Disruption of Ca 2+ i Homeostasis and Cx43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in PKP2-Deficient Mice

Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO") 14 days post-tamoxifen (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Most properties of PKP2cKO left ventricular (PKP2cKO-LV) myocytes were not different from control; in contrast, PKP2cKO right ventricular (PKP2cKO-RV) myocytes showed increased amplitude and duration of Ca transients, increased [Ca ] in the cytoplasm and sarcoplasmic reticulum (SR), increased frequency of spontaneous Ca release events (sparks) even at comparable SR load, and dynamic Ca accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, RyR2 in PKP2cKO-RV cells presented enhanced Ca sensitivity and preferential phosphorylation in a domain known to modulate Ca gating. RNAseq at 14 days post-TAM showed no relevant difference in transcript abundance between RV and LV, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca entry into the cell. Cx43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca , increased frequency of sparks and early stages of RV dysfunction. Cx43 hemichannel block with GAP19 normalized [Ca ] homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Cx43-dependent membrane conduit and repressed by PKC inhibitors. Given that asymmetric Ca dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca handling in RV myo