Abstract P1014: Cardiomyocyte Energetic Capacity Governs Responses To Cell Cycle Stimulation

Abstract

Background: Induction of cardiomyocyte (CM) proliferation is a promising strategy for cardiac regeneration. We previously showed that the overexpression of four cell cycle factors (4F), i.e. cyclin B1, CDK1, cyclin D1 and CDK4, induces CM proliferation. Temporal profiling of the single cell transcriptomes of 4F- and LacZ-overexpressing CMs showed a specific population with higher levels of CD36 that was more responsive to the 4F cell cycle induction. In addition, P7 primary CMs isolated from CD36 KO mice demonstrated 50% less response to the 4F compared with the WT controls (Abouleisa et al., Circulation, 2022). These data indicate that CD36 may be required to prime CMs to proliferate. CD36 is known to internalize fatty acids to be utilized for energy production. Here, we aim to investigate the direct effect of CM energetic capacity on their response to cell cycle stimulation. Methods and Results: Carnitine palmitoyl transferase -1 (CPT-1) is a rate limiting enzyme for the β-oxidation of fatty acids. Knockdown of CPT-1 using siRNA significantly reduced mitochondrial ATP production by 10% and led to a significant reduction in cell cycle markers (PHH3, EDU) in response to the 4F (PHH3; control siRNA+4F:24±1% vs CPT1 siRNA+4F:15±1%, EDU; control siRNA+4F:22±2% vs CPT1siRNA+4F:16±1%, P<0.01, n=4). Small molecule inhibition of CPT1 using etomoxir significantly decreased mitochondrial ATP production and suppressed CM responses to the 4F. Furthermore, overexpression of CPT-1 using viral vectors resulted in significant increase in ATP production and increased response to cell cycle stimulation using the 4F. Supplementing the culture medium with (1μmol) L-carnitine significantly increased mitochondrial ATP production and enhanced the CM response to the 4F. Finally, to increase ATP availability in CMs directly, we overexpressed a light-activated mitochondrial proton pump (MT-ON). CMs expressing MT-ON showed a significant increase in mitochondrial ATP levels and enhanced cell cycle induction in CMs transduced with 4F (PHH3; 4F:18±2% vs 4F+MT-ON:27±3%, EDU; 4F:18±1% vs 4F+MT-ON:30±4 %, P<0.01, n=4). Conclusions: These findings suggest that an energy threshold is required for CMs to progress into the cell cycle.