Abstract
RATIONALE: Phosphorylation of sarcomeric proteins has been implicated in heart failure with preserved ejection fraction (HFpEF); such changes may contribute to diastolic dysfunction by altering contractility, cardiac stiffness, Ca(2+)-sensitivity, and mechanosensing. Treatment with cardiosphere-derived cells (CDCs) restores normal diastolic function, attenuates fibrosis and inflammation, and improves survival in a rat HFpEF model. OBJECTIVE: Phosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome were analyzed. METHODS AND RESULTS: Dahl salt-sensitive rats fed a high-salt diet, with echocardiographically verified diastolic dysfunction, were randomly assigned to either intracoronary CDCs or placebo. Dahl salt-sensitive rats receiving low salt diet served as controls. Protein and phosphorylated Ser, Thr, and Tyr residues from left ventricular tissue were quantified by mass spectrometry. HFpEF hearts exhibited extensive hyperphosphorylation with 98% of the 529 significantly changed phospho-sites increased compared with control. Of those, 39% were located within the sarcomeric subproteome, with a large group of proteins located or associated with the Z-disk. CDC treatment partially reverted the hyperphosphorylation, with 85% of the significantly altered 76 residues hypophosphorylated. Bioinformatic upstream analysis of the differentially phosphorylated protein residues revealed PKC as the dominant putative regulatory kinase. PKC isoform analysis indicated increases in PKC α, β, and δ concentration, whereas CDC treatment led to a reversion of PKCβ. Use of PKC isoform specific inhibition and overexpression of various PKC isoforms strongly suggests that PKCβ is the dominant kinase involved in hyperphosphorylation in HFpEF and is altered with CDC treatment. CONCLUSIONS: Increased protein phosphorylation at the Z-disk is associated with diastolic dysfunction, with PKC isoforms driving most quantified phosphorylation changes. Because CDCs reverse the key abnormalities in HFpEF and selectively reverse PKCβ upregulation, PKCβ merits being classified as a potential therapeutic target in HFpEF, a disease notoriously refractory to medical intervention.