The present application is a request, to extend the Pi's MERIT Award for a further 5 years of support. The goal of this project is to elucidate mechanisms by which Ca2+, intermolecular cooperativity, and protein phosphorylations regulate myocardial contraction in health and disease. The objective of this proposal is to determine the roles of myosin binding protein-C (cMyBP-C), with emphasis on the regulation of contraction by cMyBP-C phosphorylation. We propose: Hypothesis 1, cMyBP-C modulates contraction by binding to myosin subfragment 2 (S2), thereby controlling the availability of cross-bridges to actin;Hypothesis 2, reduced systolic function in our cMyBP-C null mouse results from accelerated cross-bridge kinetics due to deletion of cMyBP-C;and Hypothesis 3, the positive inotropy induced by B-adrenergic agonists is due in part to PKA-mediated phosphorylation of cMyBP-C. Considerable progress has been made in the current grant period in testing each of these principal hypotheses, including the following results: 1) cMyBP-C is the primary regulator of myofibrillar contractile kinetics due to PKA phosphorylation of contractile proteins during )B-adrenergic stimulation of myocardium, 2) cMyBP-C binds to myosin along the long axis of the thick filament, thereby refuting the widely held "collar model" of cMyBP-C binding, 3) phosphorylation or ablation of cMyBP-C causes cross-bridges to move toward the thin filament, and 4) CAMKII phosphorylation of cMyBP-C mediates the positive force-frequency response in myo-cardium. These and other results set the stage for studies of the molecular mechanisms of these findings. We will develop new mouse lines expressing phosphorylation mutants of cMyBP-C to identify the PKA and CAMKII sites in cMyBP-C, the order of these phosphorylations, and the effects of each on myocardial function. We will determine whether the effects of cMyBP-C are due to its interactions with myosin or if, as proposed by some, its putative binding to actin is also involved. Further studies will focus on the functional and structural roles of cMyBP-C in living nnuscle by studying the effects of its ablation or phosphorylation on contraction in vivo and in isolated muscle. The possibility that the disease phenotypes of cMyBP-C knock-out and phosphorylation mutant mice are due in part to compensatory mechanisms will be studied by reconstitution of null myocardium with wild-type and mutant proteins, by conditional expression of null and mutant alleles, and by re-expression of wild-type alleles. These results promise to provide insights into the mechanisms by which contractile state is modulated in healthy myocardium and also the basis for functional deficits in diseased hearts.

Public Health Relevance

Work from this laboratory and others has demonstrated that myosin binding protein C is a critical regulator of heart muscle function in health and in diseases such as heart failure and hypertrophic cardiomyopathies. The studies proposed in this project are designed to understand the types of cardiac function that are mediated by this protein and also the changes in cardiac function due to disease mutations in the gene encoding the protein. Elucidating the molecular mechanisms of action of myosin binding protein C will lead to the identification of new therapeutic targets and strategies for the treatment of diseases of heart muscle and the heart.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL082900-08
Application #
8452100
Study Section
Special Emphasis Panel (NSS)
Program Officer
Adhikari, Bishow B
Project Start
2006-04-15
Project End
2016-01-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
8
Fiscal Year
2013
Total Cost
$618,581
Indirect Cost
$199,541
Name
University of Wisconsin Madison
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Golob, Mark; Moss, Richard L; Chesler, Naomi C (2014) Cardiac tissue structure, properties, and performance: a materials science perspective. Ann Biomed Eng 42:2003-13
Patel, Jitandrakumar R; Pleitner, Jonathan M; Moss, Richard L et al. (2012) Magnitude of length-dependent changes in contractile properties varies with titin isoform in rat ventricles. Am J Physiol Heart Circ Physiol 302:H697-708
Rybakova, Inna N; Greaser, Marion L; Moss, Richard L (2011) Myosin binding protein C interaction with actin: characterization and mapping of the binding site. J Biol Chem 286:2008-16
de Lange, W J; Hegge, L F; Grimes, A C et al. (2011) Neonatal mouse-derived engineered cardiac tissue: a novel model system for studying genetic heart disease. Circ Res 109:8-19
Zhang, Jiang; Guy, Moltu J; Norman, Holly S et al. (2011) Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure. J Proteome Res 10:4054-65
Roberts, Arthur G; Cheesman, Matthew J; Primak, Andrew et al. (2010) Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: implications for substrate binding. Biochemistry 49:8700-8
Hatzakis, Emmanuel; Okamoto, Keika; Yang, Danzhou (2010) Thermodynamic stability and folding kinetics of the major G-quadruplex and its loop isomers formed in the nuclease hypersensitive element in the human c-Myc promoter: effect of loops and flanking segments on the stability of parallel-stranded intramolecul Biochemistry 49:9152-60
Moss, Richard L; Fitzsimons, Daniel P (2010) Regulation of contraction in mammalian striated muscles--the plot thick-ens. J Gen Physiol 136:21-7
Colson, Brett A; Locher, Matthew R; Bekyarova, Tanya et al. (2010) Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development. J Physiol 588:981-93
Chen, Peter P; Patel, Jitandrakumar R; Rybakova, Inna N et al. (2010) Protein kinase A-induced myofilament desensitization to Ca(2+) as a result of phosphorylation of cardiac myosin-binding protein C. J Gen Physiol 136:615-27

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