AAV-Mediated Delivery of Plakophilin-2a Arrests Progression of Arrhythmogenic Right Ventricular Cardiomyopathy in Murine Hearts: Preclinical Evidence Supporting Gene Therapy in Humans.

BACKGROUND: Pathogenic variants in PKP2 (plakophilin-2) cause arrhythmogenic right ventricular cardiomyopathy, a disease characterized by life-threatening arrhythmias and progressive cardiomyopathy leading to heart failure. No effective medical therapy is available to prevent or arrest the disease. We tested the hypothesis that adeno-associated virus vector-mediated delivery of the human PKP2 gene to an adult mammalian heart deficient in PKP2 can arrest disease progression and significantly prolong survival. METHODS: Experiments were performed using a PKP2-cKO (cardiac-specific, tamoxifen-activated PKP2 knockout murine model). The potential therapeutic, adeno-associated virus vector of serotype rh.74 (AAVrh.74)-PKP2a (PKP2 variant A; RP-A601) is a recombinant AAVrh.74 gene therapy viral vector encoding the human PKP2 variant A. AAVrh.74-PKP2a was delivered to adult mice by a single tail vein injection either before or after tamoxifen-activated PKP2-cKO. PKP2 expression was confirmed by molecular and histopathologic analyses. Cardiac function and disease progression were monitored by survival analyses, echocardiography, and electrocardiography. RESULTS: Consistent with prior findings, loss of PKP2 expression caused 100% mortality within 50 days after tamoxifen injection. In contrast, AAVrh.74-PKP2a-mediated PKP2a expression resulted in 100% survival for >5 months (at study termination). Echocardiographic analysis revealed that AAVrh.74-PKP2a prevented right ventricle dilation, arrested left ventricle functional decline, and mitigated arrhythmia burden. Molecular and histological analyses showed AAVrh.74-PKP2a-mediated transgene mRNA and protein expression and appropriate PKP2 localization at the cardiomyocyte intercalated disc. Importantly, the therapeutic benefit was shown in mice receiving AAVrh.74-PKP2a after disease onset. CONCLUSIONS: These preclinical data demonstrate the potential for AAVrh.74-PKP2a (RP-A601) as a therapeutic for PKP2-related arrhythmogenic right ventricular cardiomyopathy in both early and more advanced stages of the disease.

Nanogold based protein localization enables subcellular visualization of cell junction protein by SBF-SEM.

Recent advances in volume electron microscopy (vEM) allow unprecedented visualization of the electron-dense structures of cells, tissues and model organisms at nanometric resolution in three dimensions (3D). Light-based microscopy has been widely used for specific localization of proteins; however, it is restricted by the diffraction limit of light, and lacks the ability to identify underlying structures. Here, we describe a protocol for ultrastructural detection, in three dimensions, of a protein (Connexin 43) expressed in the intercalated disc region of adult murine heart. Our protocol does not rest on the expression of genetically encoded proteins and it overcomes hurdles related to pre-embedding and immunolabeling, such as the penetration of the label and the preservation of the tissue. The pre-embedding volumetric immuno-electron microscopy (pre-embedding vIEM) protocol presented here combines several practical strategies to balance sample fixation with antigen and ultrastructural preservation, and penetration of labeling with blocking of non-specific antigen binding sites. The small 1.4 nm gold along with surrounded silver used as a detection marker buried in the sample also serves as a functional conductive resin that significantly reduces the charging of samples. Our protocol also presents strategies for facilitating the successful cutting of the samples during serial block-face scanning electron microscopy (SBF-SEM) imaging. Our results suggest that the small gold-based pre-embedding vIEM is an ideal labeling method for molecular localization throughout the depth of the sample at subcellular compartments and membrane microdomains.

Connexin hemichannels as candidate targets for cardioprotective and anti-arrhythmic treatments.

