VDAC – A New Player in Cardiac Calcium Handling

The regulation of cardiac rhythmicity and contractility critically depends on a tight regulation of calcium within the myocardium. Although the fundamental steps of cardiac calcium cycling are well understood, little is known about precise regulatory mechanisms, and still new players in cardiac calcium handling are newly discovered. The zebrafish tremblor is a loss of function mutant for the cardiac specific isoform of the Na2+/Ca 2+ exchanger NCX1h that is required to extrude Ca2+ from the cardiomyocytes after a contraction cycle. Loss of function of NCX1h in tremblor disrupts normal Ca2+ homeostasis in the heart and leads to cardiac fibrillation. In a small molecule suppressor screen a substance, OK-F7, was identified, which potently suppressed cardiac fibrillation and restored rhythmic cardiac contractions in tremblor. A pull-down assay identified the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial matrix as the molecular target of OK-F7. Knocking down VDAC2 activity blocked the rescue effect of OK-F7 and overexpression of VDAC2 in tremblor embryos restored rhythmic contractions comparable to OK-F7 treatment. In cultured NIH 3T3 cells, application of OK-F7 potently increased Ca2+ uptake into mitochondria. These experiments suggest a hitherto unknown mechanism of cardiac Ca2+ handling involving VDAC. It is conceivable that the lack of Ca2+ extrusion in NCX1h-null tremblor embryos results in an accumulation of Ca2+ in the cardiomyocytes and thereby leads to a higher SR Ca2+ load and a sensitization of the Ca2+-sensitive RyR. The combination of these effects increases the frequency of spontaneously propagating Ca2+ waves and thus offers a cellular basis of the fibrillation phenotype observed in tremblor embryos. We hypothesize that activation of VDAC (by either overexpression or the treatment with OK-F7) leads to an enhanced uptake of spontaneously released Ca2+ into mitochondria and thus prevents the formation of spontaneous Ca2+ waves and suppresses cardiac fibrillation in tremblor embryos. In this project two lines of studies are presented to test this hypothesis by investigating the mechanism by which VDAC contributes to cellular Ca2+ handling and how this process is modulated by OK-F7. First, it will be examined whether OK-F7 could enhance mitochondrial Ca2+ uptake in response to SR Ca2+ release by measuring changes in mitochondrial Ca2+ in isolated mammalian cardiomyocytes in the presence or absence of OK-F7. It will further be evaluated if OK-F7 treatment can influence the number of spontaneous propagating Ca2+ waves in cultured cardiomyocytes. Finally, biophysical features of VDAC2 required for the regulation of cardiac rhythmicity will be investigated in a structure-function approach. The success of the proposed studies will provide insights into the regulation of cardiac Ca2+ homeostasis by VDAC and will serve as a first important step to establish VDAC as a therapeutic target for cardiac arrhythmia.