Myosin is the molecular motor in skeletal muscle thick filaments that binds to actin-containing thin filaments to produce mechanical force and displacement. All vertebrates express a multigene family of the two myosin subunits; myosin heavy chain (MHC) and myosin light chain (MLC). Variations in expression of both MHC and MLC isoforms is known to be a primary determinant of a muscle's mechanical properties. However, the influence of MHC and MLC isoforms on the various parts of the force-velocity relationship remains controversial and has never been determined in intact muscle cells. In addition, individual fibers are often found to express more than one isoform of MHC. The mechanical consequences, and hence the functional purpose of this coexpression, is virtually unknown. Frog muscle provides an excellent model for studies of myosin structure/function because it is the only organism from which single intact """"""""living"""""""" fibers can be isolated that retain complete mechanical stability. This mechanical stability permits high resolution measurements of mechanical function, sarcomere length transients and myosin cross-bridge kinetics. Studies of myosin structure/function in frog single fibers have been limited to date because of inadequate definition of MHC and MLC isoforms. Recently, we have cloned four novel adult frog MHCs, identified the four corresponding protein isoforms and established their expression pattern in individual muscle cells. In this proposal we will use a similar approach (including molecular cloning) to identify and determine the expression patter of MLCs in the various fiber types. This precise identification of both MHC and MLC isoforms across the full range of fiber types will allow us to complete the following two major aims of the proposal: (1) A precise correlation will be established between MHC and MLC isoform content and mechanical function of single intact frog muscle fibers. Single muscle fibers will be isolated from adult Rana pipiens and the force-velocity properties will be measured within 1 mm segments along the full length of the fiber. Following the mechanics experiments, the MHC and MLC isoform composition will be quantified in each segment using SDS-PAGE. This analysis will include a determination of how coexpression of multiple isoforms of MHC and MLC in individual fibers affects mechanical performance. (2) We will determine how MHC and MLC isoforms influence mechanical function under conditions experienced during normal locomotion. Single frog fibers will driven through in vivo fiber length excursions and stimulus conditions that occur during jumping while measuring force production and sarcomere length transients. This paradigm will improve our understanding of how slow and fast fiber types function in the generation of stereotypical ballistic movements. Altered myosin isoform expression patterns occur in response to muscle disuse/overuse, direct trauma, denervation and aging. A fundamental understanding of how MHC and MLC isoforms influence mechanical function in an intact cellular environment will be useful for understanding potential consequences of these altered phenotypes. Further, a fundamental understanding of how contractile function is influenced by coexpression and non-uniform expression of myosin isoforms along the length of fibers is important, because a majority of fibers in a given muscle may coexpress multiple myosin isoforms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR045631-02
Application #
6171168
Study Section
Respiratory and Applied Physiology Study Section (RAP)
Program Officer
Lymn, Richard W
Project Start
1999-09-01
Project End
2002-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
2
Fiscal Year
2000
Total Cost
$230,749
Indirect Cost
Name
Veterans Medical Research Fdn/San Diego
Department
Type
DUNS #
933863508
City
San Diego
State
CA
Country
United States
Zip Code
92161
Robinson, D A; Bremner, S N; Sethi, K et al. (2005) In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle. Gene Ther 12:347-57
Lutz, Gordon J; Sirsi, Shashank R; Shapard-Palmer, Sarah A et al. (2002) Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens. Am J Physiol Cell Physiol 282:C835-44
Lutz, Gordon J; Lieber, Richard L (2002) Studies of myosin isoforms in muscle cells: single cell mechanics and gene transfer. Clin Orthop Relat Res :S51-8
Lutz, G J; Bremner, S N; Bade, M J et al. (2001) Identification of myosin light chains in Rana pipiens skeletal muscle and their expression patterns along single fibres. J Exp Biol 204:4237-48