Connexins are crucial cardiac proteins that form hemichannels and gap junctions. Gap junctions are responsible for the propagation of electrical and chemical signals between myocardial cells and cells of the specialized conduction system in order to synchronize the cardiac cycle and steer cardiac pump function. Gap junctions are normally open, while hemichannels are closed, but pathological circumstances may close gap junctions and open hemichannels, thereby perturbing cardiac function and homeostasis. Current evidence demonstrates an emerging role of hemichannels in myocardial ischemia and arrhythmia, and tools are now available to selectively inhibit hemichannels without inhibiting gap junctions as well as to stimulate hemichannel incorporation into gap junctions. We review available experimental evidence for hemichannel contributions to cellular pro-arrhythmic events in ventricular and atrial cardiomyocytes, and link these to insights at the level of molecular control of connexin-43-based hemichannel opening. We conclude that a double-edged approach of both preventing hemichannel opening and preserving gap junctional function will be key for further research and development of new connexin-based experimental approaches for treating heart disease.

The intercalated disc: a unique organelle for electromechanical synchrony in cardiomyocytes.

The intercalated disc (ID) is a highly specialized structure that connects cardiomyocytes via mechanical and electrical junctions. Although described in some detail by light microscopy in the 19th century, it was in 1966 that electron microscopy images showed that the ID represented apposing cell borders and provided detailed insight into the complex ID nanostructure. Since then, much has been learned about the ID and its molecular composition, and it has become evident that a large number of proteins, not all of them involved in direct cell-to-cell coupling via mechanical or gap junctions, reside at the ID. Furthermore, an increasing number of functional interactions between ID components are emerging, leading to the concept that the ID is not the sum of isolated molecular silos but an interacting molecular complex, an "organelle" where components work in concert to bring about electrical and mechanical synchrony. The aim of the present review is to give a short historical account of the ID's discovery and an updated overview of its composition and organization, followed by a discussion of the physiological implications of the ID architecture and the local intermolecular interactions. The latter will focus on both the importance of normal conduction of cardiac action potentials as well as the impact on the pathophysiology of arrhythmias.

Outlining cardiac ion channel protein interactors and their signature in the human electrocardiogram.

Protein-protein interactions are essential for normal cellular processes and signaling events. Defining these interaction networks is therefore crucial for understanding complex cellular functions and interpretation of disease-associated gene variants. We need to build a comprehensive picture of the interactions, their affinities and interdependencies in the specific organ to decipher hitherto poorly understood signaling mechanisms through ion channels. Here we report the experimental identification of the ensemble of protein interactors for 13 types of ion channels in murine cardiac tissue. Of these, we validated the functional importance of ten interactors on cardiac electrophysiology through genetic knockouts in zebrafish, gene silencing in mice, super-resolution microscopy and patch clamp experiments. Furthermore, we establish a computational framework to reconstruct human cardiomyocyte ion channel networks from deep proteome mapping of human heart tissue and human heart single-cell gene expression data. Finally, we integrate the ion channel interactome with human population genetics data to identify proteins that influence the electrocardiogram (ECG). We demonstrate that the combined channel network is enriched for proteins influencing the ECG, with 44% of the network proteins significantly associated with an ECG phenotype. Altogether, we define interactomes of 13 major cardiac ion channels, contextualize their relevance to human electrophysiology and validate functional roles of ten interactors, including two regulators of the sodium current (epsin-2 and gelsolin). Overall, our data provide a roadmap for our understanding of the molecular machinery that regulates cardiac electrophysiology.

Loss of Nuclear Envelope Integrity and Increased Oxidant Production Cause DNA Damage in Adult Hearts Deficient in PKP2: A Molecular Substrate of ARVC.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by high propensity to life-threatening arrhythmias and progressive loss of heart muscle. More than 40% of reported genetic variants linked to ARVC reside in the PKP2 gene, which encodes the PKP2 protein (plakophilin-2). METHODS: We describe a comprehensive characterization of the ARVC molecular landscape as determined by high-resolution mass spectrometry, RNA sequencing, and transmission electron microscopy of right ventricular biopsy samples obtained from patients with ARVC with PKP2 mutations and left ventricular ejection fraction >45%. Samples from healthy relatives served as controls. The observations led to experimental work using multiple imaging and biochemical techniques in mice with a cardiac-specific deletion of Pkp2 studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: Samples from patients with ARVC present a loss of nuclear envelope integrity, molecular signatures indicative of increased DNA damage, and a deficit in transcripts coding for proteins in the electron transport chain. Mice with a cardiac-specific deletion of Pkp2 also present a loss of nuclear envelope integrity, which leads to DNA damage and subsequent excess oxidant production (O2.- and H2O2), the latter increased further under mechanical stress (isoproterenol or exercise). Increased oxidant production and DNA damage is recapitulated in human induced pluripotent stem cell-derived PKP2-deficient myocytes. Furthermore, PKP2-deficient cells release H2O2 into the extracellular environment, causing DNA damage and increased oxidant production in neighboring myocytes in a paracrine manner. Treatment with honokiol increases SIRT3 (mitochondrial nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-3) activity, reduces oxidant levels and DNA damage in vitro and in vivo, reduces collagen abundance in the right ventricular free wall, and has a protective effect on right ventricular function. CONCLUSIONS: Loss of nuclear envelope integrity and subsequent DNA damage is a key substrate in the molecular pathology of ARVC. We show transcriptional downregulation of proteins of the electron transcript chain as an early event in the molecular pathophysiology of the disease (before loss of left ventricular ejection fraction <45%), which associates with increased oxidant production (O2.- and H2O2). We propose therapies that limit oxidant formation as a possible intervention to restrict DNA damage in ARVC.

