Probing relaxed myosin states in hypertrophic cardiomyopathy by second harmonic-generation microscopy

In this thesis, we developed a label-free imaging method based on Second-Harmonic Generation microscopy in polarization (pSHG) to probe and quantify distinct conformations of myosin in relaxed muscle. Specifically, we investigated whether pSHG is sensitive enough to distinguish between the disordered-relaxed (DRX) and super-relaxed (SRX) states—two functionally distinct configurations that regulate myosin’s availability and energy consumption. This conformational balance is often disrupted in cardiac diseases such as human hypertrophic cardiomyopathy (HCM), contributing to mechanical and energetic dysfunction. We validated the sensitivity of pSHG using pharmacological agents—deoxyATP and Mavacamten—to shift the DRX/SRX equilibrium. We then applied this technique to a minipig model carrying the R403Q mutation, associated with HCM. Our results show that this mutation increases the population of myosin in the DRX state and enhances intrinsic ATPase activity. By combining pSHG structural data with energetic measurements, this work offers new insights into the molecular mechanisms underlying sarcomeric cardiomyopathies and highlights SHG microscopy as a valuable tool for probing myosin structural dynamics in intact muscle.