Sarcomeric dysfunction plays a central role in reduced cardiac pump function in heart failure. This review focuses on the alterations in sarcomeric proteins in diseased myocardium that range from altered isoform expression to post-translational protein changes such as proteolysis and phosphorylation. Recent studies in animal models of heart failure and human failing myocardium converge and indicate that sarcomeric dysfunction, including altered maximum force development, Ca(2+) sensitivity, and increased passive stiffness, largely originates from altered protein phosphorylation, caused by neurohumoral-induced alterations in the kinase-phosphatase balance inside the cardiomyocytes. Novel therapies, which specifically target phosphorylation sites within sarcomeric proteins or the kinases and phosphatases involved, might improve cardiac function in heart failure.

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HF dys ) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HF dys ; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HF dys cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). We found that GSK-3β was deactivated in HF dys and reactivated by CRT. Mass spectrometry of myofilament proteins from HF dys animals incubated with GSK-3β confirmed GSK-3β-dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HF dys , but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HF dys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca 2+ -sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca 2+ -sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66±1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca 2+ -sensitivity (pCa 50 ) to 5.50±0.02 relative to the donor control value (pCa 50 =5.43±0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca 2+ -sensitivity relative to the failing control (pCa 50 =5.60±0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca 2+ -sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono-and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue.These differences in Ca 2+ -sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca 2+ -sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca 2+ -sensitivity.

Introduction Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted donor and end-stage failing hearts. Methods The first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX) fractionation method (10 pooled samples, technical replicates = 4) and the second employed a sodium dodecylsulfate polyacrylamide gel electrophoresis method (n = 10; technical replicates = 2). Each subsequently underwent titanium dioxide (TiO2) affinity chromatography to enrich for the tryptic phosphopeptides, which were analyzed using an LTQ-Orbitrap mass spectrometer. Moreover, recombinant C0-C2 fragment of mouse cMyBP-C incubated with PKA, PKC, CaMKII and CK2 was analyzed to identify the kinases involved with phosphorylation of cMyBP-C. Results Seventeen phosphorylation sites on cMyBP-C were identified in vivo, with the majority localized in the N-terminal domains C0-C2. The three most abundant phosphorylated sites, Ser284, Ser286 and Thr290, are located in the regulatory M-domain of cMyBP-C. Ser284 showed a significant reduction in phosphorylation in HF. Conclusion This study demonstrates that cMyBP-C harbours more phosphorylation sites than previously known, with a total of 17 (9 novel) identified phosphorylation sites in vivo. Most sites were primarily located within the N-terminal side of the protein. The most highly phosphorylated site on cMyBP-C was Ser284 and this site showed decreased phosphorylation in the failing heart, which implicate importance for fine-tuning contractility. To date, the functional importance of Ser286 and Thr290 is unknown. In addition, 16 sites were identified after in vitro kinase incubation. The data have been deposited to the ProteomeXchange with identifier PXD000158