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 [
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.Phospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slow-twitch 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 intracellular Ca
2+ concentration) stimuli.Previous studies identified three PLN mutations in families of patients with hereditary dilated cardiomyopathy. These mutations, the substitution of Cys for Arg9 (R9C) (
4), Arg14 deletion (RΔ14) (
5), and the substitution of TGA for TAA in the Leu39 codon, creating a stop codon (L39stop) (
6), also lead to dilated cardiomyopathy in transgenic mice. At the cellular level, ectopically expressed RΔ14 and L39stop PLN mutants localize at the plasma membrane in HEK-293T cells, cultured mouse neonatal cardiomyocytes, and cardiac fibroblasts, whereas wild-type and the R9C mutant reside within the endoplasmic reticulum (ER)/SR (
6,
7). These data, together with a recent study by Sharma et al. (
8), suggest a highly ordered trafficking of PLN, ultimately ensuring correct localization, and thus function, within the SR. However, PLN trafficking and degradation mechanisms in mammalian cardiomyocytes have not been clearly established.Protein degradation and clearance of damaged organelles are critical for cellular physiology, and failure in proper clearance has been shown to have pathological repercussions (
9). Autophagy is a major mechanism that mediates protein and organelle degradation in response to external and internal signals. External stimulation through pharmacological agonists, such as metformin and rapamycin, promotes autophagy via AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signal pathways, whereas amino acid starvation and an increased intracellular AMP/ATP ratio serve as internal signals to promote autophagy via the Ca
2+/Calmodulin-dependent kinase kinase-β (
10). Steps in the autophagy pathway involve nucleation of targeted macromolecules on the ER membrane, trafficking of autophagosomes to lysosomes and, finally, fusion of the autophagosome-lysosome, resulting in targeted protein degradation (
11). In the heart, autophagy plays a crucial role in response to insults, in part by relieving ER stress (
12) and removing damaged mitochondria (
13). Loss of autophagy could result in irreversible apoptosis and reduced cardiac functioning (
14).To characterize PLN degradation, we conducted a series of assays in cultured mouse neonatal cardiomyocytes (CMNCs) and the hearts of HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1 (Hace1)-null mice. Our results show that PLN degradation required both polyubiquitinylation and p62-mediated selective autophagy in CMNCs. Loss of HACE1 was associated with increased PLN levels, supporting the notion that selective autophagy modulates PLN degradation in vivo. Metformin promoted wild-type and R9C PLN degradation through autophagic pathways, resulting in metformin-induced inotropic enhancement.
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