Abstract Body: Introduction: Vascular cell adhesion molecule-1 (Vcam1), a known target of Forkhead box O (FoxO) transcription factors, plays important roles in endothelial cell homeostasis. However, its role in cardiomyocyte homeostasis, including in ischemic heart disease, remains incompletely understood.

Methods: Experiments were performed using cardiomyocyte-specific Vcam1 or FoxO1 knockout mice and control littermates subjected to ischemia-reperfusion (I/R) or SHAM surgery. Echocardiography was employed to assess cardiac contractile function, and hearts were harvested for further analyses. In-vitro experiments were performed using NRVMs subjected to simulated I/R.

Results: We report that FoxO1 and Vcam1 levels were significantly reduced in wild-type (WT) mouse hearts following I/R injury, suggesting a role for the FoxO1/Vcam1 axis in ischemic heart disease. To test for a cardiomyocyte-specific contribution of this pathway, we generated cardiomyocyte-specific FoxO1 (FoxO1-cKO) and Vcam1 (Vcam1-cKO) knockout mice. Cardiomyocyte-specific loss of FoxO1 resulted in reduced Vcam1 expression compared with WT littermates. Following I/R injury, both FoxO1-cKO and Vcam1-cKO mice manifested exacerbated cardiac injury, including reduced ventricular contractile performance, increased cardiomyocyte hypertrophy, and enhanced cardiac fibrosis. These data highlight a protective role of the FoxO1/Vcam1 axis in I/R-induced myocardial injury. Indeed, Vcam1-cKO hearts demonstrated increased cardiomyocyte apoptosis, evidenced by elevated cleaved caspase-3 levels in remote and border zones, and increased TUNEL-positive cardiomyocytes, suggesting a role of Vcam1 in ameliorating oxidative stress-induced cell death. Furthermore, Vcam1-cKO hearts exhibited reduced leukocyte infiltration and markedly attenuated phosphorylation of Ezrin, a pro-survival signaling protein, following I/R injury, indicating impaired cardiomyocyte-leukocyte-dependent pro-survival signaling. Consistently, I/R-induced phosphorylation of ERK1/2 and Akt, known downstream effectors of Ezrin, were blunted in Vcam1-cKO hearts. In vitro, siRNA-mediated knockdown of Vcam1 transcript in NRVMs exacerbated simulated I/R-induced cell death and apoptosis and markedly attenuated early reperfusion-induced Ezrin and Akt phosphorylation.

Conclusion: Findings reported here establish a novel mechanism whereby the VCAM-1/Ezrin axis activates a pro-survival pathway to protect cardiomyocytes from ischemia-induced cell death.