Faculty Bibliography

Mitochondrial connexin 43 (Cx43) plays a key role in cardiac cytoprotection caused by repeated exposure to short periods of non-lethal ischemia/reperfusion, a condition known as ischemic preconditioning. Cx43 also forms calcium (Ca2+)-permeable hemichannels that may potentially lead to mitochondrial Ca2+ overload and cell death. Here, we studied the role of Cx43 in facilitating mitochondrial Ca2+ entry and investigated its downstream consequences. To that purpose, we used various connexin-targeting peptides interacting with extracellular (Gap26) and intracellular (Gap19, RRNYRRNY) Cx43 domains, and tested their effect on mitochondrial dye- and Ca2+-uptake, electrophysiological properties of plasmalemmal and mitochondrial Cx43 channels, and cell injury/cell death. Our results in isolated mice cardiac subsarcolemmal mitochondria indicate that Cx43 forms hemichannels that contribute to Ca2+ entry and may trigger permeability transition and cell injury/death. RRNYRRNY displayed the strongest effects in all assays and inhibited plasma membrane as well as mitochondrial Cx43 hemichannels. RRNYRRNY also strongly reduced the infarct size in ex vivo cardiac ischemia-reperfusion studies. These results indicate that Cx43 contributes to mitochondrial Ca2+ homeostasis and is involved in triggering cell injury/death pathways that can be inhibited by RRNYRRNY peptide.

Arrhythmogenic cardiomyopathy, or its most well-known subform arrhythmogenic right ventricular cardiomyopathy (ARVC), is a cardiac disease mainly characterised by a gradual replacement of the myocardial mass by fibrous and fatty tissue, leading to dilatation of the ventricular wall, arrhythmias and progression towards heart failure. ARVC is commonly regarded as a disease of the intercalated disk in which mutations in desmosomal proteins are an important causative factor. Interestingly, the Dutch founder mutation PLN R14Del has been identified to play an additional, and major, role in ARVC patients within the Netherlands. This is remarkable since the phospholamban (PLN) protein plays a leading role in regulation of the sarcoplasmic reticulum calcium load rather than in the establishment of intercellular integrity. In this review we outline the intracellular cardiac calcium dynamics and relate pathophysiological signalling, induced by disturbed calcium handling, with activation of calmodulin dependent kinase II (CaMKII) and calcineurin A (CnA). We postulate a thus far unrecognised role for Ca2+ sensitive signalling proteins in maladaptive remodelling of the macromolecular protein complex that forms the intercalated disk, during pro-arrhythmic remodelling of the heart.

AIMS: Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) is often associated with desmosomal mutations. Recent studies suggest an interaction between the desmosome and sodium channel protein Nav1.5. We aimed to determine the prevalence and biophysical properties of mutations in SCN5A (the gene encoding Nav1.5) in ARVD/C. METHODS AND RESULTS: We performed whole-exome sequencing in six ARVD/C patients (33% male, 38.2 +/- 12.1 years) without a desmosomal mutation. We found a rare missense variant (p.Arg1898His; R1898H) in SCN5A in one patient. We generated induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) from the patient's peripheral blood mononuclear cells. The variant was then corrected (R1898R) using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology, allowing us to study the impact of the R1898H substitution in the same cellular background. Whole-cell patch clamping revealed a 36% reduction in peak sodium current (P = 0.002); super-resolution fluorescence microscopy showed reduced abundance of NaV1.5 (P = 0.005) and N-Cadherin (P = 0.026) clusters at the intercalated disc. Subsequently, we sequenced SCN5A in an additional 281 ARVD/C patients (60% male, 34.8 +/- 13.7 years, 52% desmosomal mutation-carriers). Five (1.8%) subjects harboured a putatively pathogenic SCN5A variant (p.Tyr416Cys, p.Leu729del, p.Arg1623Ter, p.Ser1787Asn, and p.Val2016Met). SCN5A variants were associated with prolonged QRS duration (119 +/- 15 vs. 94 +/- 14 ms, P < 0.01) and all SCN5A variant carriers had major structural abnormalities on cardiac imaging. CONCLUSIONS: Almost 2% of ARVD/C patients harbour rare SCN5A variants. For one of these variants, we demonstrated reduced sodium current, Nav1.5 and N-Cadherin clusters at junctional sites. This suggests that Nav1.5 is in a functional complex with adhesion molecules, and reveals potential non-canonical mechanisms by which Nav1.5 dysfunction causes cardiomyopathy.

With recent advances in stem cell technology, it is becoming efficient to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, which can subsequently be used for myriad purposes, ranging from interrogating mechanisms of cardiovascular disease, developing novel cellular therapeutic approaches, as well as assessing the cardiac safety profile of compounds. However, the relative inability to acquire abundant pure and mature cardiomyocytes still hinders these applications. Recently, it was reported that glucose-depleted culture medium supplemented with lactate can facilitate purification of hPSC-derived cardiomyocytes. Here, we report that fatty acid as a lactate replacement has not only a similar purification effect but also improves the electrophysiological characteristics of hPSC-derived cardiomyocytes. Glucose-depleted culture medium supplemented with fatty acid and 3,3',5-Triiodo-l-thyronine (T3) was used during enrichment of hPSC-derived cardiomyocytes. Compared to untreated control cells, the treated cardiomyocytes exhibited enhanced action potential (AP) maximum upstroke velocity (as shown by a significant increase in dV/dtmax), action potential amplitude, as well as AP duration at 50% (APD50) and 90% (APD90) of repolarization. The treated cardiomyocytes displayed higher sensitivity to isoproterenol, more organized sarcomeric structures, and lower proliferative activity. Expression profiling showed that various ion channel and cardiac-specific genes were elevated as well. Our results suggest that the use of fatty acid and T3 can facilitate purification and maturation of hPSC-derived cardiomyocytes.

