The structural proximity and functional coupling between the SR (sarcoplasmic reticulum) and mitochondria have been suggested to occur in the heart. However, the molecular architecture involved in the SR-mitochondrial coupling remains unclear. In the present study, we performed various genetic and Ca2+-probing studies to resolve the proteins involved in the coupling process. By using the bacterial 2-hybrid, glutathione transferase pull-down, co-immunoprecipitation and immunocytochemistry assays, we found that RyR2 (ryanodine receptor type 2), which is physically associated with VDAC2 (voltage-dependent anion channel 2), was co-localized in SR-mitochondrial junctions. Furthermore, a fractionation study revealed that VDAC2 was co-localized with RyR2 only in the subsarcolemmal region. VDAC2 knockdown by targeted short hairpin RNA led to an increased diastolic [Ca2+] (calcium concentration) and abolishment of mitochondrial Ca2+ uptake. Collectively, the present study suggests that the coupling of VDAC2 with RyR2 is essential for Ca2+ transfer from the SR to mitochondria in the heart.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an arrhythmogenic disease characterized by stress-triggered syncope and sudden death. Most of the CPVT mutations are concentrated in RyR ''hotspots'': the C terminus, the central and the N terminal domains. Although the phenotypic manifestations are common, it is likely that the intrinsic mechanism of RyR dysfunction varies with the location of the mutation within the RyR. Previously, we characterized RyR dysfunction in a CPVT mutation located at the C terminal domain, identifying an increased cytosolic Ca 2þ sensitivity as the causative of a gain-of-funcion defect. Recently, we have identified a mutation in a family with CPVT syndrome, which is located in the N terminal portion: RyR2 R420Q . To analyze the function of the mutated RyR channel, we have created an expression plasmids of RyR2 R420Q and RyR2 WT . Heterologous expression in HEK cells showed decreased caffeine sensitivity, suggesting loss-offunction defect. In order to be able to further characterize this mutation in a cardiac context, we expressed both RyR2 R420Q and RyR2 WT in cultured neonatal rat cardiac myocytes and analyzed the amplitude and frequency of spontaneous [Ca 2þ ] i transients and Ca 2þ sparks by confocal microscopy. We found that the spontaneous [Ca 2þ ] i transients were more frequent but of lower amplitude in RyR2 R420Q expressing cells. Further experiments are being conducted to fully understand the underlying mechanism.