Developmentally regulated and structurally distinct protein isoforms self-asssemble into thick and thin filaments in the cardiac sarcomere. The properties and interactions of these filaments will be investigated through biophysical and biochemical methods to comprehend the interplay of dynamic forces that control and produce cardiac contraction. The role of the regulatory light chain, LC2, in cardiac myosin ATPase will be further analyzed and its relationship to C-protein determined using HMM to complement the work done with myosin. Actin- activated ATPase assays will also be performed using rat and dog heart myosin subfragments to delineate the kinetic parameters that differentiate these myosin isoforms. Pure and regulated actin will be used in binding experiments with S1 and HMM and in kinetic analysis of ATP hydrolysis to clarify the factors involved and the mechanism that regulate actomyosin interactions in the heart. These studies will be executed under a number of ionic conditions and varying concentrations of calcium, as a function of LC2 and troponin phosphorylation. To provide the structural correlates to the results obtained from activity and binding measurements, the structure of cardiac myosin will be probed by monoclonal antibodies raised against the hinge and the functional domains in S1. Such an approach will permit a direct test of the involvement of the S2 region in force generation in the myocardium and identification of the actin-binding, light chain- binding and ATPase domains within the myosin heads. Also, cardiac TnT (the tropomyosin-binding subunit of troponin (will be isolated, its shape determined by electron microscopy and cleaved into discrete fragments, T1 and T2, either by a-chymotrypsin or cyanogen bromide. The functional characteristics of each fragment with respect to tropomyosin and other troponin subunits will be analyzed from the crystalline and paracrystalline aggregtes they generate. Furthermore, the reasons for the observed impairment in regulation in myopathic hamsters will be studied by analyzing the structure of the regulatory factor complex with particular attention to the integrity of TnT and the ability of troponin to bind calcium. Removal of cardiac LC2 by the myopathic hamster protease was shown to destabilize thick filament structure. This, together with the results from analysis of the regulatory factor function may provide a molecular explanation for cardiomyopathy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL026569-08
Application #
3338662
Study Section
Cardiovascular Study Section (CVA)
Project Start
1981-04-01
Project End
1990-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
8
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Montefiore Medical Center (Bronx, NY)
Department
Type
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10467
Margossian, S S; White, H D; Lefford, J et al. (1993) Functional effects of LC1-reassociation with cardiac papain Mg.S1. J Muscle Res Cell Motil 14:3-14
Margossian, S S; Hatcher, V B; Taylor, S (1993) Kinetics of hydrolysis of cardiac S1 heavy chain isoforms and identification of light chain and actin binding sites. Cardiovasc Res 27:216-21
Margossian, S S; White, H D; Caulfield, J B et al. (1992) Light chain 2 profile and activity of human ventricular myosin during dilated cardiomyopathy. Identification of a causal agent for impaired myocardial function. Circulation 85:1720-33
Margossian, S S; Krueger, J W; Sellers, J R et al. (1991) Influence of the cardiac myosin hinge region on contractile activity. Proc Natl Acad Sci U S A 88:4941-5
Margossian, S S; McPhie, P; Howard, R J et al. (1990) Physical characterization of histidine-rich protein from Plasmodium lophurae. Biochim Biophys Acta 1038:330-7
Margossian, S S; Sellers, J R; Watkins, S C et al. (1989) Formation of new quasi-crystalline ordered aggregates by gizzard myosin. J Muscle Res Cell Motil 10:413-26
Margossian, S S; Huiatt, T W; Slayter, H S (1987) Control of filament length by the regulatory light chains in skeletal and cardiac myosins. J Biol Chem 262:5791-6
Margossian, S S; Slayter, H S (1987) Electron microscopy of cardiac myosin: its shape and properties as determined by the regulatory light chain. J Muscle Res Cell Motil 8:437-47
Hatcher, V B; Fadl-Allah, N; Levitt, M A et al. (1986) Isolation and partial characterization of endothelial cell extracellular complexes. J Cell Physiol 128:353-61
Malhotra, A; Margossian, S S; Slayter, H S (1986) Physico-chemical properties of rat and dog cardiac alpha-actinin. Biochim Biophys Acta 874:347-54

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