The overall goal of this research is to elucidate molecular mechanisms by which Ca2+, cooperation, and protein phosphorylations regulate contraction of mammalian myocardium. The objective of this proposal is to elucidate the roles of myosin binding protein-C (cMyBP-C) in myocardium, with particular emphasis on the regulation of contraction and the effects of cMyBP-C phosphorylation on force and the kinetics of force development. (1) We hypothesize that cMyBP-C modulates contraction by binding to myosin subfragment 2 (S2), thereby physically controlling the availability of cross-bridges to actin. This idea will be tested by assessing the functional effects of interventions designed to disrupt cMyBP-C/S2 interactions in mouse skinned myocardium and using X-ray diffraction and electron microscopy to assess the structural effects of these interventions. (2) We hypothesize that the altered systolic function we have observed in our cMyBP-C null mouse results from accelerated cross-bridge kinetics and stretch activation due to deletion of cMyBP-C. We will test these ideas by measuring the activation dependence of the rate of rise of force and stretch activation in null myocardium and assessing the reversibility of these effects by reconstituting null myocardium with cMyBP-C. (3) We hypothesize that at least some of the positive inotropy induced by (alpha-adrenergic agonists is due to protein kinase A-mediated phosphorylation of cMyBP-C. We will test this idea by assessing the effects of PKA on the force and kinetics of force development (i) in mouse myocardium expressing mutant cardiac Tnl that cannot be phosphorylated by PKA, (ii) in cMyBP-C null myocardium expressing non-phosphorylatable cTnl, and (iii) in myocardium expressing mutants of cMyBP-C in which phosphorylatable serines are replaced with alanines or with aspartates. The idea that the effects of phosphorylation are due to alterations in cross-bridge availability to actin will be assessed by X-ray diffraction. The possibility that the contractile phenotypes of knock-out and transgenic mice are due in part to compensatory mechanisms will be studied both by reconstitution of null myocardium with wild-type and mutant proteins and by conditional expression of null and mutant alleles. Results should provide new information about mechanisms by which contractile state is modulated in healthy myocardium and new insights as to the basis for functional deficits in diseased hearts.

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 #
5R37HL082900-02
Application #
7221963
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Adhikari, Bishow B
Project Start
2006-04-15
Project End
2011-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
2
Fiscal Year
2007
Total Cost
$543,283
Indirect Cost
Name
University of Wisconsin Madison
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Kensler, Robert W; Craig, Roger; Moss, Richard L (2017) Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments. Proc Natl Acad Sci U S A 114:E1355-E1364
Moss, Richard L; Lynch 4th, Thomas L; Fitzsimons, Daniel P (2017) Acting on an impulse (or two): Advantages of high-frequency tetanic onset in skeletal muscle. J Gen Physiol 149:297-300
Moss, Richard L (2016) Cardiac myosin-binding protein C: A protein once at loose ends finds its regulatory groove. Proc Natl Acad Sci U S A 113:3133-5
Rosas, Paola C; Liu, Yang; Abdalla, Mohamed I et al. (2015) Phosphorylation of cardiac Myosin-binding protein-C is a critical mediator of diastolic function. Circ Heart Fail 8:582-94
Chen, Yi-Chen; Sumandea, Marius P; Larsson, Lars et al. (2015) Dissecting human skeletal muscle troponin proteoforms by top-down mass spectrometry. J Muscle Res Cell Motil 36:169-81
Moss, Richard L; Fitzsimons, Daniel P; Ralphe, J Carter (2015) Cardiac MyBP-C regulates the rate and force of contraction in mammalian myocardium. Circ Res 116:183-92
Tong, Carl W; Wu, Xin; Liu, Yang et al. (2015) Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or ?1-Adrenergic Stimulation. Circ Heart Fail 8:595-604
Theis, Jeanne L; Zimmermann, Michael T; Larsen, Brandon T et al. (2014) TNNI3K mutation in familial syndrome of conduction system disease, atrial tachyarrhythmia and dilated cardiomyopathy. Hum Mol Genet 23:5793-804
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

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