The kinetic and structural mechanisms by which myosin and other motor proteins convert the chemical energy of ATP hydrolysis into force and motion are far from being understood. In generally accepted theories of myosin cross-bridge function, Pi release is associated with the force-producing power stroke, but steps associated with MgADP release rate are thought to be rate limiting for unloaded muscle shortening velocity and oscillatory work production. However, recent evidence suggests that MgADP release is not the only step of the cycle that influences muscle shortening velocity, and our recent data suggest the optimal frequency of oscillatory work production by very fast Drosophila myosin is set by the Pi release rate rather than the MgADP release rate (Swank et al., 2006). Therefore, we will test our KINETIC HYPOTHESIS that unloaded velocity and oscillatory work production by very fast myosins are limited by steps associated with Pi release while slower myosins are limited by steps associated with MgADP release rate.
SPECIFIC AIMS : (1) Test our kinetic hypothesis for oscillatory work production by varying MgATP, Pi and MgADP levels in indirect flight muscle (IFM) transgenically expressing four Drosophila myosin isoforms, which vary 9-fold in velocity, to determine critical cross-bridge rate constants including the rate limiting step. (2) Test if Pi or ADP release limits unloaded velocity at the fiber level by varying MgATP, Pi and MgADP levels in the bathing solution of skinned Drosophila jump muscle transgenically expressing the same four myosin isoforms. Our kinetic hypothesis will also be tested at the molecular level using the actin sliding filament assay. (3) Test our STRUCTURAL HYPOTHESIS that the myosin converter is the primary region responsible for determining cross-bridge rate constant values critical for setting the shortening velocity of myosin isoforms. We will test this hypothesis by performing the same molecular and fiber experiments as described in Aims 1 and 2 on myosin chimeras made by replacing the IFM myosin converter with the other 4 native versions of the Drosophila converter region. (4) Test our MECHANISTIC HYPOTHESIS that the degree of hydrophobicity in the converter is critical to its function by substituting amino acids that decrease the IFM myosin isoform's hydrophobicity. SIGNIFICANCE: We will determine how the converter region influences MgADP release and/or Pi release. This will be highly significant as very little is known about the structural mechanisms by which motor proteins set Pi and MgADP release rates. Details about the converter's mechanism for setting velocity will help test recent hypotheses regarding how at least 8 different mutations in the converter cause either familial hypertrophic cardiomyopathy (FHC) or dilated cardiaomyopathy (DCM). FHC is an inherited genetic disease that is a major cause of sudden death among young adults. Swank, D.M. Project Narrative By studying the mechanics and biochemistry of the molecular motor myosin, which powers heart muscle contraction, we will learn how mutations in myosin cause two types of heart disease, familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DCM). FHC is the leading cause of sudden death in athletes and young adults (Morita et al., 2005). In contrast, DCM results in heart failure through a loss of muscle mass from the heart and a detrimental increase in heart volume.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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Skeletal Muscle and Exercise Physiology Study Section (SMEP)
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Boyce, Amanda T
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Rensselaer Polytechnic Institute
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Eldred, Catherine C; Katzemich, Anja; Patel, Monica et al. (2014) The roles of troponin C isoforms in the mechanical function of Drosophila indirect flight muscle. J Muscle Res Cell Motil 35:211-23
Wang, Qian; Newhard, Christopher S; Ramanath, Seemanti et al. (2014) An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight. J Exp Biol 217:290-8
Koppes, Ryan A; Swank, Douglas M; Corr, David T (2014) A new experimental model to study force depression: the Drosophila jump muscle. J Appl Physiol (1985) 116:1543-50
Zhao, Cuiping; Swank, Douglas M (2013) An embryonic myosin isoform enables stretch activation and cyclical power in Drosophila jump muscle. Biophys J 104:2662-70
Eldred, Catherine C; Naber, Nariman; Pate, Edward et al. (2013) Conformational changes at the nucleotide site in the presence of bound ADP do not set the velocity of fast Drosophila myosins. J Muscle Res Cell Motil 34:35-42
Swank, Douglas M (2012) Mechanical analysis of Drosophila indirect flight and jump muscles. Methods 56:69-77
Ramanath, Seemanti; Wang, Qian; Bernstein, Sanford I et al. (2011) Disrupting the myosin converter-relay interface impairs Drosophila indirect flight muscle performance. Biophys J 101:1114-22
Wang, Qian; Zhao, Cuiping; Swank, Douglas M (2011) Calcium and stretch activation modulate power generation in Drosophila flight muscle. Biophys J 101:2207-13
Clark, Kathleen A; Lesage-Horton, Heather; Zhao, Cuiping et al. (2011) Deletion of Drosophila muscle LIM protein decreases flight muscle stiffness and power generation. Am J Physiol Cell Physiol 301:C373-82
Purcell, Thomas J; Naber, Nariman; Franks-Skiba, Kathy et al. (2011) Nucleotide pocket thermodynamics measured by EPR reveal how energy partitioning relates myosin speed to efficiency. J Mol Biol 407:79-91

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