Abstract Background Plakophilin-2 (PKP2) is classically defined as a protein of the desmosome, an intercellular adhesion structure that also acts as a signaling hub to maintain structural and electrical homeostasis. 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 that occur consequent to its mutation. Here we sought to captureearly molecular/cellular events that can act as nascent substrates for subsequent arrhythmic/cardiomyopathic phenotypes. Methods We used multiple quantitative imaging modalities, as well as biochemical and high-resolution mass spectrometry methods to study the functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (“PKP2cKO”). Studies were carried out 14 days post-tamoxifen injection, a time point preceding an overt electrical or structural phenotype.Myocytes from right or left ventricular free wall were studied separately, to detect functional/structural asymmetries. Results Most properties of PKP2cKO left ventricular (LV) myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+transients, increased frequency of spontaneous Ca2+release events, increased [Ca2+] in the cytoplasm and sarcoplasmic reticulum compartments, and dynamic Ca2+accumulation in mitochondria. In addition, RyR2 in RV presented enhanced sensitivity to Ca2+and preferential phosphorylation in a domain known to modulate Ca2+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, suggesting that in the earliest stage, [Ca2+]i dysfunction is not transcriptional. Rather, we found an RV-predominant increase in membrane permeability that can permit Ca2+entry into the cell. Cx43 ablation mitigated the increase in membrane permeability, the accumulation of cytoplasmic Ca2+and the early stages of RV dysfunction. Conclusions Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy and that is, at least in part, mediated by a Cx43-dependent membrane conduit. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.