Advanced Morpho-Functional Analysis on Ventricular and Atrial Tissue Reveals Cross-Bridge Kinetics Alterations and Sarcomere Energetic Impairment in Hcm Patients

Mutations in cardiac myosin-binding protein-C (cMyBP-C), are the most common cause of Hypertrophic CardioMyopathy (HCM). The E258K-cMyBP-C is a penetrant missense mutation with poorly understood molecular mechanisms. Mechanics and kinetics of contraction as well the energy cost of tension generation were investigated using left ventricular (LV) and atrial tissue from E258K HCM patients and donor hearts. Kinetics of tension generation and relaxation were measured in LV and atrial myofibrils while ATPase and isometric active tension were simultaneously measured in permeabilized LV and atrial strips. The rate of tension generation following maximal Ca2+-activation was faster in both LV and atrial E258K myofibrils compared to donors. The rate of isometric relaxation was also faster in E258K myofibrils, suggesting faster cross-bridge detachment and increased energy cost of tension generation. Direct measurements in skinned LV and atrial strips confirmed that tension cost was higher in E258K preparations compared to controls. To check whether cardiomyocyte disarray, typical of HCM hearts, may have contributed to artificially increase the tension cost measured in the HCM preparations, the strips used for mechanical investigations were clarified, immunostained and imaged at mesoscale level. An advanced tissue clearing method in combination with two-photon microscopy was employed to reconstruct the 3D image of the strips at sub-micrometer spatial resolution. A 3D cytoarchitecture analysis tool based on 3D Fourier Transform was developed and applied to determine cardiomyocyte orientation across and along the strips. Both global and local statistics of spatial disarray were derived and correlated to mechanical and energetic data. The results did not highlight structural differences between donor and HCM strips strengthening the conclusion that the E258K mutation primarily alters apparent cross-bridge kinetics and impairs sarcomere energetics. Acknowledgement: H2020EU SILICOFCM grant agreement 777204.