships have been described between ion channels and gap junction channels, expression and function of Na+ channels is not affected by the absence of Cx43.     

Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43

Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1+/-0.13 versus 1.66+/-0.06; P:<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.     

Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.     

Functional properties of mouse connexin30.2 expressed in the conduction system of the heart

Gap junction channels composed of connexin (Cx) 40, Cx43, and Cx45 proteins are known to be necessary for impulse propagation through the heart. Here, we report mouse connexin30.2 (mCx30.2) to be a new cardiac connexin that is expressed mainly in the conduction system of the heart. Antibodies raised to the cytoplasmic loop or the C-terminal regions of mCx30.2 recognized this protein in mouse heart as well as in HeLa cells transfected with wild-type mCx30.2 or mCx30.2 fused with enhanced green fluorescent protein (mCx30.2-EGFP). Immunofluorescence analyses of adult hearts yielded positive signals within the sinoatrial node, atrioventricular node, and A-V bundle of the cardiac conduction system. Dye transfer studies demonstrated that mCx30.2 and mCx30.2-EGFP channels discriminate poorly on the basis of charge, but do not allow permeation of tracers >400 Da. Both mCx30.2 and mCx30.2-EGFP gap junctional channels exhibited weak sensitivity to transjunctional voltage (Vj) and a single channel conductance of approximately 9 pS, which is the lowest among all members of the connexin family measured in HeLa cell transfectants. HeLa mCx30.2-EGFP transfectants when paired with cells expressing Cx40, Cx43, or Cx45 formed functional heterotypic gap junction channels that exhibited low unitary conductances (15 to 18 pS), rectifying open channel I-V relations and asymmetric Vj dependence. The electrical properties of homo- and hetero-typic junctions involving mCx30.2 may contribute to slow propagation velocity in nodal tissues and directional asymmetry of excitation spread in the AV nodal region.     

Cx43 gap junctions in cardiac development

Studies utilizing knockout and transgenic mouse models revealed an important role for connexin 43 (Cx43) gap junctions in cardiac development. This may involve a quantitative requirement for gap junctions in modulating the development of cardiac crest cells. In addition, studies in humans and Xenopus indicate that Cx43 gap junctions also may play a role in regulating heart laterality. Together, these findings indicate that the perturbation of Cx43 function could play a significant role in specific congenital heart malformations.