Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca²⁺ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca²⁺ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca²⁺ entry and coupling to Ca²⁺ release microdomains at the intercalated disc, resulting in enhanced Ca²⁺ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca²⁺ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure.

Background: Patients with chronic kidney disease (CKD) are at increased risk of developing cardiac arrhythmogenesis and sudden cardiac death; however, the basis for this association is incompletely known. Methods: Here, using murine models of CKD, we examined interactions between kidney disease progression and structural, electrophysiological, and molecular cardiac remodeling. Results: C57BL/6 mice with adenine supplemented in their diet developed progressive CKD. Electrocardiographically, CKD mice developed significant QT prolongation and episodes of bradycardia. Optical mapping of isolated-perfused hearts using voltage-sensitive dyes revealed significant prolongation of action potential duration with no change in epicardial conduction velocity. Patch-clamp studies of isolated ventricular cardiomyocytes revealed changes in sodium and potassium currents consistent with action potential duration prolongation. Global transcriptional profiling identified dysregulated expression of cellular stress response proteins RBM3 (RNA-binding motif protein 3) and CIRP (cold-inducible RNA-binding protein) that may underlay the ion channel remodeling. Unexpectedly, we found that female sex is a protective factor in the progression of CKD and its cardiac sequelae. Conclusions: Our data provide novel insights into the association between CKD and pathologic proarrhythmic cardiac remodeling. Cardiac cellular stress response pathways represent potential targets for pharmacologic intervention for CKD-induced heart rhythm disorders.

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Na _v 1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1 , TBX3 , TBX18 ) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CONCLUSIONS: CS-associated gain-of-function HRAS ^G12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

It’s been decades since the synthetic version of an atrial anti-arrhythmic peptide AAP10 and its D-isomer rotigaptide were developed and demonstrated to increase myocardial gap junction conductance (g _j ). This increase in cardiomyocyte electrical communication was linked to PKC activation by ELISA assays, PKC activation by phorbol esters, or attenuation by biochemical inhibitors of PKCa. Studies in connexin43, -45, or -40 transfected cell lines further demonstrated that the effects of these gap junction agonists on cardiac g _j were predominantly due to gating effects on Cx43, with some on Cx45, and little or no effect on Cx40 gap junctions. Since Cx43 is known to be regulated by phosphorylation and ³² P was demonstrated to be incorporated into Cx43 by AAP10, the prevailing hypothesis has been that these gap junction agonists alter the gating of Cx43 gap junctions by PKC-dependent phosphorylation mechanisms. However, there are conflicting reports of PKC-downregulation of Cx43 gap junction communication and the carboxyl tail domain of Cx43 is known to contain at least 19 phosphorylation sites associated with no less than six different protein kinases. Preliminary studies in primary neonatal mouse ventricular myocyte (NMVM) cultures demonstrate that PKA and JNK inhibition prevents the acute increase in gap junction conductance (g _j ) in NMVM cell pairs. JNK inhibition or 100 nM rotigaptide treatment both attenuated the 90% decline in NMVM g _j observed during perfusion with 10% normal glucose (0.1 g/L) saline solution. Together, these new preliminary observations suggest the involvement of a PKA-dependent JNK inhibitory signaling pathway in the enhancement of cardiac g _j by rotigaptide.

BACKGROUND: Alterations in microtubule dynamics have been shown to affect cardiomyocyte membrane stiffness and modulate ion channels, including the cardiac sodium channel. While conditions, such as heart failure and Duchenne muscular dystrophy, are associated with increased detyrosination of microtubules and reduced sodium current, a potential role for microtubule detyrosination in arrhythmogenic cardiomyopathy has not been explored. We here investigated the impact of microtubule detyrosination on membrane stiffness, cardiac sodium channel distribution, and function in mouse and human models of arrhythmogenic cardiomyopathy. METHODS: Isolated ventricular cardiomyocytes from mice with cardiomyocyte-specific, tamoxifen-activated knockout of PKP2 (plakophilin-2), as well as PKP2 -c.2013delC, and isogenic control human-induced pluripotent stem cell–derived-cardiomyocytes were incubated for 2 to 4 hours with compounds known to decrease microtubule detyrosination (parthenolide, 10 µmol/L; EpoY, 20 µmol/L) or vehicle (dimethyl sulfoxide). Immunocytochemistry, mechano-scanning ion conductance microscopy, patch-clamp analysis, and stochastic optical reconstruction microscopy were performed. RESULTS: Cardiomyocyte-specific, tamoxifen-activated knockout of PKP2 cardiomyocytes displayed increased microtubule detyrosination and membrane stiffness, which were both attenuated by parthenolide treatment. Parthenolide significantly increased whole-cell sodium current density in cardiomyocyte-specific, tamoxifen-activated knockout of PKP2 mouse cardiomyocytes, with macropatch measurements demonstrating that this increase occurred both at the intercalated disc and lateral membrane. Stochastic optical reconstruction microscopy analysis revealed that parthenolide increased cardiac sodium channel cluster density at the intercalated disc of cardiomyocyte-specific, tamoxifen-activated knockout of PKP2 mouse cardiomyocytes. In contrast, parthenolide had no effect on sodium current, cardiac sodium channel cluster size, or density in cardiomyocytes from control mice. PKP2 -c.2013delC human-induced pluripotent stem cell–derived-cardiomyocytes displayed increased microtubule detyrosination and reduced sodium current compared with isogenic control human-induced pluripotent stem cell–derived-cardiomyocytes, which were both prevented by parthenolide and EpoY. CONCLUSIONS: Increased microtubule detyrosination secondary to loss of PKP2 impacts cardiomyocyte (dys)function beyond the desmosome, contributing to both electrical and mechanical alterations in the setting of arrhythmogenic cardiomyopathy. Our findings identify microtubule detyrosination as a novel therapeutic target in pathophysiological conditions, such as arrhythmogenic cardiomyopathy, aimed at improving both contractile and electrical function.