Abstract 187: Metformin Increases Degradation of Ubiquitinylated Phospholamban via Autophagy in Cardiomyocytes

Abstract

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) in striated muscles. Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [35S]-methionine pulse-chase experiments along with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3 (LC3), became increasingly co-localized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in Hace1-null mouse hearts was associated with increased PLN levels determined by immunoblot and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.