Engineered heart tissues to investigate the role of mechanical loading and injury in cardiomyocyte proliferation

Myocardial infarction is one of the most severe acute pathologies of the cardiovascular system. The adult mammalian heart is indeed unable to regenerate most of the lost cardiomyocytes (CMs) after cardiac injury. The loss of cardiomyocytes and the myocardial scarring after myocardial infarction eventually compromise contractility of the remaining myocardium, leading to heart failure. Therefore, promoting heart regeneration is one of the most crucial therapeutic targets in cardiovascular medicine. The lack of regenerative response is due to the loss of proliferative capacity of adult CMs which in mice occurs seven days after birth. One of the events which occur at birth in neonatal hearts is a sudden increase in mechanical loading that may contribute to switching mammal CMs phenotype from neonatal proliferative to adult postmitotic. Therefore, understanding the role of mechanotransduction in regulating the balance between CM proliferation and maturation may bring us to the identification of unknown mediators and new potential strategies to induce cardiac regeneration. Regulation of mechanical load in bi-dimensional cultures of CMs can be achieved in different ways, however, the poor degree of CM maturation that can be reached in a culture dish together with the lack of a tridimensional structure represent a major limitation to performing mechanotransduction studies. In our work we developed a novel system to study mechanotransduction of CMs based on 3D culture of cardiac cells, called engineered heart tissues (EHTs), that allow us to reduce or increase mechanical loading easily. We show that the three-dimensional setting of the culture leads to an improvement of CM maturation that may be reversed by mechanical unloading inducing cell proliferation. On the other hand, a persisting overload stimulus eventually induces CM switch to a more mature phenotype with a low degree of proliferation. Also, we have focused our work on developing an EHT-based model able to recapitulate the adult infarct injury in order to investigate the biology of cardiac regeneration in this setting. Specifically, we set up a cryoinjury protocol that is relatively easy and reproducible. Cryoinjury produces a localized injury without compromising EHT’s structural integrity. Indeed, all the EHTs subjected to cryoinjury preserved their contractile activity and did not show any significant change in shape. Considering that EHTs are unpurified cardiac culture rich in fibroblast and endothelial cells, we observed that cryoinjury induce fibroblast proliferation and activation together with a lack of proliferative response of the cardiomyocytes which is, on the other hand, present in the early phase of EHT’s development, similarly to what has been shown in mice and rats after myocardial infarction, highlighting the robustness of our cryoinjury approach as a model to investigate cardiac regeneration.