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-XL 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 approximately 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.
Brady
Nathan R
NR
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.
Hamacher-Brady
Anne
A
Yuan
Hua
H
Gottlieb
Roberta A
RA
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001343:Autophagy; D000071186:Beclin-1; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D003470:Culture Media; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000502:physiology*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000502:physiology*; D011485:Protein Binding; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D002614:Chelating Agents; D003470:Culture Media; D008869:Microtubule-Associated Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium