Introduction:The potentially unlimited number of cardiomyocyte (CMs) derived from human induced pluripotent stem cells (hiPSCs)in vitrofacilitates high throughput applications like cell transplantation for myocardial repair, disease modelling, and cardiotoxicity testing during drug development. Despite promising progress in these areas, a major disadvantage that limits the use of hiPSC derived CMs (hiPSC-CMs) is their immaturity.Methods: Three hiPSC lines (PCBC-hiPSC, DP3-hiPSCs, and MLC2v-mEGFP hiPSC) were differentiated into CMs (PCBC-CMs, DP3-CMs, and MLC2v-CMs, respectively) with or without retinoic acid (RA). hiPSC-CMs were either maintained up to day 30 of contraction (D30C), or D60C, or purified using lactate acid and used for experiments. Purified hiPSC-CMs were cultured in basal maturation medium (BMM) or BMM supplemented with ascorbic acid (AA) for 14 days. The AA treated and non-treated hiPSC-CMs were characterized for sarcomeric proteins (MLC2v, TNNI3, and MYH7), ion channel proteins (Kir2.1, Nav1.5, Cav1.2, SERCA2a, and RyR), mitochondrial membrane potential, metabolomics, and action potential. Bobcat339, a selective and potent inhibitor of DNA demethylation, was used to determine whether AA promoted hiPSC-CM maturation through modulating DNA demethylation.Results:AA significantly increased MLC2v expression in PCBC-CMs, DP3-CMs, MLC2v-CMs, and RA induced atrial-like PCBC-CMs. AA treatment significantly increased mitochondrial mass, membrane potential, and amino acid and fatty acid metabolism in PCBC-CMs. Patch clamp studies showed that AA treatment induced PCBC-CMs and DP3-CMs adaptation to a ventricular-like phenotype. Bobcat339 inhibited MLC2v protein expression in AA treated PCBC-CMs and DP3-CMs. DNA demethylation inhibition was also associated with reduced TET1 and TET2 protein expressions and reduced accumulation of the oxidative product, 5 hmC, in both PCBC-CMs and DP3-CMs, in the presence of AA.Conclusions:Ascorbic acid induced MLC2v protein expression and promoted ventricular-like CM subtype in hiPSC-CMs. The effect of AA on hiPSC-CM was attenuated with inhibition of TET1/TET2 mediated DNA demethylation.