Abstract 31: Regulation of Acetylation Restores Proteolytic Function in Diseased Myocardium

Abstract

RATIONALE: Proteasome complexes play essential roles in maintaining cardiac protein homeostasis in normal and stressed conditions. However, proteasomal function is often compromised in diseased myocardium; and the regulation of proteasomal dynamics remains poorly understood. METHODS AND RESULTS: We studied cardiac proteolytic function and its regulation by acetylation in murine and human models of heart disease. Proteasomes, from both normal and diseased myocardium, were treated with histone de-acetylases (HDACs) inhibitors and they exhibited enhanced proteolytic capacity in vitro and in vivo. This unique regulatory paradigm was first examined in a murine model of ischemic injury (n=5); where diminished proteolytic function in the ischemic hearts was restored by HDACs inhibition (either by SAHA or by Sodium Valproate) in a dose-dependent fashion. Importantly, this phenomenon was validated in human samples, where inhibition of HDACs augmented proteolytic functions in the diseased myocardium (n=5). Using high resolution LC-MS/MS coupled with a combined collision-induced dissociation and electron-transfer dissociation approach, the acetylome (N-terminal and lysine) of cardiac 20S proteasomes was delineated. Targeted enrichment strategy of the posttranslationally modified peptides enabled the capture of eight lysine and nine N-terminal acetylation sites in the murine heart, contributing to the first comprehensive acetylome map for the cardiac 20S proteasomes. Among them, at least six lysine acetylation sites were inducible by HDACs inhibitors. Furthermore, parallel investigations using cardiac 20S proteasomes pinpointed the functional impact of HDAC inhibitions to specific acetylation sites on the 20S proteasomal subunits. CONCLUSIONS: Proteasomal biology is modulated by acetylation modifications in the heart. This regulatory mechanism is punctual and potent; and it was observed both in an acutely pathological murine model of ischemia-reperfusion injury as well as in a chronic human disease of end-stage heart failure. These findings demonstrate the utility of pharmacological interventions (e.g., HDAC inhibition) to restore damaged proteolytic function in diseased myocardium.