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      Novel control of cardiac myofilament response to calcium by S-glutathionylation at specific sites of myosin binding protein C

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          Our previous studies demonstrated a relation between glutathionylation of cardiac myosin binding protein C (cMyBP-C) and diastolic dysfunction in a hypertensive mouse model stressed by treatment with salt, deoxycorticosterone acetate, and unilateral nephrectomy. Although these results strongly indicated an important role for S-glutathionylation of myosin binding protein C as a modifier of myofilament function, indirect effects of other post-translational modifications may have occurred. Moreover, we did not determine the sites of thiol modification by glutathionylation. To address these issues, we developed an in vitro method to mimic the in situ S-glutathionylation of myofilament proteins and determined direct functional effects and sites of oxidative modification employing Western blotting and mass spectrometry. We induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers) with oxidized glutathione (GSSG). Immuno-blotting results revealed increased glutathionylation with GSSG treatment of a protein band around 140 kDa. Using tandem mass spectrometry, we identified the 140 kDa band as cMyBP-C and determined the sites of glutathionylation to be at cysteines 655, 479, and 627. Determination of the relation between Ca 2+-activation of myofibrillar acto-myosin ATPase rate demonstrated an increased Ca 2+-sensitivity induced by the S-glutathionylation. Force generating skinned fiber bundles also showed an increase in Ca-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol (DTT). Our data demonstrate that a specific and direct effect of S-glutathionylation of myosin binding protein C is a significant increase in myofilament Ca 2+-sensitivity. Our data also provide new insights into the functional significance of oxidative modification of myosin binding protein C and the potential role of domains not previously considered to be functionally significant as controllers of myofilament Ca 2+-responsiveness and dynamics.

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          Most cited references 34

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          Probability-based protein identification by searching sequence databases using mass spectrometry data

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            Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes.

             P Matsudaira (1987)
            Small amounts (7-250 pmol) of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride (PVDF) membranes, stained with Coomassie Blue, and sequenced directly. The membranes are not chemically activated or pretreated with Polybrene before usage. The average repetitive yields and initial coupling of proteins spotted or blotted into PVDF membranes ranged between 84-98% and 30-108% respectively, and were comparable with the yields measured for proteins spotted onto Polybrene-coated glass fiber discs. The results suggest that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.
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              In the thick of it: HCM-causing mutations in myosin binding proteins of the thick filament.

              In the 20 years since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM), an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins, including the myosin essential and regulatory light chains and cardiac myosin binding protein (cMyBP)-C. However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM-causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin-driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes.

                Author and article information

                Front Physiol
                Front Physiol
                Front. Physiol.
                Frontiers in Physiology
                Frontiers Media S.A.
                09 October 2013
                20 November 2013
                : 4
                Department of Physiology and Biophysics and Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago Chicago, IL USA
                Author notes

                Edited by: Aikaterini Kontrogianni-Konstantopoulos, University of Maryland, School of Medicine, USA

                Reviewed by: Han-Zhong Feng, Wayne State University School of Medicine, USA; P. Bryant Chase, The Florida State University, USA

                *Correspondence: R. John Solaro, Department of Physiology and Biophysics (M/C 901), College of Medicine, University of Illinois at Chicago, 835 Sourth Wolcott Ave., Chicago, IL 60612, USA e-mail: solarorj@

                This article was submitted to Striated Muscle Physiology, a section of the journal Frontiers in Physiology.

                Copyright © 2013 Patel, Wilder and Solaro.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 6, Tables: 1, Equations: 0, References: 41, Pages: 10, Words: 6995
                Original Research Article

                Anatomy & Physiology

                sarcomeres, oxidative stress, cardiac relaxation, c-protein


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