Role of ryanodine receptor dysfunction after spinal cord injury

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Muscles paralyzed by spinal cord injury (SCI) atrophy extensively, are weaker, fatigue more quickly and generate lower specific tension (force produced per unit of cross-sectional area), such that they generate less force in response to maximal motor neuron activation. For individuals with motor-incomplete lesions, interventions that improve specific force and/or fatigue resistance would be obvious opportunities to improve function. A newly recognized cause of impaired specific force production is oxidation/nitrosylation of ryanodine receptors (RyR) which results in dissociation of calstabin and spontaneous opening of RyR thereby impairing RyR gating; the ultimate physiological effect of RyR dysfunction is diminished force of muscle contraction and lower muscle endurance. Small molecules such as S107 bind oxidized/nitrosylated RyR and improve specific force by as much as 50%. Our preliminary data demonstrates extensive oxidation/nitrosylation of RyR in muscle after SCI thus implicating RyR dysfunction in reduced specific force production and endurance of paralyzed muscle after SCI. These changes in RyR are associated with increased expression of NADH oxidase 4 (Nox4) which is a potent source of reactive oxygen species (ROS) that has been linked to RyR. A direct link between Nox4 and oxidation/nitrosylation of RyR is supported by findings that binding of Nox4 to RyR is also increased in muscle after SCI. The overarching objectives of this application are to test the possibility that administration of S107 or a Nox4 inhibitor after SCI will improve skeletal muscle specific tension and endurance, and hence function, and to investigate the role of Nox4 in oxidation/nitrosylation of RyR in skeletal muscle after SCI. Aim 1. To determine the role of elevated Nox4 expression in the oxidation/nitrosylation of RyR in muscle after spinal cord transection. Hypothesis: oxidation/nitrosylation of RyR after SCI results from increased expression and activity of Nox4. Approach. Aim 1A. We will compare SCI and sham-operated groups over time after a spinal cord transection at various times between 1 and 56 days after SCI. We will examine the temporal relationships between oxidation/nitrosylation of RyR and dissociation of calstabin from RyR with changes in Nox4 expression. Aim 1B: Effects of a conditional knockout of Nox4 in skeletal muscle on muscle strength, specific force, fatigue, RyR oxidation after SCI will be determined. Aim 1C: We will compare muscle force production, specific force, fatigue, RyR oxidation/nitrosylation and binding of RyR to calstabin between spinal cord transected mice treated with a Nox4 inhibitor or vehicle. Aim 2) To test the effects of S107 on muscle force production and RyR-calstabin binding interactions after spinal cord transection. Hypothesis: administration of S107 will improve muscle specific force and endurance and increase calstabin binding to RyR but will not alter RyR oxidation/nitrosylation. Approach: We will compare muscle specific force, endurance, binding of calstabin to RyR, and RyR oxidation/nitrosylation between SCI groups administered S107 or vehicle. Aim 3. To A) determine whether S107 or a Nox4 inhibitor improves functional recovery after a contusion SCI. Hypothesis: S107 or a Nox4 inhibitor improves functional recovery in a graded contusion model of mid-thoracic SCI in mice. Approach: Following a mild, moderate or severe contusion spinal cord injury, mice will be treated with the most effective drug (either S107 or GKT137831) from Aims 1 and 2 or vehicle. Effects of the treatment on locomotor function and gait will be determined. Expected Outcomes and Benefits to Veterans. Knowledge gained from such studies may result in new pharmacologic or rehabilitation treatments to improve function after SCI.