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.     

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.     

Loss of cadherin-binding proteins β-catenin and plakoglobin in the heart leads to gap junction remodeling and arrhythmogenesis

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome, including plakoglobin (JUP). We previously reported that cardiac tissue-specific plakoglobin (PG) knockout (PG CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin (CTNNB1), showed increased association with the gap junction protein connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and β-catenin specifically in the heart (i.e., double knockout [DKO]). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD, consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and β-catenin, are indispensable for maintaining mechanoelectrical coupling in the heart.     

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.     

Age- and myopathy-dependent changes in connexins of normal and cardiomyopathic Syrian hamster ventricular myocardium

The conduction of cardiac action potentials depends on the flow of excitation through gap junctions, which are hexameric protein associations of connexins (Cxs). The major Cx reported in the heart is Cx43, although some Cx40 and Cx45 are also present. There is some evidence for altered Cx content in heart failure. In heart failure, conduction is depressed and slowed conduction may contribute to arrhythmogenesis and (or) the maintenance of arrhythmia. Cx content and distribution were determined in ventricular tissues from normal and cardiomyopathic Syrian hamsters, an animal model of heart failure which has reproducible age-specific cardiomyopathy resulting in heart failure and age-matched controls in three groups: young (3-5 weeks), adult (13-18 weeks), and old (>45 weeks). Frozen, unfixed sections of ventricular tissues were immunofluorescently stained using antibodies against Cx43, Cx40, and Cx45. Cx43 was the predominant Cx detected in all samples. In normal hamsters, Cx43 was localized predominantly at the intercalated disc region, while in myopathic myocytes, it was scattered. In Western blots, Cx43 content of normal hamster hearts was highest in the adult hearts compared with young and old hamster hearts. In contrast, Cx43 content was significantly lower in adult cardiomyopathic hamster hearts compared with all other groups. The alterations of content and distribution of gap junction Cx43 may contribute to diminished conduction, pump function, and arrhythmogenesis in heart failure.     

Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac alpha-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.     

Altered connexin43 expression produces arrhythmia substrate in heart failure

Recently, we found that repolarization heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias in failing myocardium. We hypothesized that the mechanism responsible for maintaining transmural action potential duration heterogeneities in heart failure is related to intercellular uncoupling from downregulation of cardiac gap junction protein connexin43 (Cx43). With the use of the canine model of pacing-induced heart failure, left ventricles were sectioned to expose the transmural surface (n = 5). To determine whether heterogeneous Cx43 expression influenced electrophysiological function, high-resolution transmural optical mapping of the arterially perfused canine wedge preparation was used to measure conduction velocity (theta(TM)), effective transmural space constant (lambda(TM)), and transmural gradients of action potential duration (APD). Absolute Cx43 expression in failing myocardium, quantified by confocal immunofluorescence, was uniformly reduced (by 40 +/- 3%, P < 0.01) compared with control. Relative Cx43 expression was heterogeneously distributed and lower (by 32 +/- 18%, P < 0.05) in the subepicardium compared with deeper layers. Reduced Cx43 expression in heart failure was associated with significant reductions in intercellular coupling between transmural muscle layers, as evidenced by reduced theta(TM) (by 18.9 +/- 4.9%) and lambda(TM) (by 17.2 +/- 1.4%; P < 0.01) compared with control. Heterogeneous transmural distribution of Cx43 in failing myocardium was associated with lower subepicardial theta(TM) (by 12 +/- 10%) and lambda(TM) (by 13 +/- 7%), compared with deeper transmural layers (P < 0.05). APD dispersion was greatest in failing myocardium, and the largest transmural APD gradients were consistently found in regions exhibiting lowest relative Cx43 expression. These data demonstrate that reduced Cx43 expression produces uncoupling between transmural muscle layers leading to slowed conduction and marked dispersion of repolarization between epicardial and deeper myocardial layers. Therefore, Cx43 expression patterns can potentially contribute to an arrhythmic substrate in failing myocardium.     

Mice Lacking Connexin45 Conditionally in Cardiac Myocytes Display Embryonic Lethality Similar to That of Germline Knockout Mice Without Endocardial Cushion Defect

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac α-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.     

