Optimisation of ATPase assay for the measurement of myosin isoform specific skinned fibers in murine skeletal muscle

Skeletal muscle constitutes a significant portion of the human body and plays a pivotal role in overall basal metabolism. While the impact of muscle tissue on metabolism during intense exercise is commonly considered, recent attention has focused on the energy consumption of resting muscles due to its substantial energetic potential. The discovery of an energy-saving mechanism within skeletal muscle myosin and its potential pharmacological modulation has garnered interest. This mechanism, termed the Super Relaxed State (SRX) of myosin, presents an opportunity to induce modest yet impactful metabolic changes in energy balance, particularly during prolonged periods of inactivity, given the abundance of muscle tissue in the body. However, the study of SRX within muscle physiology and pathology has been limited. This thesis explores the adaptation and application of an assay used in our laboratory to render it compatible and functional with murine skeletal muscle fibers. The murine model offers a valuable platform for investigating various pathologies, including Amyotrophic Lateral Sclerosis (ALS). In our lab, a single fiber ATPase assay has been developed on rabbit muscle tissue. In thi work, I will explore the technical enhancement of this assay to allow the measurement of murine skinned muscle fibers. Our findings suggest that KH2PO4 marginally enhances the extraction process, while further investigation is necessary to determine the effectiveness of Latrunculin-b (lat-b) in depolymerizing actin under the desired conditions.