With the support of collaborators (Monzino hospital, Milan, Italy), we obtained the biopsy material of a patient affected by lamin cardiomyopathy and created stem cells which can be directed to develop into heart-specific cells. We also used a control line from a healthy donor. To generate the cardiac microtissues we first created individual cellular types that are most frequent in the heart, namely cardiomyocytes, fibroblasts and endothelial cells. First, we found that there was a strong reduction in Lamin AC in the nucleus of the patient cells, compared to the healthy control cells. We also noticed that the nuclei of different heart cell types were less round than in control conditions, suggesting that structural integrity is compromised in diseased conditions. In addition, the DNA, that is normally tightly packed around the nuclear borders helped by its anchoring to the Lamin AC protein, was instead in less compact state in the diseased cardiac cells. This effect is particularly evident in cardiomyocytes, which are the contracting cells in the heart and therefore subjected to mechanical stress. We also examined calcium cycling within the cell and nucleus, as calcium plays a central role in contraction. Our analysis revealed changes in calcium dynamics that suggest contractility is impaired in the mutated cells. To recreate the native environment of the heart, that is a three-dimensional (3D) organ composed of many different cell types, we built microtissues with the three cardiac cell types and saw that the traits previously observed in cardiomyocytes alone hold true also in cardiomyocytes within the 3D environment and are even augmented. The work done so far has laid the foundations to perform additional assessments related to the lamin mutations phenotype. To explore further the link between the changes in the DNA regions that are linked to the Lamin AC protein we will analyze the cardiac microtissues at the single cell level. Altogether, this work has created the lay ground to generate a disease platform that can be used not only for better understanding cardiac laminopathies, but also to identify novel therapeutic approaches.
