Abstract 4114625: Upregulation of Palmitoylating Enzymes, DHHC2 and DHHC5, in Failing Human Hearts Protects Impulse Propagation but Endangers Myocyte Survival after Ischemia/Reperfusion (I/R) Injury

Background: Palmitoylation 'Palm* catalyzed by DHHC enzymes promotes protein binding to membranes, impacting protein translocation and function. Palm* dysregulation has been linked to cancers and neurogenerative disorders; its impact on cardiac function is unclear. We have used global purification of palmitoylated proteins from human and animal hearts followed by proteomic analysis to build 'cardiac palmitoylome', whose 454 constituents are involved in organizing/stabilizing cardiomyocyte microdomains (intercalated discs 'ICD', dyads, and costameres) critical for myocyte functions/homeostasis. We confirmed 11 of the 23 DHHC genes are expressed in human&animal hearts. In particular, DHHC2 and DHHC5 were upregulated in human failing hearts. DHHC enzymes are membrane-embedded, limiting their substrates to enzymes' 'territories' in cells. Objective: To identify DHHC2 and DHHC5 distribution patterns and substrates in myocytes, and to understand how upregulation of DHHC2 or DHHC5 affects myocyte function. Methods: Track adenovirus-expressed mCherry-DHHC2 and -DHHC5 (mChr-DHHCx, x=2 or 5, Adv-mChr as control) in ventricular myocytes from adult Sprague-Dawley rats. Globally purify palmitoylated proteins for proteomic analysis/quantification. Use high-resolution quantitative imaging to analyze how mChr-DHHCx affects myocyte response to ischemia/reperfusion (I/R) injury and the expression/organization of palmitoylated proteins at ICD and lateral surface. Results: mChr-DHHC2 is mainly present in cytoplasmic vesicles bearing markers of recycling or autophagy, with a minor presence along lateral surface, dyads, and ICD. mChr-DHHC5 is mainly present along the lateral surface, dyads and ICD, with a minor presence in small endosomal vesicles. Palmitoylome normalized by proteome identifies DHHC2 and DHHC5 substrates that overlap with cardiac palmitoylome, and match the enzymes' distribution patterns. I/R injury increases endocytosis in myocytes, quantified by uptake of plasma-membrane marker, wheat germ agglutinin. I/R-induced endocytosis is exacerbated by mChr-DHHC2 but, surprising, alleviated by mChr-DHHC5. mChr-DHHC2 and -DHHC5 increase internalization of ICD and lateral membrane proteins, n-cadherin and NCX1 respectively. Conclusions: DHHC2 and DHHC5 have non-overlapping distribution patterns, consistent with varying substrate groups. DHHC2, but not DHHC5, appears involved in the previously reported palm*-dependent massive endocytosis in I/R-stressed cardiomyocytes.