Mukhopadhyay, D.; Varanasi, S. M.; Sabharwal, A.; Kar, R.; Magnano, C.; Dorairaj, A.; Wang, E.; Dutta, S. K.; Das, P.; Ekker, S. C.; Wang, Y.; Singh Angom, R.

Background: Myocardial infarction (MI) remains a leading cause of mortality worldwide. Recent studies suggest a cardioprotective role for vascular endothelial growth factor-B (VEGF-B) in MI. However, the molecular mechanisms of VEGF-B-mediated signaling via its co-receptor Neuropilin-1 (NRP1) in MI are poorly understood. In this study, we investigated the intricate signaling mechanisms involving VEGF-B and NRP1 in cardiomyocytes (CMs) using ischemic injury as a model of MI. Methods: We utilized both in vitro and in vivo approaches to elucidate the role of VEGF-B and NRP1 signaling in MI and how it manifests a protective role in mitochondrial functions and cardiac regeneration following ischemic injury. We used two different cardiomyocyte cell lines, H9c2 (rat ventricular cardiomyocytes) and HL-1 (mouse ventricular cardiomyocytes), and induced hypoxia conditions, to mimic the myocardial infarction-induced ischemic injury in the heart. In addition, we developed a novel heat shock inducible zebrafish model of a cardiomyocyte-specific VEGF-B overexpression system to further examine the protective role of VEGF-B in vivo. Results: Our findings indicate that both VEGF-B and NRP1 are co-expressed in heart tissue compared to other tissues, and their expression is altered in response to hypoxia/ischemic injury. VEGF-B treatment prior to hypoxia enhances cardiomyocyte survival, by improving mitochondrial function, while NRP1 knockdown abolishes this protective effect, highlighting a prominent role of NRP1 signaling in VEGF-B-mediated cardio protection. Lastly, using our VEGF-B transgenic zebrafish model, we demonstrated that VEGF-B overexpression in zebrafish cardiomyocytes protects the heart from ischemic injury and enhances cardiac regeneration in an NRP1-dependent manner. Conclusion: Our study has uncovered an important role of VEGF-B-NRP1 signaling axis in VEGF-B mediated cell survival and in beneficial mitochondrial functions in the CMs. We further demonstrated that VEGF-B/NRP1 axis protects against ischemic injury in vivo using a novel zebrafish model.