Background: Arrhythmogenic cardiomyopathy (also known as "ARVC") is an inherited disease characterized by fibrous or fibrofatty infiltration of the heart muscle, commonly of right ventricular (RV) predominance, ventricular arrhythmias, and high propensity for sudden death. Sudden cardiac arrest frequently associates with exercise and most often occurs in early adulthood during the subclinical ("concealed") phase of the disease. Understanding electrical remodeling in the early stage of the disease is paramount to understand sudden death mechanisms. Methods: We generated a cardiomyocyte-specif-ic, tamoxifen-activated, PKP2 knockout murine line (alphaMHC-Cre-ERT2/PKP2 fl/fl) which allowed us to control the onset of PKP2 loss of expression, limit it to adult cardiomyocytes, and establish a time line for progression of molecular and functional events. Results: The first consequence of PKP2 loss was RV mechanical dysfunction (14 days post-tamoxifen injection, 14 dpi), followed by fibrosis of RV predominance and RV dilation (21 dpi), then biventricular dilated cardiomyopa-thy and left ventricular (LV) failure (28 dpi and beyond). End-stage failure and death occurred between 30 and 49 dpi. Isoproterenol (ISO)-induced ventricular arrhythmias were first detected prior to LV dysfunction (17/17 mice), and ISO-induced fatal ventricular fibrillation was observed only at 16 dpi, i.e., during the concealed stage (3/9). Differential tran-scriptome analysis at 21 dpi revealed reduced transcript levels for a gene network involved in intracellular calcium ([Ca²⁺]i) cycling, most critically genes encoding Ca²⁺ channel proteins (RyR2 and CaV1.2) and structural molecules that scaffold the dyad (ankyrin-B and triadin). Nanoscale imaging (3D super-resolution microscopy, SICM, and FIB-SEM) showed preservation of T-tubular structure, reduced size and increased separation of CaV1.2 clusters, and displacement of functional CaV1.2 channels from the T-tubular domain. Calcium imaging showed disruption of [Ca²⁺]i homeostasis, potentially causative of ventricular arrhythmias. Flecainide i.p. prevented ISO-induced arrhythmias in all animals. Retrospective analysis of clinical cases showed instances of sudden cardiac arrest without structural disease and suspect diagnosis of catechol-aminergic polymorphic ventricular tachycardia (CPVT) later revealed to foster PKP2 nonsense mutations. Conclusions: Our data provide the first evidence that PKP2 deficiency in adult ventricular myocytes is sufficient to cause an arrhythmo-genic cardiomyopathy of RV predominance. Adrenergic-induced arrhythmias and sudden death occur before the onset of overt structural disease and can mimic a CPVT phenotype. Our data also document a transcript-based [Ca²⁺]i dysfunction as a new key mechanism of arrhythmias in PKP2-deficient hearts and suggest flecainide as potential effective antiarrhyth-mic treatment

Rapid impulse propagation in the heart is a defining property of pectinated atrial myocardium (PAM) and the ventricular conduction system (VCS) and is essential for maintaining normal cardiac rhythm and optimal cardiac output. Conduction defects in these tissues produce a disproportionate burden of arrhythmic disease and are major predictors of mortality in heart failure patients. Despite the clinical importance, little is known about the gene regulatory network that dictates the fast conduction phenotype. Here, we have used signal transduction and transcriptional profiling screens to identify a genetic pathway that converges on the NRG1-responsive transcription factor ETV1 as a critical regulator of fast conduction physiology for PAM and VCS cardiomyocytes. Etv1 was highly expressed in murine PAM and VCS cardiomyocytes, where it regulates expression of Nkx2-5, Gja5, and Scn5a, key cardiac genes required for rapid conduction. Mice deficient in Etv1 exhibited marked cardiac conduction defects coupled with developmental abnormalities of the VCS. Loss of Etv1 resulted in a complete disruption of the normal sodium current heterogeneity that exists between atrial, VCS, and ventricular myocytes. Lastly, a phenome-wide association study identified a link between ETV1 and bundle branch block and heart block in humans. Together, these results identify ETV1 as a critical factor in determining fast conduction physiology in the heart.

BACKGROUND: Mutations in SCN2B, encoding voltage-gated sodium channel beta2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that beta2-subunits play critical roles in the establishment or maintenance of normal cardiac electric activity in vivo. METHODS AND RESULTS: To understand the pathophysiological roles of beta2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current densities in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional reentry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild-type ventricles, as were levels of Nav1.5 protein, suggesting that similar to the previous work in neurons, the major function of beta2-subunits in the ventricle is to chaperone voltage-gated sodium channel alpha-subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared with wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than wild-type littermates. CONCLUSIONS: Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.

Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.