RPN6‐Ser14 Phosphorylation Is Responsible for Proteasome Activation by PKA and Protects against Pathological Cardiac Hypertrophy and Malfunction in Mice

Abstract

Proteasome (Psm) functional insufficiency (PFI) contributes to the genesis of a large subset of heart failure (HF). Measures to correct PFI promise to benefit HF treatment but the search for such measures requires a better understanding on how Psm activity is regulated. Phosphorylation of Rpn6/PSMD11 at Ser14 (pS14‐Rpn6) underlines activation of the 26S Psm by the cAMP‐dependent protein kinase (PKA) in cultured cells but this remains to be confirmed in vivo. Moreover, the in vivo (patho)physiological requirement of Psm phosphoregulation by PKA, or by any kinases for that matter, has not been established. To address these critical gaps, we sought to test the novel hypothesis that pS14‐Rpn6 protects the heart under stress conditions. (1) We created two mouse models in which the Rpn6/Psmd11 gene is edited so that its Ser14 is mutated to either Ala (S14A) or Asp (S14D) to block or mimic phosphorylation, respectively. Increases in myocardial pS14‐Rpn6 and in 26S Psm activities by stimulating PKA are completely lost in S14A mice and, conversely, S14D mouse hearts show significantly higher basal 26S Psm activities than wild type (WT) mouse hearts. For the first time, these studies unequivocally establish Ser14 of Rpn6 as the primary, if not the only, phosphosite responsible for the activation of 26S Psms by PKA in vivo. (2) A small cohort of S14A and S14D mice are used for a preliminary aging study. To date, they have reached 22 months of age but none has died or shown gross abnormalities. (3) β‐Adrenergic stimulation with isoproterenol (5 mg/kg × 15 days, s.c.) induced significantly greater myocardial ubiquitin conjugate accumulation and cardiac hypertrophy in S14A mice than in WT mice. (4) Myocardial pS14‐Rpn6 levels were markedly increased in WT mice subject to coronary artery ligation induced myocardial infarction (MI) or transverse aorta constriction (TAC). (5) Adult female S14A, S14D, and age‐matched WT mice were subject to MI. Serial echocardiography (Echo) at 2, 4, 8, and 12 weeks post‐MI revealed that post‐MI left ventricular chamber dilatation was significantly less in the S14D MI group (vs. WT MI and S14A MI groups). By 8 weeks post‐MI, the reduction of fractional shortening and ejection fraction became more severe in the S14A MI group than in the other two MI groups, indicating that pS14‐Rpn6 protects against post‐MI cardiac remodeling and malfunction. (6) Serial Echo revealed that TAC induced greater cardiac hypertrophy at the earlier time points (1 and 2 weeks) but more rapid cardiac function decompensation in S14A mice than in WT mice. TAC‐induced greater increases of cardiac ubiquitin conjugates in S14A mice than in WT mice. Thus, through unequivocal genetic interrogations, we have established for the first time that pS14‐Rpn6, the primary mechanism for PKA to activate the Psm, is indispensable for the heart to handle various stresses and that increases in pS14‐Rpn6 promote new cardiac proteostasis and protect against cardiac hypertrophy and function decompensation in diseased hearts.Support or Funding InformationNIH grant R01 HL072166