Abstract 17407: A Small-molecule Screening Strategy to Promote Cardiomyocyte Proliferation That Protects the Heart From Ischemic Injury

Abstract

Restoring the ability of adult cardiac myocytes (ACMs) to proliferate and promote heart regeneration have been proposed as a promising therapeutic strategy. We have demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases ACM cell cycle activity, but proliferation was limited. To improve this, we performed a small-molecule screening in combination with KDM4D on neonatal rat cardiomyocytes (NRVMs), using a library of kinase inhibitors targeting different signaling pathways. Our study revealed that compounds targeting the PI3K and JAK signaling pathways significantly potentiated the effects of KDM4D and promote NRVM proliferation in vitro. Specifically, a JAK inhibitor (JAKi2) in combination with Ad-KDM4D treatment resulted in approximately 100%, 34%, and 62% increase in EdU+, phospho-H3+, and total cell number compared to control, respectively. To validate these findings in vivo, we tested JAKi2 in an inducible KDM4D transgenic mouse model (iKDM4D). The animals underwent ischemia/reperfusion (I/R) to induce infarction, and JAKi2 was administrated orally for up to 8 weeks. We found both early and late effects. 48 hours after I/R, the infarct size in iKDM4D+JAKi2 hearts was approximately 4-fold smaller than that in vehicle-treated or JAKi2 alone groups, and the combination increased EdU+, Ki67+, and phospho-H3+ CM number separately by 3.2-fold, 2.9-fold, and 6.5-fold. At 2 weeks post-I/R, the scar size was smaller in iKDM4D+JAKi2 hearts compared to vehicle-treated mice (9.0% vs 1.8%, P <0.05), and the combination resulted in a 3-fold, 5.5-fold, and 8.5-fold increases in EdU+, Ki67+, and phospho-H3+ CMs, respectively. Moreover, the iKDM4D+JAKi2 hearts demonstrated progressive improvement in left ventricular ejection fraction (26.8%, 27.3%, and 25.5%) and fraction shorten (37.9%, 38.1%, and 35.0%) at 2 weeks, 4 weeks, and 8 weeks post-I/R. The increased estimated ACM number and mononucleated CMs at 8 weeks post-I/R support that CM division was increased in the iKDM4D+JAKi2 hearts. These findings suggest that a combination therapy involving JAKi2, along with KDM4D, promotes robust CM proliferation and protects the heart after ischemic injury.