Heart and head defects in mice lacking pairs of connexins

Gene ablation studies in mice have revealed roles for gap junction proteins (connexins) in heart development. Of the 20 connexins in vertebrates, four are expressed in developing heart: connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). Although each cardiac connexin has a different pattern of expression, some heart cells coexpress multiple connexins during cardiac morphogenesis. Since different connexins could have overlapping functions, some developmental phenotypes may only become evident when more than one connexin is ablated. In this study, we interbred Cx40(-/-) and Cx43(-/-) mice to generate mice lacking both Cx40 and Cx43. Cx40(-/-)Cx43(-/-) mice die around embryonic day 12.5 (E12.5), much earlier than either Cx40(-/-) or Cx43(-/-) mice, and they exhibit malformed hearts with ventricles that are abnormally rotated, suggesting a looping defect. Some Cx40(-/-)Cx43(-/-) animals also develop head defects characteristic of exencephaly. In addition, we examined mice lacking both Cx40 and Cx37 and found a high incidence of atrial and ventricular septal defects at birth. These results provide further evidence for the importance of gap junctions in embryonic development. Moreover, ablating different pairs of cardiac connexins results in distinct heart defects, suggesting both common and unique functions for Cx40, Cx43, and Cx37 during cardiac morphogenesis.     

Inducible Cx40-Cre expression in the cardiac conduction system and arterial endothelial cells

The Connexin-40 (Cx40) gene encodes a gap junction protein that plays an important role in cell-cell communication in cardiomyocytes of the atria and cardiac conduction system and endothelial cells of large arteries. During embryonic development, Cx40 expression is tightly regulated and correlates with progressive ventricular conduction system (VCS) differentiation and vessel function. We have generated Cx40(Cre) mice carrying a CreERT2-IRESmRFP cassette by targeted recombination. In Cx40(Cre) mice, the pattern of expression of RFP is identical to that of the endogenous Cx40 gene and a Cx40(GFP) allele. Using a LacZ-based Cre reporter mouse line, tamoxifen dependent Cre recombination was observed throughout the spatio-temporal profile of Cx40 expression in the VCS and arterial endothelial cells. Cx40(Cre) mice can therefore be used to direct inducible genetic modification in Cx40 expressing cells.     

Functional consequences of abnormal Cx43 expression in the heart

The major gap junction protein expressed in the heart, connexin43 (Cx43), is highly remodeled in the diseased heart. Usually, Cx43 is down-regulated and heterogeneously redistributed to the lateral sides of cardiomyocytes. Reverse remodeling of the impaired Cx43 expression could restore normal cardiac function and normalize electrical stability. In this review, the reduced and heterogeneous Cx43 expression in the heart will be addressed in hypertrophic, dilated and ischemic cardiomyopathy together with its functional consequences of conduction velocity slowing, dispersed impulse conduction, its interaction with fibrosis and propensity to generate arrhythmias. Finally, different therapies are discussed. Treatments aimed to improve the Cx43 expression levels show new potentially anti-arrhythmic therapies during heart failure, but those in the context of acute ischemia can be anti-arrhythmogenic at the cost of larger infarct sizes. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

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.     

Atrial tachycardia/fibrillation in the connexin 43 G60S mutant (Oculodentodigital dysplasia) mouse

Atrial fibrillation (AF), the most common cardiac arrhythmia seen in general practice, can be promoted by conduction slowing. Cardiac impulse conduction depends on gap junction channels, which are composed of connexins (Cxs). While atrial Cx40 and Cx43 are equally expressed, AF studies have primarily focused on Cx40 reductions. The G60S Cx43 mutant (Cx43(G60S/+)) mouse model of Oculodentodigital dysplasia has a 60% reduction in Cx43 in the atria. Cx43(G60S/+) mice were compared with Cx40-deficient (Cx40(-/-)) mice to determine the role of Cxs in atrial tachycardia/fibrillation (AT/F). Intracardiac electrophysiological studies were done in 6-mo-old male C57BL/6 Cx43(G60S/+) mutant, littermate (Cx43(+/+)), Cx40(-/-), and C57BL/6 wild-type (WT) mice. AT/F induction used an extra stimulus during sinus rhythm, programmed electrical stimulation, or burst pacing (1-ms pulses, 50-Hz, 400-ms train) in the absence and presence of carbachol (CCh). Atrial effective refractory periods did not differ between strains. Cx43(G60S/+) mice were more susceptible to induction of sustained AT/F (duration >2 min, 9 of 12; maximum >35 min) compared with Cx43(+/+) mice (3 of 11; χ(2) = 5.24; P = 0.02). CCh enhanced sustained AT/F susceptibility in WT (from 1 of 12 without, to 7 of 10 with CCh; χ(2) = 8.98; P < 0.01) but not in Cx40(-/-) mice (1 of 13 without vs. 2 of 9 with CCh; χ(2) = 0.95; P = NS). The pattern of epicardial recordings during AT/F in Cx43(G60S/+) mice was left preceding right, with left atrial fractionated activation patterns consistent with clinical observations of AF. In conclusions, while Cx43(G60S/+) mice had severe AT/F, Cx40(-/-) mice were resistant to CCh-induced AT/F.     