"Orphan" Connexin43 in Plakophilin-2 Deficient Hearts Revealed by Volume Electron Microscopy.

Previous studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythmogenic/cardiomyopathic phenotype. The latter has translational implications to the molecular mechanisms of inheritable arrhythmogenic right ventricular cardiomyopathy (ARVC). Despite functional evidence, visualization of these "orphan" (i.e., non-paired in a gap junction configuration) Cx43 hemichannels remains lacking. Immuno-electron microscopy (IEM) remains an extremely powerful tool to localize, with nanometric resolution, a protein within its native structural landscape. Yet, challenges for IEM are to preserve the antigenicity of the molecular target and to provide access for antibodies to reach their target, while maintaining the cellular/tissue ultrastructure. Fixation is important for maintaining cell structure, but strong fixation and vigorous dehydration (as it is routine for EM) can alter protein structure, thus impairing antigen-antibody binding. Here, we implemented a method to combine pre-embedding immunolabeling (pre-embedding) with serial block-face scanning electron microscopy (SBF-SEM). We utilized a murine model of cardiomyocyte-specific, Tamoxifen (TAM) activated knockout of PKP2. Adult hearts were harvested 14 days post-TAM, at this time hearts present a phenotype of concealed ARVC (i.e., an arrhythmogenic phenotype but no overt structural disease). Thick (200 µm) vibratome slices were immunolabelled for Cx43 and treated with nanogold or FluoroNanogold, coupled with a silver enhancement. Left or right ventricular free walls were dissected and three-dimensional (3D) localization of Cx43 in cardiac muscle was performed using SBF-SEM. Reconstructed images allowed us to visualize the entire length of gap junction plaques, seen as two parallel, closely packed strings of Cx43-immunoreactive beads at the intercalated disc. In contrast, in PKP2-deficient hearts we observed bulging of the intercellular space, and entire areas where only one of the two strings could be observed, indicating the presence of orphan Cx43. We conclude that pre-embedding and SBF-SEM allowed visualization of cardiac Cx43 plaques in their native environment, providing for the first time a visual complement of functional data indicating the presence of orphan Cx43 hemichannels resulting from loss of desmosomal integrity in the heart.

The Genetics of Brugada Syndrome.

Brugada syndrome is a heritable channelopathy characterized by a peculiar electrocardiogram (ECG) pattern and increased risk of cardiac arrhythmias and sudden death. The arrhythmias originate because of an imbalance between the repolarizing and depolarizing currents that modulate the cardiac action potential. Even if an overt structural cardiomyopathy is not typical of Brugada syndrome, fibrosis and structural changes in the right ventricle contribute to a conduction slowing, which ultimately facilitates ventricular arrhythmias. Currently, Mendelian autosomal dominant transmission is detected in less than 25% of all clinical confirmed cases. Although 23 genes have been associated with the condition, only SCN5A, encoding the cardiac sodium channel, is considered clinically actionable and disease causing. The limited monogenic inheritance has pointed toward new perspectives on the possible complex genetic architecture of the disease, involving polygenic inheritance and a polygenic risk score that can influence penetrance and risk stratification.

