Phospho-Proteomic Analysis of Cardiac Dyssynchrony and Resynchronization Therapy

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Abstract

Cardiac dyssynchrony arises from conduction abnormalities during heart failure and worsens morbidity and mortality. Cardiac Resynchronization Therapy (CRT) re-coordinates contraction using bi-ventricular pacing, but the cellular and molecular mechanisms involved remain largely unknown. We aimed to determine how dyssynchronous heart failure (HF dys) alters the phospho-proteome and how CRT interacts with this unique phospho-proteome by analyzing Ser/Thr and Tyr phosphorylation. Phospho-enriched myocardium from dog models of Control, HF dys, and CRT was analyzed via mass spectrometry. There were 209 regulated phospho-sites among 1,761 identified sites. Compared to Con and CRT, HF dys was hyper-phosphorylated and tyrosine phosphorylation was more likely to be involved in signaling that increased with HF dys and was exacerbated by CRT. For each regulated site, the most-likely targeting-kinase was predicted, and CK2 was highly specific for sites that were “fixed” by CRT, suggesting activation of CK2 signaling occurs in HF dys that is reversed by CRT, which was supported by western blot analysis. These data elucidated signaling networks and kinases that may be involved and deserve further study. Importantly, we have identified a possible role for CK2 modulation in CRT. This may be harnessed in the future therapeutically to compliment CRT, improving its clinical effects.

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Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.

Kirti Sharma, Stefka Tyanova, Christoph Schaab … (2014)

Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Calcium cycling proteins and heart failure: mechanisms and therapeutics.

Andrew Marks (2013)

Ca2+-dependent signaling is highly regulated in cardiomyocytes and determines the force of cardiac muscle contraction. Ca2+ cycling refers to the release and reuptake of intracellular Ca2+ that drives muscle contraction and relaxation. In failing hearts, Ca2+ cycling is profoundly altered, resulting in impaired contractility and fatal cardiac arrhythmias. The key defects in Ca2+ cycling occur at the level of the sarcoplasmic reticulum (SR), a Ca2+ storage organelle in muscle. Defects in the regulation of Ca2+ cycling proteins including the ryanodine receptor 2, cardiac (RyR2)/Ca2+ release channel macromolecular complexes and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a)/phospholamban complex contribute to heart failure. RyR2s are oxidized, nitrosylated, and PKA hyperphosphorylated, resulting in "leaky" channels in failing hearts. These leaky RyR2s contribute to depletion of Ca2+ from the SR, and the leaking Ca2+ depolarizes cardiomyocytes and triggers fatal arrhythmias. SERCA2a is downregulated and phospholamban is hypophosphorylated in failing hearts, resulting in impaired SR Ca2+ reuptake that conspires with leaky RyR2 to deplete SR Ca2+. Two new therapeutic strategies for heart failure (HF) are now being tested in clinical trials: (a) fixing the leak in RyR2 channels with a novel class of Ca2+-release channel stabilizers called Rycals and (b) increasing expression of SERCA2a to improve SR Ca2+ reuptake with viral-mediated gene therapy. There are many potential opportunities for additional mechanism-based therapeutics involving the machinery that regulates Ca2+ cycling in the heart.