Age-Dependent Regulation of Excitation-Contraction Coupling

Wake Forest University Health Sciences, Winston-Salem NC

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Abstract

DESCRIPTION (provided by applicant): This project's goal is to elucidate the mechanisms responsible for the decline in skeletal muscle performance with aging. We propose that JP-45 regulates SR Ca2+ release elicited by DHPR-RyR1 coupling and that the JP-45/DHPR11 complex is crucial to the age-dependent decrease in muscle specific force. Hypotheses: The specific aims will test the hypotheses that JP-45 plays a role in DHPR11 subunit expression and that it's age-dependent down regulation leads to decreased DHPR11 expression, contributing to the decline in SR Ca2+ release and specific force in skeletal muscle fibers from senescent mice. Aim 1. To establish that JP-45 KO leads to decreased DHPR11 subunit expression by increasing its rate of degradation in skeletal muscle from adult mice. We have previously demonstrated that JP-45 KO leads to decreased DHPR11 subunit expression, charge movement, SR Ca2+ release, and muscle fiber contraction force. However, the mechanism is unknown. Here, we propose that JP-45 down regulation or ablation results in increased 11 subunit degradation meditated by an ubiquitin-proteasome proteolytic pathway. Aim 2. To determine that age-dependent down regulation of JP-45 expression contributes to excitation- contraction uncoupling (ECU) in senescent mice. These experiments will test the hypothesis that JP45 is downregulates with senescence, leading to decreased DHPR11 expression and ECU. We created a JP-45- viral construct targeted exclusively to skeletal muscle, which can either prevent or restore age-dependent down regulation of JP-45 and, hence, DHPR11 down regulation, rescuing the aging ECC phenotype. Aim 3. To demonstrate that age-dependent decrease in JP-45 expression contributes to reduced single intact muscle fiber specific force without affecting contractile myofilament sensitivity to Ca2+. In the same single fiber, first intact and then skinned, we will test voltage-dependent and independent properties, respectively, to answer the following questions: (1) Can JP-45 rescue age-dependent loss in contraction specific force? (2) Can it rescue decreases in peak intracellular Ca2+ mobilization with aging? (3) Is the decrease in fiber specific force a consequence of changes in contractile myofilament sensitivity to Ca2+? Aim 4. To demonstrate that JP-45 gene transcription declines with aging, that insulin-like growth factor-1 (IGF-1) regulates JP-45 gene transcription and that IGF-1-induced prevention or restoration of the decline in DHPR 11 subunit expression in aging skeletal muscle requires JP-45. We propose that: (1) JP-45 gene transcription decreases with aging, (2) IGF-1 regulates JP-45 gene transcription, and (3) IGF-1- induced 11 subunit upregulation is precluded by age-dependent JP-45 down regulation and JP-45 KO, supporting the theory that JP-45 protects 11 subunits from poly-ubiquitination and proteasome degradation. We will test this concept using S1S2, a transgenic mouse model that over-expresses IGF-1 in skeletal muscle, crossbred with JP-45KO mice or an IGF-1bidir viral vector.