The primary goals of this project are (a) to provide the applicant with extensive training in muscle biochemistry, physiology, and biophysics, toward completion of a PhD thesis in the molecular biophysics of muscle aging, and (b) to prepare the applicant for the next step along the path to becoming and independent investigator. This training program focuses on a research project that is central to the sponsor's NIA-funded research on the aging of muscle. The specific goal of this research is to determine the effect of aging on force-dependent structural changes in the catalytic domain (CD) and light chain domain (LCD) of the myosin head in rat muscle fibers. The fundamental hypothesis for this research is that aging affects the structural coupling between the CD and LCD, disrupting the weak-to-strong (W-to-S) structural transition in myosin. This hypothesis will be tested by completing the following aims: (1) Develop improved techniques for detecting force-dependent structural changes in the two domains of myosin in rat skeletal muscle fibers. This includes development of procedures for site-directed spin labeling of the CD and LCD in rat semitendinosus fibers. Additionally, a novel EPR resonator, designed specifically for muscle, will be constructed and used to improve the efficiency, sensitivity, and orientational capabilities of muscle fiber EPR. (2) Determine the age-dependent effects of light-chain exchange on structural changes in the CD.
This aim emphasizes the W-to-S transition, and the simultaneous measurement of EPR and force to determine whether age-dependent changes in the CD can be reversed by RLC exchange. (3) Determine the age-dependent effects on structural changes in the LCD. In this case, RLC exchange will be used for labeling of the LCD, and the hypothesis is that age-dependent changes in CD structure will be propagated to the LCD and thus to the force-producing lever arm. By completing these three aims, the applicant will have directly tested hypotheses about the age-dependent coupling of structural changes in the two domains of myosin.
The primary significance of this work is to advance understanding of the molecular basis of muscle aging, which will provide the foundation for future clinical efforts. The technical advances in Aim 1 will have significance not only for the field of aging, but for muscle biophysics in general.
Aim 2 has implications for therapeutic approaches, since it will reveal whether youthful function and structure can be restored, at least in part, in aging muscle by the replacement of a single subunit of myosin.
| Mello, Ryan N; Thomas, David D (2012) Three distinct actin-attached structural states of myosin in muscle fibers. Biophys J 102:1088-96 |
