Muscarinic 2 receptors modulate cardiac proteasome function in a protein kinase G-dependent manner

Proteasome function insufficiency and inadequate protein quality control are strongly implicated in a large subset of cardiovascular disease and may play an important role in their pathogenesis. Protein degradation by the ubiquitin proteasome system can be physiologically regulated. Cardiac muscarinic 2 (M2) receptors were pharmacologically interrogated in intact mice and cultured neonatal rat ventricular myocytes (NRVMs). Proteasome-mediated proteolysis was measured with a surrogate misfolded protein, proteasome peptidase assay, and by characterizing key proteasome subunits. Successful M2 receptor manipulation in cardiomyocytes was determined by measuring an endogenous protein substrate, and in mice, the cardiovascular physiological response. M2 receptor stimulation was associated with increased proteasome-mediated proteolysis and enhanced peptidase activities, while M2 receptor inhibition yielded opposing results. Additionally, M2 receptor manipulation did not alter abundance of the key proteasome subunits, Rpt6 and β5, but significantly shifted their isoelectric points. Inhibition of protein kinase G abrogated the stimulatory effects on proteasome-mediated proteolysis from M2 receptor activation. We conclude that M2 receptor stimulation enhances, whereas M2 receptor inhibition reduces, proteasome-mediated proteolysis likely through posttranslational modifications. Protein kinase G appears to be the mediator of the M2 receptors actions.