Fangyu Liu

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.

The organization of adherens junctions and desmosomes at the cardiac intercalated disc is independent of gap junctions

Adherens junctions and desmosomes are responsible for mechanically coupling myocytes in the heart and are found closely apposed to gap junction plaques at the intercalated discs of cardiomyocytes. It is not known whether loss of cardiac gap junctions, such as described in cardiac disease states, may influence the expression patterns of other intercalated disc-associated proteins. We investigated whether the major cardiac gap junction protein connexin43 (Cx43) may be responsible for regulating adherens junctions, desmosomes and their associated catenins, in terms of abundance and localization at the intercalated discs of cardiomyocytes. In order to study the effect of loss of cardiac gap junctions on the intercalated disc-associated proteins, we used a combination of immunoblotting, immunofluorescence with confocal microscopy and electron microscopy to evaluate heart tissue from mice with cardiac-specific conditional knockout of Cx43. We found that the cardiac adherens junctions, desmosomes and their associated catenins, as well as vinculin and ZO-1, maintain their normal abundance, structural appearance and localization in the absence of Cx43. We conclude from these data that Cx43 is not required for the organization of the cell adhesion junctions and their associated catenins at the intercalated disc in the adult cardiac myocyte.

Purkinje Cells From RyR2 Mutant Mice Are Highly Arrhythmogenic But Responsive to Targeted Therapy

Rationale The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca2+ release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. Objective We sought to determine the frequency and severity of spontaneous Ca2+ release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2R4496C/+ CPVT mutant mice and littermate controls. Methods and Results We crossed RyR2R4496C/+ knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP−) and Purkinje cells (EGFP+) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca2+ dynamics recorded by microfluorimetry. Both wild-type and RyR2R4496C/+ mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and &tgr;decay in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2R4496C/+ mutant Purkinje cells were also most likely to develop spontaneous Ca2+ release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca2+i events, the RyR2R4496C/+ Purkinje cells responded with the most profound abnormalities in intracellular Ca2+ handling, including a significant increase in the frequency of unstimulated Ca2+i events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca2+ release events with flecainide, whereas in RyR2R4496C/+ mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes. Conclusions Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca2+ handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca2+ release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.

Phosphatase-Resistant Gap Junctions Inhibit Pathological Remodeling and Prevent Arrhythmias

Rationale: Posttranslational phosphorylation of connexin43 (Cx43) has been proposed as a key regulatory event in normal cardiac gap junction expression and pathological gap junction remodeling. Nonetheless, the role of Cx43 phosphorylation in the context of the intact organism is poorly understood. Objective: To establish whether specific Cx43 phosphorylation events influence gap junction expression and pathological remodeling. Methods and Results: We generated Cx43 germline knock-in mice in which serines 325/328/330 were replaced with phosphomimetic glutamic acids (S3E) or nonphosphorylatable alanines (S3A). The S3E mice were resistant to acute and chronic pathological gap junction remodeling and displayed diminished susceptibility to the induction of ventricular arrhythmias. Conversely, the S3A mice showed deleterious effects on cardiac gap junction formation and function, developed electric remodeling, and were highly susceptible to inducible arrhythmias. Conclusions: These data demonstrate a mechanistic link between posttranslational phosphorylation of Cx43 and gap junction formation, remodeling, and arrhythmic susceptibility.

Cell Coupling Between Ventricular Myocyte Pairs From Connexin43-Deficient Murine Hearts

Abstract— Mice with cardiac-restricted inactivation of the connexin43 gene (CKO mice) have moderate slowing of ventricular conduction and lethal arrhythmias. Mechanisms through which propagation is maintained in the absence of Cx43 are unknown. We evaluated gap junctional conductance in CKO ventricular pairs using dual patch clamp methods. Junctional coupling was reduced to 4±2 nS (side-to-side) and 11±2 nS (end-to-end), including 21% of cell-pairs with no detectable coupling, compared with 588±104 nS (side-to-side) and 558±92 nS (end-to-end) in control cell-pairs. Voltage dependence of control gap junctions was characteristic of Cx43. CKO conductance showed increased voltage dependence, suggesting low-level expression of other connexin isoforms. From theoretical models, this degree of CKO coupling is not expected to support levels of conduction persisting in vivo, suggesting the possibility that there are additional mechanisms for maintained propagation when gap junctional conductance is severely reduced.

