Using Novel Techniques in Fluorescent Microscopy to Investigate the Energy Consumption of HCM-Related Proteins in Striated Muscles

Hypertrophic Cardiomyopathy (HCM) affects 1 in 500 people worldwide and in severe cases can be a cause of sudden cardiac death. This genetic condition can be linked to protein mutations that regulate the energy consumption in cardiac muscle tissues. To develop a treatment, one must identify the underline cause and correct it. Unfortunately, many current treatments, like β-blockers, involve medications treating the symptoms but not underlying conditions and thus a broader and deeper understanding of the mechanism by which muscles suffer HCM is needed. This thesis was focused around sarcomeric proteins, mutations in which, were shown to be the main causes of HCM. Furthermore, an investigation was conducted into multiple novel small molecule myosin ATPase inhibitors that were developed as a promising treatments for HCM. The mechanism of action of these novel treatments was focus on the correction of misregulation of myosin heads ATP consumption where a more ATP consuming disordered relaxed state (DRX) was transferred into a more energy-saving super relaxed state (SRX). To evaluate the effects of abovementioned pharmaceutical agents, a single-molecule approach was utilised to monitor the ATP consumption of different muscle tissues. Firstly, a methodology was established to image fast-twitch skeletal muscles (rabbit psoas) and evaluated both the effects of phosphorylation on HCM-related protein, myosin binding protein-C (MyBP-C), and the effect of a small molecule myosin inhibitor Mavacamten on ATPase rates of Cy3ATP in relaxed psoas tissue. Following the PKA treatment there was an observed increase in myosin activity and a reduction in the number of active myosin heads following the Mavacamten treatment. The effects of Mavacamten on skeletal tissue were limited even at higher, fully saturated, concentrations thus showing the cardiac specificity of Mavacamten. The study of skeletal tissue was followed up with a more clinically relevant study of porcine cardiac left ventricle (β-myosin). After an initial optimisation, the effects of phosphorylation were established on both regulatory light chain (RLC) and cardiac myosin binding protein C (cMyBP-C), mutations in which together account for ~75 % all HCM cases. This work was the first single-molecule imaging for ATPase activity for β-myosin thus providing an interesting insight into the energy consumption of ventricular muscle. Finally, the method was developed for imaging multiple samples quasi-simultaneously using the bulk fluorescence imaging approach, for imaging of large quantities of patient or model organism data. In this method, a self-developed moving stage was employed to locate and move though different samples (cardiomyocytes), which could be contained in separate buffer solutions. Unfortunately, due to the time limitations, the only reliable dataset produced was that of porcine cardiac muscle and not of mutated and/or patient tissues as was initially planned. Using this novel approach, two novel small molecule myosin ATPase inhibitors Aficamten and Mavacamten were assessed, along with the myosin activator 2’-deoxy-ATP.