Fig. 5.

HiPSC-cardiomyocytes with short telomeres have poor cardiac health and mitochondrial function. A Cell Index curves indicating the initial adhesion of CRISPRi TERT hiPSC-cardiomyocytes with long (black line) and short (red line) telomeres until first 3 days followed by declining cell index of the hiPSC-cardiomyocytes with short telomeres (red line) until day 6. The curve represents the mean Cell Index value ± SD (n = 6–7 wells from one differentiation round); B Statistical analyses for cell index values performed at the end-point (day 7) between hiPSC-cardiomyocytes with long (black line) and short (red line) telomeres (n = 6–7 wells from one differentiation round). C Assessment of contractility (beats per minute, bpm) on day 7 of the RTCA analysis between hiPSC-cardiomyocytes with long (black line) and short (red line) telomeres (n = 6–7 wells from one differentiation round) with Mann–Whitney test. D Level of caspase activity at the basal level of CRISPRi TERT hiPSC-cardiomyocytes with long (black bar) and short telomeres (red bar) (n = 9 samples per group, triplicates from 3 independent experiments). E Doxorubicin (1 µM) treatment further enhances the caspase activity in CRISPRi TERT hiPSC-cardiomyocytes with shorter telomeres (+ Doxycycline) (n = 4–6 samples per group, triplicates from 2 independent differentiation experiments). F Analysis of mitochondrial metabolism using Seahorse XFe96 Analyzer in presence (black line) or absence (red line) of long telomeres in CRISPRi TERT hiPSC-cardiomyocytes. The CRISPRi TERT hiPSC-cardiomyocytes were further treated with Doxorubicin (1 µM, 48 h) to investigate the effect of mitochondrial function in hiPSC-cardiomyocytes with long (black line with dot) or short (red line with dot) telomeres. Oxygen consumption rate (OCR) was measured continuously at baseline and after addition of Oligomycin (2 µM), FCCP (1 µM) and R/A (0.5 µM) (n = 5–12 wells per group with 50,000 cells in each well). G–I The levels of basal respiration, maximal respiration, and ATP production before and after Doxorubicin (1 µM, 48 h) treatment. The mitochondrial metabolism reduces after doxorubicin treatment, but mitochondrial function is significantly poor in hiPSC-cardiomyocytes with short telomeres. All data are mean fold change relative to control ± SEM; hiPSC-CM = human induced pluripotent stem cell derived cardiomyocyte; Oligo = oligomycin; FCCP = carbonyl cyanide-4-phenylhydrazone; R/A = rotenone and antimycinA. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test was performed to calculate significance between 2 groups, One-way ANOVA, Kruskal–Wallis test with Dunn’s multiple comparison test