Abstract Body (Do not enter title and authors here): Background: Adult mammalian cardiomyocytes (CMs) exhibit limited regenerative capacity after myocardial infarction (MI). Yes-associated protein (YAP) has been shown to stimulate CMs cell-cycle reentry in the adult mouse heart. TT-10, a fluorinated small-molecule YAP activator, effectively promotes CMs proliferation in MI mouse. However, its clinical translation is limited by poor myocardial retention and rapid systemic clearance.
Methods: To address these limitations, we formulated TT-10-loaded poly-lactic-co-glycolic acid nanoparticles (TT-10 NPs). In a porcine model of myocardial ischemia-reperfusion induced by 1-hour LAD coronary artery ligation followed by reperfusion, intramyocardial injection of TT-10 at the time of reperfusion enabled targeted, sustained, and stable cardiac delivery (N=8). In vitro, the TT-10 NPs’ activation of YAP pathway and proliferation-promoting effect were examined in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs, Day 60 after beating initiated), and in pig studies (Day 7 after TT-10 NPs treatment; N=3). The effects of TT-10 NPs on LV function, infarct size and hypertrophy were examined 4 weeks after TT-10 NPs treatment (N=5) compared to control group (PBS, N=6).
Results: TT-10 NPs exhibited favorable physicochemical characteristics and prolonged drug release (Figure 1). In vitro, in response to TT-10 NPs treatment, total YAP protein was highly expressed in post-mitotic CMs (Figure 2A and 2B), accompanied by activation of cell cycle and proliferation markers, including Ki67(Figure 2C), phospho-histone H3 (PH3) ( Figure2C), and Aurora B kinase (AuB) (Figure 2C), as well as an increase in CMs number(Figure 2D). In vivo studies, the TT-10 NPs treatment results in significantly increased total YAP protein expression and nuclear YAP localization at 7 days post-treatment (Figure3G and 3H), accompanied by a marked increase in CMs proliferation (eg. Ki67, PH3 and AuB) (Figure 3I) in the border zone of the LV infarct. Four weeks after the TT-10 NPs treatment myocardial infarct size was significantly reduced (Figure 3B and 3C), , which was accompanied by the improved LV function(Figure 3D-3F), and decreased myocardial hypertrophy compared to the control group of pigs (Figure 3J).
Conclusion: TT-10 NPs activates CMs proliferation via YAP signaling pathway, which in turn promotes myocardial regeneration, leading to cardiac structure and function recovery of hearts with acute myocardial infarction in pigs.