A unique feature of striated muscle is its enormous diversity of fiber types. Much of the functional diversity is due to a range of myosin isoforms that help determine muscle speed. Sequence comparisons and functional studies at the molecular level have suggested specific structural domains of the myosin heavy chain modulate kinetic steps of the crossbridge cycle that presumably affect muscle speed. We will exploit unique features of the Drosophila system for investigating relationships between isoform differences in myosin head structure and muscle kinetics. A major advance will be the use of myofibrillar preparations that allow precise manipulation of sarcomere length and ion concentrations (calcium, MgATP, MgADP and phosphate), from which we can deduce kinetic constants of the acto-myosin cross-bridge cycle with high precision. The research addresses 3 basic questions: (i) what step(s) of the crossbridge cycle establishes the major kinetic differences between a very fast muscle and a very slow muscle? (ii) do fiber types with intermediate speeds have crossbridge rate constants between those of the extremes? and (iii) what steps of the myosin crossbridge cycle are affected by changes in specific variable region(s) between myosin isoforms? To answer these questions (framed as hypotheses), we will contrast kinetic schemes obtained from myofibrils of indirect flight muscle (IFM, an extremely fast muscle), jump muscle (a fast muscle), IFM myofibrils transgenically expressing myosin from embryonic body wall muscle (EMB, a very slow muscle), and IFM myofibrils expressing chimeras of IFM and EMB myosin. By correlating kinetic differences to specific structural regions (including the converter region), we will deduce molecular mechanisms based on the latest structural models of myosin. The major strengths of this investigation are the fully integrated approach (from single molecules to whole animal) made possible by employing Drosophila, and the use of myofibrils which bridges a gap between experiments with isolated myosin and skinned fiber experiments.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR049425-04
Application #
7116918
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Nuckolls, Glen H
Project Start
2003-08-01
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2008-07-31
Support Year
4
Fiscal Year
2006
Total Cost
$240,402
Indirect Cost
Name
University of Vermont & St Agric College
Department
Physiology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Tanner, Bertrand C W; Farman, Gerrie P; Irving, Thomas C et al. (2012) Thick-to-thin filament surface distance modulates cross-bridge kinetics in Drosophila flight muscle. Biophys J 103:1275-84
Miller, Mark S; Dambacher, Corey M; Knowles, Aileen F et al. (2009) Alternative S2 hinge regions of the myosin rod affect myofibrillar structure and myosin kinetics. Biophys J 96:4132-43
Yang, Chaoxing; Ramanath, Seemanti; Kronert, William A et al. (2008) Alternative versions of the myosin relay domain differentially respond to load to influence Drosophila muscle kinetics. Biophys J 95:5228-37
Swank, Douglas M; Vishnudas, Vivek K; Maughan, David W (2006) An exceptionally fast actomyosin reaction powers insect flight muscle. Proc Natl Acad Sci U S A 103:17543-7
Maughan, David; Vigoreaux, Jim (2005) Nature's strategy for optimizing power generation in insect flight muscle. Adv Exp Med Biol 565:157-66; discussion 167, 371-7