Morpho-functional investigation of electro-mechanical dysfunction in cardiac hypertrophy by advanced optical methods and tissue transformation

During my PhD, I performed functional and structural investigations on the heart; I optimized clearing and staining protocols on cardiac tissue, and I exploited advanced imaging techniques to perform high-resolution studies working at two different projects. In the first project, the energetics of demembranated multicellular cardiac muscle strips were investigated from three Hypertrophy Cardiomyopathy (HCM) patients with the E258K mutation. Energetic measurements in multicellular preparations may suffer from artefacts, related to the density and the orientation of the contractile material. Alterations of cardiomyocytes organization or disarray, a common HCM histopathological feature at whole heart level, may decrease isometric tension while increasing the isometric ATPase of multicellular preparations, thus leading to an artificial increase in tension cost (TC). To exclude this hypothesis, a new protocol that combines a novel tissue clearing technique, previously used to clear the mouse brain 1, and an advanced optical microscopy, two-photon fluorescence microscope (TPFM), was developed. With this approach it was possible to perform a three-dimensional (3D) cytoarchitecture analysis of the myofibril orientation, with a micron-scale resolution, on a subset of human ventricular strips previously used for mechanical and energetical experiments. In the second project, action potential propagation of entire hearts was investigated from a transgenic mouse model of HCM with a mutation on the gene coding for cardiac Troponin T (cTnT). The wide field system used to perform this investigation has already been utilized to study electrical activity 2. Subsequently, on the same hearts employed for functional studies, clearing techniques were applied to make the tissue transparent and to homogenize the refractive index 3. This isnecessary to label entire samples with fluorescent proteins, used to visualize cardiac fiber structure, and to execute high resolution imaging with light-sheet microscopy for large volume acquisitions. Finally, cytoarchitecture analysis based on cardiomyocytes and myofilaments alignment was applied to correlate electro-mechanical dysfunction with structural alterations. These innovative experimental approaches will allow to dissect the morphological causes leading to alterations of electrical conduction and to electro-mechanical dysfunction, and, more generally, will represent a whole new paradigm for diagnostic and therapeutic investigations.