Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. 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. Methods: 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 injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca 2+ transients, increased Ca 2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca 2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca 2+ 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, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca 2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca 2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca 2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca 2+ , increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca 2+ ] i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca 2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca 2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca 2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

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Key pointsr Cardiac myocytes are subjected to fluid shear stress during the cardiac cycle and haemodynamic disturbance.r A longitudinally propagating, regenerative Ca 2+ wave is initiated in atrial myocytes under shear stress.r Here we determine the cellular mechanism for this shear-induced Ca 2+ wave using two-dimensional confocal Ca 2+ imaging combined with pressurized fluid flow.r Our data suggest that shear stress triggers the Ca 2+ wave through ryanodine receptors via P2Y 1 purinoceptor-phospholipase C-type 2 inositol 1,4,5-trisphosphate receptor signal transduction in atrial myocytes, and that this mechanotransduction is activated by gap junction hemichannel-mediated ATP release.r Shear-specific mechanotransduction and the subsequent regenerative Ca 2+ wave may be one way for atrial myocytes to assess mechanical stimuli directly and alter their Ca 2+ signalling accordingly.Abstract Atrial myocytes are exposed to shear stress during the cardiac cycle and haemodynamic disturbance. In response, they generate a longitudinally propagating global Ca 2+ wave. Here, we investigated the cellular mechanisms underlying the shear stress-mediated Ca 2+ wave, using two-dimensional confocal Ca 2+ imaging combined with a pressurized microflow system in single rat atrial myocytes. Shear stress of ß16 dyn cm −2 for 8 s induced ß1.2 aperiodic longitudinal Ca 2+ waves (ß79 μm s −1 ) with a delay of 0.2−3 s. Pharmacological blockade of ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate receptors (IP 3 Rs) abolished shear stress-induced Ca 2+ wave generation. Furthermore, in atrial myocytes from type 2 IP 3 R (IP 3 R2) knock-out mice, shear stress failed to induce longitudinal Ca 2+ waves. The phospholipase C (PLC) inhibitor U73122, but not its inactive analogue U73343, abolished the shear-induced longitudinal Ca 2+ wave. However, pretreating atrial cells with blockers for stretch-activated channels, Na + −Ca 2+ exchanger, transient receptor potential melastatin subfamily 4, or nicotinamide adenine dinucleotide phosphate oxidase did not suppress wave generation under shear stress. The P2 purinoceptor inhibitor suramin, and the potent P2Y 1 receptor antagonist MRS 2179, both suppressed the Ca 2+ wave, whereas the P2X receptor antagonist, iso-PPADS, did not alter it. Suppression of gap junction hemichannels permeable to ATP or extracellular application of ATP-metabolizing apyrase inhibited the wave. Removal of external Ca 2+ to enhance hemichannel opening facilitated the wave generation. Our data suggest that longitudinally propagating, regenerative Ca 2+ release through RyRs is triggered by P2Y 1 -PLC-IP 3 R2 signalling that is activated by gap junction hemichannel-mediated ATP release in atrial myocytes under shear stress.

This study examines whether fluid pressure (FP) modulates the L-type Ca(2+) channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (approximately 16 dyn/cm(2)) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca(2+) current (I(Ca)) and cytosolic Ca(2+) signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I(Ca) (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I(Ca). The level of I(Ca) suppression by FP depended on the level and duration of pressure. The Ba(2+) current through the Ca(2+) channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca(2+) channel during FP stimulation. The cytosolic Ca(2+) transients and the basal Ca(2+) in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I(Ca) and on the Ca(2+) transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca(2+) buffer, eliminated the FP-induced acceleration of I(Ca) inactivation and reduced the inhibitory effect of the FP on I(Ca) by approximately 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca(2+) release, eliminated the accelerating effect of FP on the I(Ca) inactivation, and they reduced the inhibitory effect of FP on the I(Ca). These results suggest that the fluid pressure indirectly suppresses the Ca(2+) channel by enhancing the Ca(2+)-induced intracellular Ca(2+) release in rat ventricular myocytes.

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