Bcl-2 and ryanodine receptors: physiology, pathophysiology and therapeutics for the heart

Anti-apoptotic Bcl-2 proteins are critical regulators of intracellular Ca2+ homeostasis and dynamics (Vervliet T. et al, Oncogene, 2016). Recently, we identified ryanodine receptors (RyRs), a class of intracellular Ca2+-release channels with important functions in excitable cells like skeletal and cardiac muscle cells as well as neurons, as novel targets for anti-apoptotic Bcl-2 family members, including Bcl-2 and Bcl-Xl (Vervliet T. et al, J Cell Sci, 2014; Vervliet T. et al, Sci Rep, 2015). The molecular determinant responsible for RyR control by Bcl-2 is the BH4 domain, which is sufficient to bind RyRs and to inhibit their Ca2+-release properties.

In this PhD project, we will focus on the regulation of the type 2 RyR (RyR2), the cardiac isoform, by Bcl-2. We will examine whether Bcl-2 can form complexes with the RyR2 channel in in vitro expression systems and in cardiac cells using co-immunoprecipitation and proximity ligation assays and whether Bcl-2 can regulate RyR2-mediated Ca2+ flux in cell systems and in electrophysiological approaches (collaboration with Sitsapesan, Oxford University, UK). For the latter, we will also apply exogenously added Bcl-2, purified from bacteria, or its BH4 domain, produced as a synthetic peptide. Hyperactivating RyR2 mutations are associated with several cardiac diseases, including catecholaminergic polymorphic ventricular tachycardia (CPVT). Some mutations are located near the Bcl-2 binding site, while others are distant from this site. The impact of these mutant RyR2 channels will be studied. A final part (in collaboration with Prof. Dr. Llewelyn Roderick) will deal with primary cardiomyocytes, in which Bcl-2 is knocked down using adeno-associated viral vectors and Ca2+-release events will be studied.