Targeting the Hippo Pathway for Heart Cell Regeneration and Survival

Heart diseases are the leading cause of death in the industrialized world and an effective cure is yet to be found. This is attributable to the very poor regenerative capability of the heart muscle cells. In the event of a heart attack, these cells are simply lost and cannot be replaced, which can lead to heart failure. In an effort to overcome the loss of cardiac cells, multiple investigations have proved that the Hippo pathway can be manipulated to benefit cardiomyocyte recovery. The Hippo pathway is a cell signaling pathway involved in cellular proliferation, stem cell differentiation, control of organ size and apoptosis. The pathway’s main effector is a transcriptional co-factor YAP (Yes-associated protein) whose activation is controlled by different regulators like SAV1 (Salvador Homolog 1), NF2 (Neurofibromin 2) and MOB1 (Mps one binder kinase activator). We investigated the use of two different RNA-based methods to manipulate the expression of regulators of the Hippo pathway and their potential in heart tissue healing and protection. We used a synthetic modified YAP(5SA) messenger RNA which overexpressed a constitutively active form of YAP on AC16 human fibroblast-cardiomyocyte fusion cells. The treatment greatly protected the cells from Staurosporine-induced apoptosis. The second RNA-based treatment we tested involves highly potent Dicer substrate siRNAs (DsiRNAs) to silence upstream core regulators of the pathway: MOB1, NF2 and SAV1. The DsiRNAs successfully lowered the level of the corresponding genes and proteins. We report that a combination of NF2 and SAV1 knockdowns is more potent than individual knockdowns. We also report for the first time MOB1 silencing as a very promising target for heart regeneration, which was more efficacious than NF2 and SAV1. Finally, we examined targeting MOB1 with a small molecule or a peptide-therapeutic to induce cell proliferation. We performed a virtual screening by docking a database of compounds into MOB1 structure. We then tested 24 hits in vitro and three drug candidates inhibited the Hippo pathway. In conclusion, in this project, we used three distinct approaches to induce cardiac cell proliferation by targeting the Hippo signaling.