Subcellular trafficking and endocytic recycling of KATP channels.

Sarcolemmal/plasmalemmal ATP-sensitive K+ (KATP) channels have key roles in many cell types and tissues. Hundreds of studies have described how the KATP channel activity and ATP sensitivity can be regulated by changes in the cellular metabolic state, by receptor signaling pathways and by pharmacological interventions. These alterations in channel activity directly translate to alterations in cell or tissue function, that can range from modulating secretory responses, such as insulin release from pancreatic ß-cells or neurotransmitters from neurons, to modulating contractile behavior of smooth muscle or cardiac cells to elicit alterations in blood flow or cardiac contractility. It is increasingly becoming apparent, however, that KATP channels are regulated beyond changes in their activity. Recent studies have highlighted that KATP channel surface expression is a tightly regulated process with similar implications in health and disease. The surface expression of KATP channels is finely balanced by several trafficking steps including synthesis, assembly, anterograde trafficking, membrane anchoring, endocytosis, endocytic recycling, and degradation. This review aims to summarize the physiological and pathophysiological implications of KATP channel trafficking and mechanisms that regulate KATP channel trafficking. A better understanding of this topic has potential to identify new approaches to develop therapeutically useful drugs to treat KATP channel-related diseases.

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts.

BACKGROUND: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. METHODS: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. RESULTS: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca2+i transient amplitude showed a differential response to ß-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. CONCLUSIONS: Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular ß-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable ß-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

Preserved cardiac performance and adrenergic response in a rabbit model with decreased ryanodine receptor 2 expression.

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/-) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/- myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.

Beta-blocker/ACE inhibitor therapy differentially impacts the steady state signaling landscape of failing and non-failing hearts.

Heart failure is a multifactorial disease that affects an estimated 38 million people worldwide. Current pharmacotherapy of heart failure with reduced ejection fraction (HFrEF) includes combination therapy with angiotensin-converting enzyme inhibitors (ACEi) and ß-adrenergic receptor blockers (ß-AR blockers), a therapy also used as treatment for non-cardiac conditions. Our knowledge of the molecular changes accompanying treatment with ACEi and ß-AR blockers is limited. Here, we applied proteomics and phosphoproteomics approaches to profile the global changes in protein abundance and phosphorylation state in cardiac left ventricles consequent to combination therapy of ß-AR blocker and ACE inhibitor in HFrEF and control hearts. The phosphorylation changes induced by treatment were profoundly different for failing than for non-failing hearts. HFrEF was characterized by profound downregulation of mitochondrial proteins coupled with derangement of ß-adrenergic and pyruvate dehydrogenase signaling. Upon treatment, phosphorylation changes consequent to HFrEF were reversed. In control hearts, treatment mainly led to downregulation of canonical PKA signaling. The observation of divergent signaling outcomes depending on disease state underscores the importance of evaluating drug effects within the context of the specific conditions present in the recipient heart.

Role of plakophilin-2 expression on exercise-related progression of arrhythmogenic right ventricular cardiomyopathy: a translational study.

AIMS: Exercise increases arrhythmia risk and cardiomyopathy progression in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients, but the mechanisms remain unknown. We investigated transcriptomic changes caused by endurance training in mice deficient in plakophilin-2 (PKP2cKO), a desmosomal protein important for intercalated disc formation, commonly mutated in ARVC and controls. METHODS AND RESULTS: Exercise alone caused transcriptional downregulation of genes coding intercalated disk proteins. The changes converged with those in sedentary and in exercised PKP2cKO mice. PKP2 loss caused cardiac contractile deficit, decreased muscle mass and increased functional/transcriptomic signatures of apoptosis, despite increased fractional shortening and calcium transient amplitude in single myocytes. Exercise accelerated cardiac dysfunction, an effect dampened by pre-training animals prior to PKP2-KO. Consistent with PKP2-dependent muscle mass deficit, cardiac dimensions in human athletes carrying PKP2 mutations were reduced, compared to matched controls. CONCLUSIONS: We speculate that exercise challenges a cardiomyocyte "desmosomal reserve" which, if impaired genetically (e.g., PKP2 loss), accelerates progression of cardiomyopathy.

