Abstract-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 (2.1Ϯ0.

The Ca 2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exerciseinduced sudden cardiac death. IntroductionPharmacological seizure models have implicated abnormalities in intracellular Ca 2+ cycling of inhibitory interneurons and/or astrocytes as a mechanism of seizure generation (1, 2), and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular calcium release channel on the ER, has been associated with seizures in mice (3). However, a causal relationship between defective intracellular calcium release channels and seizures has not been reported. Calcium stored within the ER contributes to neuronal signaling and is controlled by intracellular Ca 2+ release channels, in particular ryanodine receptors (RyRs) (4-6) and IP3Rs (7,8). To explore the underlying mechanism for seizures in CPVT we generated mice that harbor a missense mutation (RyR2-R2474S) that has been linked to exercise-induced cardiac arrhythmias in humans (9-12).More than 50 distinct RYR2 mutations have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic cardiomyopathy (13-15). CPVT patients experience syncope and sudden cardiac death (SCD) from the toddler to adult ages, and by 35 years age the mortality is up to 50% (13,16,17).

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell–cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.

Abstract-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 (PϽ0.01), 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. Key Words: connexin43 Ⅲ arrhythmia Ⅲ electrophysiology Ⅲ heart Ⅲ mice V entricular tachyarrhythmias are a frequent cause of sudden cardiac death in ischemic and nonischemic heart disease. Despite intense investigation, molecular mechanisms underlying the propensity of diseased myocardium to initiate and propagate lethal arrhythmias are incompletely understood. In recent years, a number of genetically engineered murine models have been developed to explore the pathophysiology of arrhythmogenesis. Although many such mice display increases in the frequency of spontaneous or inducible ventricular ectopy, in almost all cases this activity is self-limited and has not been shown to be the proximate cause of death. 1-4 Several other mutant mouse models are able to support sustained ventricular arrhythmias, but these require provocative stimuli, such as anesthesia or exercise with administration of adrenergic agents. [5][6][7][8] To date, only the heartspecific connexin43 (Cx43) conditional knockout (CKO) mouse has been shown to die prematurely from spontaneous sustained ventricular tachyarrhythmias. 9 Because of the arrhythmic propensity of the Cx43 CKO mouse and the ease of inducible sustained arrhythmias, 10 it has served as an ideal model for the study of basic mechanisms of arrhythmia.In myopathic hearts, abnormal expression of Cx43 (gap junction remodeling) may contri...

The cardiac conduction system is a network of cells responsible for the rhythmic and coordinated excitation of the heart. Components of the murine conduction system, including the peripheral Purkinje fibers, are morphologically indistinguishable from surrounding cardiomyocytes, and a paucity of molecular markers exists to identify these cells. The murine conduction system develops in close association with the endocardium. Using the recently identified CCS-lacZ line of reporter mice, in which lacZ expression delineates the embryonic and fully mature conduction system, we tested the ability of several endocardial-derived paracrine factors to convert contractile cardiomyocytes into conduction-system cells as measured by ectopic reporter gene expression in the heart. In this report we show that neuregulin-1, a growth and differentiation factor essential for ventricular trabeculation, is sufficient to induce ectopic expression of the lacZ conduction marker. This inductive effect of neuregulin-1 was restricted to a window of sensitivity between 8.5 and 10.5 days postcoitum. Using the whole mouse embryo culture system, neuregulin-1 was shown to regulate lacZ expression within the embryonic heart, whereas its expression in other tissues remained unaffected. We describe the electrical activation pattern of the 9.5-days postcoitum embryonic mouse heart and show that treatment with neuregulin-1 results in electrophysiological changes in the activation pattern consistent with a recruitment of cells to the conduction system. This study supports the hypothesis that endocardial-derived neuregulins may be the major endogenous ligands responsible for inducing murine embryonic cardiomyocytes to differentiate into cells of the conduction system.

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A sensitive method that accurately measures local CVs throughout the ventricles revealed no changes in Cx43+/- mice, which is consistent with the demonstration that ECG parameter values in the heterozygous mice are the same as those in wild-type mice.