Douglas M. Swank - US grants

This investigation will determine the function of a domain of the myosin heavy chain (MHC) that spans from the base of the head to the region where the light chains bind. This domain is likely important in the conformational change that causes the power stroke which results in muscle contraction. It also contains known mutation sites that cause familial hypertrophic cardiomyopathy (FHC), a leading cause of sudden death in young adults. The investigation will be accomplished by expressing a myosin heavy chain transgene in a Drosophila myosin-null mutant. The transgene will encode normal adult MHC isoforms, but will encode the embryonic form of the domain under study. This should result in quantitative changes in mechanical function because the contractile properties of adult fibers differ dramatically from embryonic muscles. The transgenic flies will be assayed for jump and flight ability. The indirect flight muscle (IFM) and tergal depressor of the trochanter muscle (TDT), a jump muscle, will be examined by electron and light microscopy for ultrastructure integrity. Myosin isolated from the animals will be assayed for ATPase activity and nucleotide binding. The ability to produce force and move actin filaments will be assessed by in vitro motility/force generation assays. Maximum velocity of shortening (Vmax) and force production will be measured on isolated IFM and TDT muscle fibers. The results of these assays will reveal the influence of this MHC region on isolated myosin properties and intact muscle function. Future site-directed mutant studies can then determine which amino acids are critical for this domain's function, and may provide insight into why some of these mutations lead to FHC and skeletal muscle central core disease.

[unreadable] DESCRIPTION (provided by applicant): The kinetic and structural mechanisms by which myosin converts the chemical energy of ATP hydrolysis to force and motion are far from being understood. Generally accepted myosin cross-bridge theories have Pi release associated with the force producing power stroke, but steps associated with ADP release rate are thought to be rate limiting for unloaded shortening velocity. However, recent evidence from myosin molecular studies suggests that ADP release is not the only step of the cycle that can influence unloaded myosin shortening velocity. Further, kinetic studies of Drosophila myosins and flight muscle fibers suggest that fast Drosophila myosins may be limited by steps associated with Pi release rather than ADP release rate. Therefore, the KINETIC HYPOTHESIS to be tested is that the unloaded velocity of very fast myosins is limited by steps associated with Pi release while slow myosin velocities are limited primarily by ADP release rate. SPECIFIC AIMS: (1) Test our kinetic hypothesis at the molecular level by varying ATP, Pi and ADP levels in the in vitro sliding filament assay. We will contrast the effect on velocity for two very fast adult Drosophila isoforms compared with two slow embryonic isoforms. (2) Test if Pi release limits unloaded velocity at the fiber level by varying ATP, Pi and ADP levels in the bathing solution of skinned TDT (jump) muscle transgenically expressing the four myosin isoforms. (3) Determine which structural region sets shortening velocity of myosin isoforms. Our STRUCTURAL HYPOTHESIS is that the myosin converter region is primarily responsible for setting differences in unloaded velocity. We will test this hypothesis by performing the same molecular and fiber experiments as described in Aims 1 and 2 on two myosin chimeras previously made by exchanging converter regions between a very fast and a very slow myosin isoform. Significance: Depending on the validity of our kinetic hypothesis, we will either be determining which region influences ADP release or which region influences Pi release. The latter would be highly significant as regions of myosin that set Pi release rate have not been identified. In either case, information on the function of the converter region will be highly informative as the converter is a hotspot for mutations that lead to familial hypertrophic cardiomyopathy (FHC). FHC is an inherited genetic disease that is a major cause of sudden death among young adults. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): 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 cardiomyopathy (DCM). FHC is an inherited genetic disease that is a major cause of sudden death among young adults. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Stretch activation (SA) is an intrinsic sarcomeric property that increases muscle force, power and economy. SA is most prominent in muscle types that rhythmically lengthen and shorten such as cardiac and insect flight muscle. Rapidly increasing the length of a muscle with significant SA causes a delayed jump in force above calcium activated force. The mechanisms behind this force increase are not known, thus limiting our understanding of a fundamental muscle property. Our long-term goal is to apply insights gained from learning how SA mechanisms modulate force, power and muscle efficiency to help devise ways to restore impaired muscle function. The immediate objective of this application is to elucidate the sarcomeric mechanisms behind SA. Our central hypothesis is that SA can be caused by any sarcomeric mechanism that increases the total number of strongly bound cross-bridges following stretch. We propose that there are at least two mechanisms by which this occurs. In moderately SA muscle types the increase occurs by a myosin based mechanism, while a thin filament mechanism is required in highly SA muscle types. Our hypotheses are based on our novel preliminary data that a myosin isoform exchange increases SA force generation in a minimally SA muscle type to be equivalent to a moderately SA muscle type, and that a specific troponin C isoform, TnC4, is necessary for SA in highly SA insect flight muscle. These findings were made possible by our development of a new Drosophila muscle fiber preparation, the jump muscle, which allows us to look for gain of SA function and not just loss of SA function in the IFM. Specific aim 1 is to determine the kinetic and structural mechanisms by which some myosin isoforms enhance SA force production. Our working hypothesis is that for SA myosin isoforms, muscle stretch increases their probability of temporarily rejoining other cross-bridges in a low force state, thus increasing the number of cross-bridges available to subsequently transition into a high force, actin bound state. We will test our hypotheses that Pi affinity and different versions of the myosin relay helix are critical for changing the sensitivityof myosin to stretch. Aim 2 is to determine mechanisms by which thin filament proteins contribute to SA. We will test our working hypothesis that TnC isoforms in SA muscle types do not fully activate the thin filament upon calcium binding compared to TnC isoforms from muscles with minimal SA. This allows for further activation of the thin filament by stretch. We will test the hypothesis that further activation occurs by direct physical movement of troponin and tropomyosin by troponin bridges which span the thick and thin filaments. The proposed research is significant because it will provide a detailed understanding of the kinetic and structural mechanisms by which myosin, TnC, and other muscle protein isoforms enable SA. We will gain new insights into fundamental mechanisms by which force, power, and energetics are modulated in different muscle types. Elucidating SA mechanisms may lead to ways of restoring or improving muscle function.