The autophagic response to nutrient deprivation in the hl-1 cardiac myocyte is modulated by Bcl-2 and sarco/endoplasmic reticulum calcium stores

Macroautophagy is a vital process in the cardiac myocyte: it plays a protective role in the response to ischemic injury, and chronic perturbation is causative in heart disease. Recent findings evidence a link between the apoptotic and autophagic pathways through the interaction of the antiapoptotic proteins Bcl-2 and Bcl-X-L with Beclin 1. However, the nature of the interaction, either in promoting or blocking autophagy, remains unclear. Here, using a highly sensitive, macroautophagy-specific flux assay allowing for the distinction between enhanced autophagosome production and suppressed autophagosome degradation, we investigated the control of Beclin 1 and Bcl-2 on nutrient deprivation-activated macroautophagy. We found that in HL-1 cardiac myocytes the relationship between Beclin 1 and Bcl-2 is subtle: Beclin 1 mutant lacking the Bcl-2-binding domain significantly reduced autophagic activity, indicating that Beclin 1-mediated autophagy required an interaction with Bcl-2. Overexpression of Bcl-2 had no effect on the autophagic response to nutrient deprivation; however, targeting Bcl-2 to the sarco/endoplasmic reticulum (S/ER) significantly suppressed autophagy. The suppressive effect of S/ER-targeted Bcl-2 was in part due to the depletion of S/ER calcium stores. Intracellular scavenging of calcium by BAPTA-AM significantly blocked autophagy, and thapsigargin, an inhibitor of sarco/endoplasmic reticulum calcium ATPase, reduced autophagic activity by similar to 50%. In cells expressing Bcl-2-ER, thapsigargin maximally reduced autophagic flux. Thus, our results demonstrate that Bcl-2 negatively regulated the autophagic response at the level of S/ER calcium content rather than via direct interaction with Beclin 1. Moreover, we identify calcium homeostasis as an essential component of the autophagic response to nutrient deprivation.