(336f) Controlling the Electrophysiological Maturation of Stem Cell-Derived Cardiomyocytes Via Nitric Oxide Supplementation

Heart disease is responsible for approximately one in four deaths annually in the United States. Because donor organs remain scarce, the ability to generate physiologically-accurate heart muscle cells (cardiomyocytes) in vitro provides an alternative tissue source for the treatment of cardiovascular disease. Pluripotent stem cells have demonstrated the capacity for developing mature cardiomyocytes for use in both clinical and diagnostic research applications. Unfortunately, current methods for generating therapeutically-relevant quantities of cardiomyocytes from stem cells are hindered by the creation of cardiac cells with immature electrophysiological phenotypes. Furthermore, inherent heterogeneities among stem cell-derived cardiomyocyte populations in individual batches may contribute a host of arrhythmogenic effects post-implantation in native myocardium. Nitric oxide (NO), a small, free radical molecule, is involved in a multitude of different cell signaling mechanisms including neurotransmission, vasodilatation, and differentiation. The supplementation of NO-donating materials into differentiation media has been shown to up-regulate processes responsible for cardiogenesis. While changes in gene and protein expression of stem cell-derived cardiomyocytes grown under such culture conditions have been shown, the electrophysiological effects of nitric oxide addition have not yet been examined. In this study, populations of embryoid bodies (EBs) were exposed to either S-nitrosothiol (CysNO, at 10, 50, or 100µM) NO-donors or nitric oxide synthase inhibitor (L-NAME, 1mM) every two days after the onset of differentiation; spontaneous contractile activity was observed and quantified over a period of 18 days. Both the normalized beating percentage and beat frequency of EBs exposed to CysNO demonstrated higher rates of spontaneous beating than either the control or L-NAME populations from day 10 to 14. Additionally, beating frequency among NO-supplemented groups increased between days 8 and 14, whereas control and L-NAME groups retained a constant beating frequency. Further work will investigate the protein and genetic expression profiles of the stem cell-derived cardiomyocytes. Furthermore, calcium transient analysis and whole cell patch clamp will elucidate the ion channel activity of maturing cardiomyocytes. The results of this study provide insight into the mechanisms of stem cell-derived cardiomyocyte maturation and will aid in establishing a platform for improving electrophysiological homogeneity of pluripotent stem cells differentiated in large-scale culture environments.