Structural and Mechanistic Bases of Nuclear Calcium Signaling in Human Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes

Figure 6

Pathways involved in Ca²⁺-regulated gene expression in hESC-VCMs. (a) As examined by Q-RT-PCR, treatment of ionomycin (2 μM, 3 hours) dramatically increased mRNA level of ANP, which was blocked by EGTA (4 mM) and CsA (10 μM), but not KN62 (10 μM), pretreatment. . (b) As revealed by western blotting, ionomycin-induced Ca²⁺ elevation caused NFAT dephosphorylation which was blocked by the pretreatment of CsA (10 μM), the calcineurin inhibitor, but not KN62 (10 μM), the CaMK inhibitor. (c) Ionomycin treatment induced NFAT translocation from cytosol to nucleus. Bar graph showed the ratio of nuclear to total NFAT fluorescence. cells, . Scale . (d) Treatment of ionomycin (2 μM) and PMA (100 ng/mL) significantly increased NFAT activity measured by luciferase assay. (e) Ionomycin treatment (2 μM, 1 minute) caused CaMKII phosphorylation. (f) Immunostaining showed the cytosolic localization of endogenous CaMKII in hESC-VCMs. (g) ATP (100 μM)-induced Ca²⁺ rise phosphorylated the endogenous CaMKII, which was blocked by the expression of cytosolic, but not nuclear, PV overexpression. , .

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