MicroRNA-23a Participates in Estrogen Deficiency Induced Gap Junction Remodeling of Rats by Targeting GJA1

Increased incidence of arrhythmias in women after menopause has been widely documented, which is considered to be related to estrogen (E₂) deficiency induced cardiac electrophysiological abnormalities. However, its molecular mechanism remains incompletely clear. In the present study, we found cardiac conduction blockage in post-menopausal rats. Thereafter, the results showed that cardiac gap junctions were impaired and Connexin43 (Cx43) expression was reduced in the myocardium of post-menopausal rats. The phenomenon was also observed in ovariectomized (OVX) rats, which was attenuated by E₂ supplement. Further study displayed that microRNA-23a (miR-23a) level was significantly increased in both post-menopausal and OVX rats, which was reversed by daily E₂ treatment after OVX. Importantly, forced overexpression of miR-23a led to gap junction impairment and Cx43 downregulation in cultured cardiomyocytes, which was rescued by suppressing miR-23a by transfection of miR-23a specific inhibitory oligonucleotide (AMO-23a). GJA1 was identified as the target gene of miR-23a by luciferase assay and miRNA-masking antisense ODN (miR-Mask) assay. We also found that E₂ supplement could reverse cardiac conduction blockage, Cx43 downregulation, gap junction remodeling and miR-23a upregulation in post-menopausal rats. These findings provide the evidence that miR-23a mediated repression of Cx43 participates in estrogen deficiency induced damages of cardiac gap junction, and highlights a new insight into molecular mechanism of post-menopause related arrhythmia at the microRNA level.     

MIR-206 regulates connexin43 expression during skeletal muscle development

Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3′-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development.     

Remodeling of the peripheral cardiac conduction system in response to pressure overload

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.     

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

Abstract

With each heartbeat, billions of cardiomyocytes work in concert to propagate the electrical excitation needed to effectively circulate blood. Regulated expression and timely delivery of connexin proteins to form gap junctions at the specialized cell–cell contact region, known as the intercalated disc, is essential to ventricular cardiomyocyte coupling. We focus this review on several regulatory mechanisms that have been recently found to govern the lifecycle of connexin 43 (Cx43), the short-lived and most abundantly expressed connexin in cardiac ventricular muscle. The Cx43 lifecycle begins with gene expression, followed by oligomerization into hexameric channels, and then cytoskeletal-based transport toward the disc region. Once delivered, hemichannels interact with resident disc proteins and are organized to effect intercellular coupling. We highlight recent studies exploring regulation of Cx43 localization to the intercalated disc, with emphasis on alternatively translated Cx43 isoforms and cytoskeletal transport machinery that together regulate Cx43 gap junction coupling between cardiomyocytes.     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.     

The connexin43 carboxyl terminus and cardiac gap junction organization

The precise spatial order of gap junctions at intercalated disks in adult ventricular myocardium is thought vital for maintaining cardiac synchrony. Breakdown or remodeling of this order is a hallmark of arrhythmic disease of the heart. The principal component of gap junction channels between ventricular cardiomyocytes is connexin43 (Cx43). Protein-protein interactions and modifications of the carboxyl-terminus of Cx43 are key determinants of gap junction function, size, distribution and organization during normal development and in disease processes. Here, we review data on the role of proteins interacting with the Cx43 carboxyl-terminus in the regulation of cardiac gap junction organization, with particular emphasis on Zonula Occludens-1. The rapid progress in this area suggests that in coming years we are likely to develop a fuller understanding of the molecular mechanisms causing pathologic remodeling of gap junctions. With these advances come the promise of novel approach to the treatment of arrhythmia and the prevention of sudden cardiac death. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Conditional gene targeting of connexin43: exploring the consequences of gap junction remodeling in the heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

MiR-30-Regulated Autophagy Mediates Angiotensin II-Induced Myocardial Hypertrophy

Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group). Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene), and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy.     

