Roles for endoplasmic reticulum associated degradation (ERAD) in Myocardial Proteostasis - Proteostasis comprises the processes governing the life cycle of proteins from synthesis to degradation. Imbalanced proteostasis contributes to numerous pathologies, including those affecting the heart. Proteostasis in the sarco/endoplasmic reticulum (ER) of cardiac myocytes is important since proteins involved in contractile Ca handling, as well as receptors and secreted proteins, are synthesized on ER-bound ribosomes. We found that cardiac pathology imbalances ER proteostasis, causing ER stress and misfolded proteins that must be degraded to avoid toxicity. This proposal concerns one such degradation process, ER associated protein degradation (ERAD), which is the mechanism responsible for proteasome-mediated degradation of ER proteins. We found that in mice cardiac ischemia/reperfusion (I/R) increases ROS and activates the ER stress response, which induces several previously uncharacterized antioxidant proteins, including the trans-ER membrane selenoprotein, Vimp, which has not been studied in the ischemic heart. We found that Vimp knockdown in mice increased I/R-generated ROS and infarct size in in vivo I/R, consistent with a role for Vimp as an antioxidant. In addition to its antioxidant activity, Vimp has been shown in model cell lines to be important for assembly of the ERAD complex, the function of which is not known to require antioxidants or Se. Vimp overexpression in cultured cardiac myocytes increased ERAD, consistent with Vimp’s function as a key regulator of ERAD in the heart. Our hypothesis is that Vimp mitigates ROS and misfolded protein accumulation, both of which protect against I/R damage. Moreover, the unique dual roles of Vimp are mechanistically linked to the antioxidant function of its Se and the protein degradation function of its ERAD domain. Finally, endogenous proteins in the heart are degraded by ERAD as part of their life cycle, which is essential for balancing proteostasis and optimizing heart function. Our specific aims are to: 1- determine the effects of AAV9-sh-RNA-mediated knockdown of endogenous Vimp, which is very effective in vivo, on cardiac structure and function, infarct size and remodeling, as well as ROS, ERAD and molecular sensors of cardiac pathology and ER protein misfolding in mice subjected to I/R, 2- mechanistically dissect roles for the Se and the ERAD-enabling domain of Vimp using AAV9 encoding wild type Vimp (Vimp-WT), Vimp lacking Se (Vimp-Se), and Vimp with a mutated, dysfunctional ERAD domain (Vimp-ERAD), allof which we have already prepared, in mice subjected to I/R, and 3- use three complementary approaches to examine how ERAD affects endogenous proteins in the mouse heart, in vivo, investigating 1- ERAD-mediated degradation of Serca2a, 2- ER proteome dynamics using ER-targeted proximity biotin labeling and quantitative proteomics, and 3- ER proteome dynamics using stable isotope labeling mass spectrometry. In terms of relevance to heart disease or significance, our concept that ERAD is critical for the protein degradation component of proteostasis is innovative. Examining this concept is expected to provide new avenues for exploring the development of novel therapies for ischemic heart disease.
