2 research outputs found

    Mapping Super-Relaxed States of Myosin Heads in Sarcomeres using Oblique Angle Fluorescent Microscopy

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    We have utilised modern methods of super-resolution fluorescent microscopy to spatially map fluorescently labelled ATP molecules in relaxed rabbit psoas skeletal muscles. For our imaging process, we have labelled ATP molecules with Rhodamine and Z-lines with Alexa488. Data from imaging these fluorophores have been collected using oblique angle fluorescent microscopy and further analysed to map super relaxed states (SRX) of myosin heads on the thick filament. Our experiments have concluded that most SRX of myosin heads were found in the C-zone of the thick filament, while other zones of thick filament had smaller populations of SRX. Further introduction of mavacamten (MAVA) to our imaging system has revealed an increase in SRX in both D and P zones, while the C zone population of SRX had remained constant. Further experiments must be conducted to establish a clear pattern and further proof our findings

    Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils

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    Muscle contraction is controlled at two levels: the thin and the thick filaments. The latter level of control involves three states of myosin heads: active, disordered relaxed (DRX), and super-relaxed (SRX), the distribution of which controls the number of myosins available to interact with actin. How these are controlled is still uncertain. Using fluorescently labeled ATP, we were able to spatially assign the activity of individual myosins within the sarcomere. We observed that SRX comprises 53% of all heads in the C-zone compared with 35% and 44% in the P- and D-zones, respectively. The recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten (mava), significantly decreased DRX, favoring SRX in both the C- and D-zones at 60% and 63%, respectively. Since thick filament regulation is in part regulated by the myosin-binding protein-C (MyBP-C), we also studied PKA phosphorylation. This had the opposite effect as mava, specifically in the C-zone where it decreased SRX to 34%, favoring DRX. These results directly show that excess concentrations of mava do increase SRX, but the effect is limited across the sarcomere, suggesting mava is less effective on skeletal muscle. In addition, we show that PKA directly affects the contractile machinery of skeletal muscle leading to the liberation of repressed heads. Since the effect is focused on the C-zone, this suggests it is likely through MyBP-C phosphorylation, although our data suggest that a further reserve of myosins remain that are not accessible to PKA treatment