期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：23
页码：7165-7170
DOI：10.1073/pnas.1508815112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificancePhospholamban (PLN) can regulate Ca2+ uptake rates in the sarcoplasmic reticulum in cardiomyocytes. However, the mechanisms that control PLN levels are not fully understood. This study shows that PLN degradation depends on ubiquitinylation of its lysine 3 residue and p62-mediated selective autophagy. Metformin was shown to accelerate autophagy and to induce PLN degradation, resulting in increased Ca2+ uptake. These results suggest that changes in PLN degradation could account for the cardiac inotropic effects of metformin. Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). 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, together 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. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.
关键词：selective autophagy ; ubiquitinylation ; protein degradation