In silico Identification of Disrupted Myocardial Calcium Homeostasis as Proarrhythmic Trigger in Arrhythmogenic Cardiomyopathy.

Background: Patients with arrhythmogenic cardiomyopathy may suffer from lethal ventricular arrhythmias. Arrhythmogenic cardiomyopathy is predominantly triggered by mutations in plakophilin-2, a key component of cell-to-cell adhesion and calcium cycling regulation in cardiomyocytes. Calcium dysregulation due to plakophilin-2 mutations may lead to arrhythmias but the underlying pro-arrhythmic mechanisms remain unclear. Aim: To unravel the mechanisms by which calcium-handling abnormalities in plakophilin-2 loss-of-function may contribute to proarrhythmic events in arrhythmogenic cardiomyopathy. Methods: We adapted a computer model of mouse ventricular electrophysiology using recent experimental calcium-handling data from plakophilin-2 conditional knock-out (PKP2-cKO) mice. We simulated individual effects of beta-adrenergic stimulation, modifications in connexin43-mediated calcium entry, sodium-calcium exchanger (NCX) activity and ryanodine-receptor 2 (RyR2) calcium affinity on cellular electrophysiology and occurrence of arrhythmogenic events (delayed-afterdepolarizations). A population-of-models approach was used to investigate the generalizability of our findings. Finally, we assessed the potential translation of proposed mechanisms to humans, using a human ventricular cardiomyocyte computational model. Results: The model robustly reproduced the experimental calcium-handling changes in PKP2-cKO cardiomyocytes: an increased calcium transient amplitude (562 vs. 383 nM), increased diastolic calcium (120 vs. 91 nM), reduced L-type calcium current (15.0 vs. 21.4 pA/pF) and an increased free SR calcium (0.69 vs. 0.50 mM). Under beta-adrenergic stimulation, PKP2-cKO models from the population of models (n = 61) showed a higher susceptibility to delayed-afterdepolarizations compared to control (41 vs. 3.3%). Increased connexin43-mediated calcium entry further elevated the number of delayed-afterdepolarizations (78.7%, 2.5-fold increase in background calcium influx). Elevated diastolic cleft calcium appeared responsible for the increased RyR2-mediated calcium leak, promoting delayed-afterdepolarizations occurrence. A reduction in RyR2 calcium affinity prevented delayed-afterdepolarizations in PKP2-cKO models (24.6 vs. 41%). An additional increase in INCX strongly reduced delayed-afterdepolarizations occurrence, by lowering diastolic cleft calcium levels. The human model showed similar outcomes, suggesting a potential translational value of these findings. Conclusion: Beta-adrenergic stimulation and connexin43-mediated calcium entry upon loss of plakophilin-2 function contribute to generation of delayed-afterdepolarizations. RyR2 and NCX dysregulation play a key role in modulating these proarrhythmic events. This work provides insights into potential future antiarrhythmic strategies in arrhythmogenic cardiomyopathy due to plakophilin-2 loss-of-function.

Coordination of endothelial cell positioning and fate specification by the epicardium.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

[Figure: see text].

Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability.

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.

Arrhythmogenic cardiomyopathy: An in-depth look at molecular mechanisms and clinical correlates.

Arrhythmogenic cardiomyopathy (ACM) is a familial disease, with approximately 60% of patients displaying a pathogenic variant. The majority of genes linked to ACM code for components of the desmosome: plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2), plakoglobin (JUP) and desmoplakin (DSP). Genetic variants involving the desmosomes are known to cause dysfunction of cell-to-cell adhesions and intercellular gap junctions. In turn, this may result in failure to mechanically hold together the cardiomyocytes, fibrofatty myocardial replacement, cardiac conduction delay and ventricular arrhythmias. It is becoming clearer that pathogenic variants in desmosomal genes such as PKP2 are not only responsible for a mechanical dysfunction of the intercalated disc (ID), but are also the cause of various pro-arrhythmic mechanisms. In this review, we discuss in detail the different molecular interactions associated with desmosomal pathogenic variants, and their contribution to various ACM phenotypes.

Structural and Functional Characterization of a Nav1.5-Mitochondrial Couplon.

RATIONALE: The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. OBJECTIVE: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. METHODS AND RESULTS: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. CONCLUSIONS: We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.