Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Abstract

Phospholamban (PLN) is an effective inhibitor of the sarco(endo) plasmic reticulum Ca 2+ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [ 35 S]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.selective autophagy | ubiquitinylation | protein degradation P hospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slowtwitch skeletal, and smooth muscle, where it exists as a monomer or pentamer. Whereas monomeric PLN physically interacts with sarco(endo)plasmic reticulum Ca 2+ ATPase type 2a (SERCA2a) to antagonize its function, pentameric PLN complexes are thought to be a reservoir of inactive PLN (1-3). The physical interaction between SERCA2a and PLN reduces the apparent affinity of SERCA2a for Ca 2+ , thereby making SERCA2a less active in transporting Ca 2+ from the cytoplasm to the lumen of the SR at the same concentration of cytoplasmic Ca 2+. The physical interaction between the two proteins is regulated by phosphorylation of PLN at Ser16 by protein kinase A or at Thr17 by Ca 2+ /calmodulin-dependent protein kinase II (2). Phosphorylation of PLN reduces its affinity for SERCA2a, thereby increasing SERCA2a activity (2). Evidence from transgenic mice also supports the inhibitory function of PLN. Although targeted PLN deletion enhances baseline cardiac performance, cardiac-specific overexpression of superinhibitory forms of PLN leads to decreases in the affinity of SERCA2a for Ca 2+ (2). These observations underscore the primary role of PLN as a regulator of SERCA2a activity and, therefore, as a crucial regulator of cardiac contractility. PLN inhibition of SERCA2a can be reversed by either external (i.e., activation of β-adrenergic receptors) or internal (i.e., increased...

The intramembrane protease SPPL 2c promotes male germ cell development by cleaving phospholamban

“…The affinity of PLN to SERCA2 is significantly influenced by phosphorylation of PLN. Furthermore, initial studies have revealed that triggered degradation of PLN by autophagy or the ubiquitin-proteasome system may play an additional regulatory role [49][50][51]. However, an involvement of intramembrane proteases in the control of PLN as we report it here has not been reported before.…”

Metformin mediates cardioprotection against aging‐induced ischemic necroptosis

“…Metformin is a first‐line antidiabetic drug that also activates autophagy and has cardiovascular protective effects (Song et al, ; Teng et al, ), although recent study has reported that metformin fails to cardioprotective effect in nonaging I/R swine models (Techiryan et al, ). In the present study, we found that metformin treatment effectively reduced I/R‐induced necroptosis in aging hearts, improved cardiac function after myocardial infarction, and improved long‐term survival.…”

Phospholamban p.Arg14del cardiomyopathy is characterized by phospholamban aggregates, aggresomes, and autophagic degradation

“…Autophagy is of paramount importance in the clearance of stress‐induced protein aggregates and aggresomes in cardiomyopathies . Recently, it has been shown, in primary cultured neonatal mouse cardiomyocytes, that PLN is degraded by selective p62‐mediated autophagy after polyubiquitinylation . p62 acts as an adaptor protein during aggregation, sequestration [hence its synonym, sequestosome‐1 (SQSTM‐1)] and degradation of misfolded proteins by the ubiquitin–proteasome pathway and selective autophagy .…”