Deoxy-ATP producing hiPSC-derived cardiomyocytes improve cardiac function in chronically infarcted hearts.

Abstract

We have demonstrated that 2-deoxy-ATP (dATP) improves contractility by activating myosin and increasing crossbridge cycling without impairing relaxations, making it a promising sarcomere targeted molecule to mitigate cardiac dysfunction. Over-expression of Ribonucleotide Reductase (RNR) elevates dATP in cardiomyocytes sufficient to increase contractile function. We have shown that dATP from RNR over-expressing cells spreads to neighboring cells via gap junctions and increases their contractile function. This suggests transplantation of RNR over-expressing human-induced pluripotent stem cell-derived cardiomyocytes (RNR-hiPSC-CMs) could significantly increase the effectiveness of cardiac cell therapy in chronic myocardial infarcted (MI) hearts. In initial studies, overexpressing a proteolytic degradation resistant form of RNR in hiPSC-CMs, using a CAG promoter (CAG-RNR-CMs), resulted in elevated levels of dATP % (4.7-fold ± 0.38), and improved cell shortening (2-fold ± 0.15) compared to WT-CMs (N=3). Upon transplantation into normal rat hearts, left ventricular (LV) % fractional shortening (FS) was significantly increased with CAG-RNR-CMs (N=4-6, p ≤ 0.05) and increased dATP levels of native myocardium. When transplanted into chronically infarcted hearts, CAG-RNR-CMs had a protective effect on LV dilation and stabilized %FS from deteriorating post-MI compared to WT-CM transplants. To maximize these benefits, we engineered a new cell line using a muscle specific CK8m promoter to drive dATP levels higher. Compared to CAG-RNR-CMs, CK8m-RNR-CMs had significantly higher RNR and dATP % (∼5-fold). At 3 months post-transplant, these CK8m-RNR-CMs demonstrated greatly increased efficacy of dATP delivery to host myocardium. This greater level of dATP translated to significantly improved LV function at 3 months post-transplantation compared to WT-CMs, with more organized grafts of similar size, and improved voluntary exercise. R01HL128368