Long form of latent TGF-β binding protein 1 (Ltbp1L) is essential for cardiac outflow tract septation and remodeling

Latent TGF-β binding protein 1 (LTBP1) is a member of the LTBP/fibrillin family of extracellular proteins. Due to the usage of different promoters, LTBP1 exists in two major forms, long (L) and short (S), each expressed in a temporally and spatially unique fashion. Both LTBP1 molecules covalently interact with latent TGF-β and regulate its function, presumably via interaction with the extracellular matrix (ECM). To explore the in vivo role of Ltbp1 in mouse development, at the time when only the L isoform is expressed, we mutated the Ltbp1L locus by gene targeting. Ltbp1L-null animals die shortly after birth from defects in heart development, consisting of the improper septation of the cardiac outflow tract (OFT) and remodeling of the associated vessels. These cardiac anomalies present as persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA), which are associated with the faulty function of cardiac neural crest cells (CNCCs). The lack of Ltbp1L in the ECM of the septating OFT and associated vessels results in altered gene expression and function of CNCCs and decreased Tgf-β activity in the OFT. This phenotype reveals a crucial role for Ltbp1L and matrix as extracellular regulators of Tgf-β activity in heart organogenesis.

Distinct cardiac malformations caused by absence of connexin 43 in the neural crest and in the non-crest neural tube

Connexin 43 (Cx43) is expressed in the embryonic heart, cardiac neural crest (CNC) and neural tube, and germline knockout (KO) of Cx43 results in aberrant cardiac outflow tract (OFT) formation and abnormal coronary deployment. Prior studies suggest a vital role for CNC expression of Cx43 in heart development. Surprisingly, we found that conditional knockout (CKO) of Cx43 in the dorsal neural tube and CNC mediated by Wnt1-Cre failed to recapitulate the Cx43-null OFT phenotype, although coronary vasculature was abnormal in this mutant line. A broader CKO mediated by P3pro (Pax3)-Cre, involving both ventral and dorsal aspects of the thoracic neural tube and CNC, resulted in infundibular bulging and coronary anomalies similar to those seen in germline Cx43-null hearts. P3pro-Cre-mediated loss of Cx43 in the neural tube was characterized by a late phase of cellular delamination from the dorsal and lateral neural tube, a markedly increased abundance of neuroepithelium-derived cells outside of the neural tube and an excess of such cells infiltrating the heart and infundibulum. Thus, expression of Cx43 in the CNC is crucial for normal coronary deployment, but Cx43 is not required in the CNC for normal OFT morphogenesis. Rather, this study suggests a novel function for Cx43 in which Cx43 acts through non-crest neuroepithelial cells to suppress cellular delamination from the neural tube and thereby preserve normal OFT development.

PCP4 regulates Purkinje cell excitability and cardiac rhythmicity

Cardiac Purkinje cells are important triggers of ventricular arrhythmias associated with heritable and acquired syndromes; however, the mechanisms responsible for this proarrhythmic behavior are incompletely understood. Here, through transcriptional profiling of genetically labeled cardiomyocytes, we identified expression of Purkinje cell protein-4 (Pcp4), a putative regulator of calmodulin and Ca2+/calmodulin-dependent kinase II (CaMKII) signaling, exclusively within the His-Purkinje network. Using Pcp4-null mice and acquired cardiomyopathy models, we determined that reduced expression of PCP4 is associated with CaMKII activation, abnormal electrophysiology, dysregulated intracellular calcium handling, and proarrhythmic behavior in isolated Purkinje cells. Pcp4-null mice also displayed profound autonomic dysregulation and arrhythmic behavior in vivo. Together, these results demonstrate that PCP4 regulates cardiac excitability through both Purkinje cell-autonomous and central mechanisms and identify this modulator of CaMKII signaling as a potential arrhythmia-susceptibility candidate.

Identification of Binding Partners for the Cytoplasmic Loop of Connexin43: A Novel Interaction with β-Tubulin

Connexin43 (Cx43), a component of gap junctions, has a relatively large carboxy-terminal region with multiple proteomic interactions. Proteomic interactions with its cytoplasmic loop, however, are poorly defined. The goal of this study is to examine proteomic interactions involving the cytoplasmic loop (CL) of Cx43. The authors utilized various techniques, including glutathione-S-transferase (GST) pull-down, immunoblot analysis, two-dimensional (2D) gel electrophoresis, and mass spectrometry, to elucidate binding partners for Cx43-CL. The authors identified novel interactions with Cx43-CL involving α- and β-tubulin, myelin basic protein, and Purα. Because tubulin interacts with the C-terminus of Cx43 (Cx43-CT), the authors further investigated the nature of the interaction between β-tubulin and Cx43-CL. β-Tubulin binds with the full length of Cx43-CL with approximately one-fifth the affinity of the interaction between Cx43-CT and β-tubulin. This study demonstrates novel proteomic interactions involving Cx43-CL that may lead to a more complete understanding of trafficking and gating of gap junction channels.

Short-term pacing in the mouse alters cardiac expression of connexin43.

BackgroundCardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time.ResultsThe subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias.ConclusionOur data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.