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre+;Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre+;Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40

Gap junction channels provide the basis for the electrical syncytial properties of the heart as a communicating electrical network. Cardiac gap junction channels are predominantly composed of connexin 40 or connexin 43. The conductance of these channels (g(j)) can be regulated pharmacologically: substances which activate protein kinase C, protein kinase A or protein kinase G may alter Cx43 gap junction conductance. However, for PKC, this seems to be subtype specific. Thus, antiarrhythmic peptides can enhance g(j) via activation of PKCepsilon, while FGF-2 reduces g(j) via PKCepsilon. Lipophilic drugs can uncouple the channels. Besides an acute regulation of g(j), the expression of the cardiac connexins can also be regulated. A decrease in Cx43 with a concomitant increase in Cx40 has been found in end-stage failing hearts, while in renovascular hypertension, an increase in Cx43 has been described. Mediators like endothelin-1, angiotensin-II, TGF-beta, VEGF, and cAMP have been shown to increase Cx43. Interestingly, endothelin-1 and angiotensin-II increased Cx43 but did not affect Cx40 expression. In contrast, in humans suffering from atrial fibrillation (AF), the content in Cx40 can be enhanced while Cx43 was unaltered, although in several other studies, other changes of the cardiac connexins were found, which might be related to the type of AF. Regarding the role of calcium, the content in both Cx40 and Cx43 was decreased in cultured neonatal rat cardiomyocytes after 24 h administration of 100 nM verapamil. Thus, gap junctional channels can be affected pharmacologically either acutely by modulating gap junction conductance or chronically by altering gap junction protein expression. Interestingly, it appears that the expression of Cx43 and Cx40 can be differentially regulated.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C₂C₁₂ cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca²⁺ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca²⁺, in the modulation of myoblast differentiation is discussed. gap junctions; connexin43     

TMEM43 Mutation p.S358L Alters Intercalated Disc Protein Expression and Reduces Conduction Velocity in Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.     

Effects of cytochrome P450 inhibitors on agonist-induced Ca2+ responses and production of NO and PGI2 in vascular endothelial cells

The gap junction protein connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43–46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).     

Altered expression of connexin43 contributes to the arrhythmogenic substrate during the development of heart failure in cardiomyopathic hamster

Heart failure is known to predispose to life-threatening ventricular tachyarrhythmias even before compromising the systemic circulation, but the underlying mechanism is not well understood. The aim of this study was to clarify the connexin43 (Cx43) gap junction remodeling and its potential role in the pathogenesis of arrhythmias during the development of heart failure. We investigated stage-dependent changes in Cx43 expression in UM-X7.1 cardiomyopathic hamster hearts and associated alterations in the electrophysiological properties using a high-resolution optical mapping system. UM-X7.1 hamsters developed left ventricular (LV) hypertrophy by ages 6 approximately 10 wk and showed a moderate reduction in LV contractility at age 20 wk. Appreciable interstitial fibrosis was recognized at these stages. LV mRNA and protein levels of Cx43 in UM-X7.1 were unaffected at age 10 wk but significantly reduced at 20 wk. The expression level of Ser255-phosphorylated Cx43 in UM-X7.1 at age 20 wk was significantly greater than that in control golden hamsters at the same age. In UM-X7.1 at age 10 wk, almost normal LV conduction was preserved, whereas the dispersion of action potential duration was significantly increased. UM-X7.1 at age 20 wk showed significant reduction of cardiac space constant, significant decrease in conduction velocity, marked distortion of activation fronts, and pronounced increase in action potential duration dispersion. Programmed stimulation resulted in sustained ventricular tachycardia or fibrillation in UM-X7.1. LV activation during polymorphic ventricular tachycardia was characterized by multiple phase singularities or wavebreaks. During the development of heart failure in the cardiomyopathic hamster, alterations of Cx43 expression and phosphorylation in concert with interstitial fibrosis may create serious arrhythmogenic substrate through an inhibition of cell-to-cell coupling.     

Regulation of Connexin-43-Mediated Growth Inhibition by a Phosphorylatable Amino-Acid is Independent of Gap Junction-Forming Ability

The ability of the gap junction phosphoprotein connexin-43 (Cx43) to inhibit DNA synthesis in primary cardiomyocytes is regulated by serine (S) 262, a protein kinase C phosphorylation site that also affects metabolic coupling. We have now examined if the S262-regulated growth suppression is operating in transformed cells and if so whether it depends on gap junction channel forming ability. Serine 262 became phosphorylated in response to protein kinase C stimulation in HEK293 cells transiently expressing either Cx43 or the non-channel-forming carboxy-terminal tail of Cx43 (Cx43CT). Expression of either wild type Cx43 or Cx43CT inhibited DNA synthesis, as did their mutated versions simulating lack of phosphorylation by carrying an S262-to-alanine substitution. The ability to inhibit DNA synthesis was eliminated when expressing mutated versions of either Cx43 or Cx43CT simulating constitutive phosphorylation by carrying an S262-to-aspartate substitution. We conclude that S262 phosphorylation cancels growth inhibition by Cx43 independently of channel-forming ability.