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

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

Solution Structures of the C-Terminal Domain of Cardiac Troponin C Free and Bound to the N-Terminal Domain of Cardiac Troponin I

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33−80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81−161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81−161) both free and bound to cTnI(33−80). No significant differences were observed between secondary structural elements determined for free and cTnI(33−80)-bound cTnC(81−161). We have determined solution structures of Ca2+-saturated cTnC(81−161) free and bound to cTnI(33−80). While the tertiary structure of cTnC(81−161) is qualitatively similar to that observed free in solution, the binding of cTnI(33−80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33−80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33−80), onto the C-terminal cTnC structure. The binding interface for cTnI(33−80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF

Sarcomere Control Mechanisms and the Dynamics of the Cardiac Cycle

This review focuses on recent developments in the molecular mechanisms by which Ca activates cardiac sarcomeres and how these mechanisms play out in the cardiac cycle. I emphasize the role of mechanisms intrinsic to the sarcomeres as significant determinants of systolic elastance and ventricular stiffening during ejection. Data are presented supporting the idea that processes intrinsic to the thin filaments may promote cooperative activation of the sarcomeres and be an important factor in maintaining and modifying systolic elastance. Application of these ideas to translational medicine and rationale drug design forms an important rationale for detailed understanding of these processes

Geodetic model of the 2016 Central Italy earthquake sequence inferred from InSAR and GPS data

We investigate a large geodetic data set of interferometric synthetic aperture radar (InSAR)and GPS measurements to determine the source parameters for the three main shocks of the 2016Central Italy earthquake sequence on 24 August and 26 and 30 October (Mw6.1, 5.9, and 6.5,respectively). Our preferred model is consistent with the activation of four main coseismic asperitiesbelonging to the SW dipping normal fault system associated with the Mount Gorzano-Mount Vettore-Mount Bove alignment. Additional slip, equivalent to aMw~ 6.1–6.2 earthquake, on a secondary (1) NEdipping antithetic fault and/or (2) on a WNW dipping low-angle fault in the hanging wall of the mainsystem is required to better reproduce the complex deformation pattern associated with the greatestseismic event (theMw6.5 earthquake). The recognition of ancillary faults involved in the sequencesuggests a complex interaction in the activated crustal volume between the main normal faults and thesecondary structures and a partitioning of strain releas

Use of a decoy peptide to purify p21 activated kinase-1 in cardiac muscle and identification of ceramide-related activation

The p21 activated kinase-1 (Pak1) is a serine-threonine protein kinase directly activated by Cdc42 and Rac1. In cardiac myocytes, Pak1 activation leads to dephosphorylation of cTnI and C-protein through upregulation of phosphatase-2A (PP2A). Pak1 activity is directly correlated with its autophosphorylation, which occurs upon binding to the small GTPases and to some small organic molecules as well. In this report, we describe a novel method for rapid purification of endogenous Pak1 from bovine ventricle muscle. The method is simple and easy to carry out. The purified Pak1 demonstrated autophosphorylation in vitro that was enhanced by D-erythro-sphingosine-1, N-acetyl-D-erythro-sphingosine (C2-ceramide), and N-hexanoyl-D-erythro-sphingosine (C6-ceramide). Dihydro-L-threo-sphingosine (saphingol) also had some effect on Pak1 autophosphorylation. The method we developed provides a useful tool to study Pak1 activity and regulation in the heart. Moreover, our results indicate a potential role of the sphingolipids as unique signaling molecules inducing a direct activation of Pak1 that may modulate different cardiac functions

Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression

Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle. © 2012 Clause et al