ZO-1 determines adherens and gap junction localization at intercalated disks

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.     

Heterogeneous connexin43 expression produces electrophysiological heterogeneities across ventricular wall

Recently we found that electrophysiological (EP) heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias. However, cell-to-cell coupling through gap junctions is expected to attenuate transmural heterogeneities between cell types spanning the ventricular wall. Because connexin43 (Cx43) is the principal ventricular gap junction protein, we hypothesized that transmural EP heterogeneities are in part produced by heterogeneous Cx43 expression across the ventricular wall. The left ventricles of eight dogs were sectioned to expose the transmural surface. To determine whether heterogeneous Cx43 expression influenced EP function, high-resolution transmural optical mapping of the arterially perfused canine wedge preparation was used to measure transmural conduction velocity (thetaTM), dV/dt(max), transmural space constant (lambdaTM), and transmural gradients of action potential duration (APD). Relative Cx43 expression, quantified by confocal immunofluorescence, was significantly lower (by 24 +/- 17%; P < 0.05) in subepicardial compared with deeper layers. Importantly, reduced subepicardial Cx43 was associated with transmural heterogeneities of EP function evidenced by selectively reduced subepicardial thetaTM (by 18 +/- 9%; P < 0.05) compared with deeper layers. In subepicardial regions, dV/dt(max) was fastest (by 19 +/- 15%) and lambdaTM was smallest (by 18.1 +/- 2%), which suggests that conduction slowing was attributable to localized uncoupling rather than reduced excitability. The maximum transmural APD gradients occurred in the same regions where Cx43 expression was lowest; this suggests that Cx43 expression patterns served to maintain APD gradients across the transmural wall. These data demonstrate that heterogeneous Cx43 expression is closely associated with functionally significant EP heterogeneities across the transmural wall. Therefore, Cx43 expression patterns can potentially contribute to arrhythmic substrates that are dependent on transmural electrophysiological heterogeneities.     

MicroRNA-1 Downregulation Increases Connexin 43 Displacement and Induces Ventricular Tachyarrhythmias in Rodent Hypertrophic Hearts

Downregulation of the muscle-specific microRNA-1 (miR-1) mediates the induction of pathologic cardiac hypertrophy. Dysfunction of the gap junction protein connexin 43 (Cx43), an established miR-1 target, during cardiac hypertrophy leads to ventricular tachyarrhythmias (VT). However, it is still unknown whether miR-1 and Cx43 are interconnected in the pro-arrhythmic context of hypertrophy. Thus, in this study we investigated whether a reduction in the extent of cardiac hypertrophy could limit the pathological electrical remodeling of Cx43 and the onset of VT by modulating miR-1 levels. Wistar male rats underwent mechanical constriction of the ascending aorta to induce pathologic left ventricular hypertrophy (LVH) and afterwards were randomly assigned to receive 10mg/kg valsartan, VAL (LVH+VAL) delivered in the drinking water or placebo (LVH) for 12 weeks. Sham surgery was performed for control groups. Programmed ventricular stimulation reproducibly induced VT in LVH compared to LVH+VAL group. When compared to sham controls, rats from LVH group showed a significant decrease of miR-1 and an increase of Cx43 expression and its ERK1/2-dependent phosphorylation, which displaces Cx43 from the gap junction. Interestingly, VAL administration to rats with aortic banding significantly reduced cardiac hypertrophy and prevented miR-1 down-regulation and Cx43 up-regulation and phosphorylation. Gain- and loss-of-function experiments in neonatal cardiomyocytes (NCMs) in vitro confirmed that Cx43 is a direct target of miR-1. Accordingly, in vitro angiotensin II stimulation reduced miR-1 levels and increased Cx43 expression and phosphorylation compared to un-stimulated NCMs. Finally, in vivo miR-1 cardiac overexpression by an adenoviral vector intra-myocardial injection reduced Cx43 expression and phosphorylation in mice with isoproterenol-induced LVH. In conclusion, miR-1 regulates Cx43 expression and activity in hypertrophic cardiomyocytes in vitro and in vivo. Treatment of pressure overload-induced myocyte hypertrophy reduces the risk of life-threatening VT by normalizing miR-1 expression levels with the consequent stabilization of Cx43 expression and activity within the gap junction.