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.

Atrial cardiomyopathy resulting from loss of plakophilin-2 expression: Response to adrenergic stimulation and implications for the exercise response.

Atrial arrhythmias occur in 20-40% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) and are associated with an increased risk of sustained ventricular arrhythmias and inappropriate implantable cardioverter-defibrillator shocks. The pathophysiology of atrial arrhythmias in ARVC remains unclear. Most cases of gene-positive ARVC are linked to pathogenic variants in the desmosomal gene plakophilin-2 (PKP2). Here, we test the hypothesis that loss of PKP2 expression leads to pro-arrhythmic changes in atrial cardiomyocytes. Atrial cells/tissue were obtained from a cardiac-specific, tamoxifen-activated model of PKP2 deficiency (PKP2cKO). By contrast to PKP2cKO ventricular myocytes, PKP2cKO atrial cardiomyocytes presented no significant differences in intracellular calcium (Ca2+ i) transient dynamics, sarcoplasmic reticulum load or action potential morphology. PKP2cKO atrial cardiomyocytes showed elevated reactive oxygen species levels, increased frequency and amplitude of Ca2+ sparks, and increased diastolic [Ca2+]i compared to control; the latter two parameters were further increased by isoproterenol exposure and reversed by exposure to ryanodine receptor blocker dantrolene. We speculate that these isoproterenol-dependent effects may impact on the exercise-related atrial arrhythmia risk in ARVC patients. Despite absence of changes in Ca2+ i transient dynamics, PKP2cKO atrial cardiomyocytes showed enhanced sarcomere shortening and impaired sarcomere relaxation. Orthogonal transcriptomic analysis of human(GTEx) and PKP2cKO atrial tissue led to identification of 41 transcripts depending on PKP2 expression. Biochemical follow-up confirmed reduced abundance of sarcomeric protein myosin binding protein C, potentially playing a role in cellular shortening and relaxation changes observed. Our findings provide novel insights into the role of PKP2 in atrial myocardium with potential implications to therapeutic management of atrial fibrillation in patients with PKP2-related ARVC. KEY POINTS: Atrial arrhythmias occur in a large group of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a cardiac disease mostly caused by pathogenic variants in the desmosomal gene plakophilin-2 (PKP2). Exercise is considered to be an independent risk factor for arrhythmias consequent to PKP2 deficiency. We show that loss of PKP2 expression affects cellular calcium handling and electrophysiology differently in left atrial vs. ventricular myocardium and causes extensive atrial fibrosis. PKP2-deficient atrial cardiomyocytes present increased spontaneous sarcoplasmic reticulum calcium release events, further enhanced by isoproterenol exposure and reversible by a ryanodine receptor blocker (dantrolene). In addition, PKP2-deficient atrial myocytes exhibit impaired relaxation and enhanced sarcomere shortening, most probably related to reduced abundance of myosin binding protein C. We speculate that cellular effects reported upon isoproterenol impact on the exercise-related atrial arrhythmia risk in ARVC patients. We further propose that therapeutic approaches aimed at mitigating ventricular damage may be effective to treat the atrial disease in ARVC.

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.

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.

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.

Plakophilin-2 Haploinsufficiency Causes Calcium Handling Deficits and Modulates the Cardiac Response Towards Stress.

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.

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.

"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.

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.

Transcriptomic Coupling of PKP2 With Inflammatory and Immune Pathways Endogenous to Adult Cardiac Myocytes.

Plakophilin-2 (PKP2) is classically defined as a component of the desmosome. Besides its role in cell-cell adhesion, PKP2 can modulate transcription through intracellular signals initiated at the site of cell-cell contact. Mutations in PKP2 associate with arrhythmogenic right ventricular cardiomyopathy (ARVC). Recent data demonstrate that inflammation plays a key role in disease progression; other results show an abundance of anti-heart antibodies in patients with confirmed diagnosis of ARVC. Here, we test the hypothesis that, in adult cardiac myocytes, PKP2 transcript abundance is endogenously linked to the abundance of transcripts participating in the inflammatory/immune response. Cardiac-specific, tamoxifen (TAM)-activated PKP2-knockout mice (PKP2cKO) were crossed with a RiboTag line to allow characterization of the ribosome-resident transcriptome of cardiomyocytes after PKP2 knockdown. Data were combined with informatics analysis of human cardiac transcriptome using GTEx. Separately, the presence of non-myocyte cells at the time of analysis was assessed by imaging methods. We identified a large number of transcripts upregulated consequent to PKP2 deficiency in myocytes, inversely correlated with PKP2 abundance in human transcriptomes, and part of functional pathways associated with inflammatory/immune responses. Our data support the concept that PKP2 is transcriptionally linked, in cardiac myocytes, to genes coding for host-response molecules even in the absence of exogenous triggers. Targeted anti-inflammatory therapy may be effective in ARVC.

Mitochondrial Dysfunction as Substrate for Arrhythmogenic Cardiomyopathy: A Search for New Disease Mechanisms.

Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease, associated with ventricular arrhythmias, fibrofatty replacement of the myocardial mass and an increased risk of sudden cardiac death (SCD). Malignant ventricular arrhythmias and SCD largely occur in the pre-clinical phase of the disease, before overt structural changes occur. To prevent or interfere with ACM disease progression, more insight in mechanisms related to electrical instability are needed. Currently, numerous studies are focused on the link between cardiac arrhythmias and metabolic disease. In line with that, a potential role of mitochondrial dysfunction in ACM pathology is unclear and mitochondrial biology in the ACM heart remains understudied. In this review, we explore mitochondrial dysfunction in relation to arrhythmogenesis, and postulate a link to typical hallmarks of ACM. Mitochondrial dysfunction depletes adenosine triphosphate (ATP) production and increases levels of reactive oxygen species in the heart. Both metabolic changes affect cardiac ion channel gating, electrical conduction, intracellular calcium handling, and fibrosis formation; all well-known aspects of ACM pathophysiology. ATP-mediated structural remodeling, apoptosis, and mitochondria-related alterations have already been shown in models of PKP2 dysfunction. Yet, the limited amount of experimental evidence in ACM models makes it difficult to determine whether mitochondrial dysfunction indeed precedes and/or accompanies ACM pathogenesis. Nevertheless, current experimental ACM models can be very useful in unraveling ACM-related mitochondrial biology and in testing potential therapeutic interventions.

Blockade of the Adenosine 2A Receptor Mitigates the Cardiomyopathy Induced by Loss of Plakophilin-2 Expression.

Background: Mutations in plakophilin-2 (PKP2) are the most common cause of familial Arrhythmogenic Right Ventricular Cardiomyopathy, a disease characterized by ventricular arrhythmias, sudden death, and progressive fibrofatty cardiomyopathy. The relation between loss of PKP2 expression and structural cardiomyopathy remains under study, though paracrine activation of pro-fibrotic intracellular signaling cascades is a likely event. Previous studies have indicated that ATP release into the intracellular space, and activation of adenosine receptors, can regulate fibrosis in various tissues. However, the role of this mechanism in the heart, and in the specific case of a PKP2-initiated cardiomyopathy, remains unexplored. Objectives: To investigate the role of ATP/adenosine in the progression of a PKP2-associated cardiomyopathy. Methods: HL1 cells were used to study PKP2- and Connexin43 (Cx43)-dependent ATP release. A cardiac-specific, tamoxifen-activated PKP2 knock-out murine model (PKP2cKO) was used to define the effect of adenosine receptor blockade on the progression of a PKP2-dependent cardiomyopathy. Results: HL1 cells silenced for PKP2 showed increased ATP release compared to control. Knockout of Cx43 in the same cells blunted the effect. PKP2cKO transcriptomic data revealed overexpression of genes involved in adenosine-receptor cascades. Istradefylline (an adenosine 2A receptor blocker) tempered the progression of fibrosis and mechanical failure observed in PKP2cKO mice. In contrast, PSB115, a blocker of the 2B adenosine receptor, showed opposite effects. Conclusion: Paracrine adenosine 2A receptor activation contributes to the progression of fibrosis and impaired cardiac function in animals deficient in PKP2. Given the limitations of the animal model, translation to the case of patients with PKP2 deficiency needs to be done with caution.

Cardiac Ca2+ signalling in zebrafish: Translation of findings to man.

Sudden cardiac death is a leading cause of death worldwide, mainly caused by highly disturbed electrical activation patterns in the heart. Currently, murine models are the most popular model to study underlying molecular mechanisms of inherited or acquired cardiac electrical abnormalities, although the numerous electrophysiological discrepancies between mouse and human raise the question whether mice are the optimal model to study cardiac rhythm disorders. Recently it has been uncovered that the zebrafish cardiac electrophysiology seems surprisingly similar to the human heart, mainly because the zebrafish AP contains a clear plateau phase and ECG characteristics show alignment with the human ECG. Although, before using zebrafish as a model to study cardiac arrhythmogenesis, however, it is very important to gain a better insight into the electrophysiological characteristics of the zebrafish heart. In this review we outline the electrophysiological machinery of the zebrafish cardiomyocytes, with a special focus on the intracellular Ca2+ dynamics and excitation-contraction coupling. We debate the potential of zebrafish as a model to study human cardiovascular diseases and postulate steps to employ zebrafish into a more 'humanized' model.

Optogenetic sensors in the zebrafish heart: a novel in vivo electrophysiological tool to study cardiac arrhythmogenesis.

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart. Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development. Results: Isoproterenol slightly increased heart rate, diastolic Ca2+ levels and Ca2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ikr blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. ICa,L blocker nifedipine caused total blockade of Ca2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile animals demonstrated the possibility of employing an older zebrafish model for in vivo cardiac electrophysiology studies. We observed differences in atrial and ventricular Ca2+ recovery dynamics between 3 dpf and 14 dpf casper fish, but not in Ca2+ upstroke dynamics. Conclusion: By introducing the optogenetic sensors chimeric VSFP-butterfly CY and GCaMP6f into the zebrafish we successfully generated an in vivo cellular electrophysiological readout tool for the zebrafish heart. Complementary use of both sensor lines demonstrated the ability to study heart rate, cardiac action potential configuration, spatiotemporal patterning of electrical activation and intracellular Ca2+ homeostasis in embryonic zebrafish. In addition, we demonstrated the first successful use of an optogenetic sensor to study cardiac function in older zebrafish. These models present a promising new research tool to study the underlying mechanisms of cardiac arrhythmogenesis.

Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm.

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.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.