Abstract 1370: Bcl-2 Regulation of Sarco/Endoplasmic Reticulum Calcium Stores Mediates the Autophagic Response to Nutrient Deprivation in the HL-1 Cardiomyocyte
Apoptotic and autophagic pathways are linked through the interaction of anti-apoptotic Bcl-2 proteins with the autophagy protein Beclin1. However, the nature of the interaction, either in promoting or blocking autophagy, remains unclear. We developed a highly sensitive methodology for quantifying nutrient-deprivation-activated autophagic flux in the HL-1 cardiomyocyte by comparing levels of GFP-LC3 labeled autophagosomal vesicles (AVs) under steady-state conditions and in the presence of lysosomal inhibitors (cumulative AVs). Expression of Beclin1 lacking the Bcl-2 binding domain (Beclin1ΔBcl2BD) significantly reduced autophagic flux, indicating that Beclin1-mediated autophagy requires an interaction with Bcl-2. In apparent contrast, overexpression of Bcl-2 slightly suppressed autophagic flux; an effect enhanced by targeting Bcl-2 to the sarco/endoplasmic reticulum (S/ER; Figure⇓). Suppression of autophagy by S/ER-targeted Bcl-2 may be in part due to depletion of S/ER calcium stores: intracellular scavenging of calcium by BAPTA-AM and treatment with thapsigargin, an inhibitor of the S/ER calcium ATPase (SERCA), significantly reduced autophagic activity. These findings reveal that Bcl-2 regulates the autophagic response at the level of S/ER calcium content rather than via direct interaction with Beclin1. Moreover, calcium homeostasis was identified as an essential component of the autophagic response to nutrient deprivation in the cardiomyocyte.