Molecular and functional evidence of HCN4 and caveolin-3 interaction during cardiomyocyte differentiation from human embryonic stem cells.

Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) is accompanied by changes in ion channel expression, with relevant electrophysiological consequences. In rodent cardiomyocytes, the properties of HCN4, a major f-channel isoform, depends on association with caveolin-3 (Cav3). To date, no information exists on changes in Cav3 expression and its associative relationship with HCN4 upon hESC-CM maturation. We hypothesize that Cav3 expression and its compartmentalization with HCN4 channels during hESC-CM maturation accounts for the progression of f-current properties toward adult phenotypes. To address this, hESC were differentiated into spontaneously beating CM and examined at ~30, ~60 and ~110 days of differentiation. Human adult and fetal CM served as references. HCN4 and Cav3 expression and localization were analyzed by qPCR and immunocyto/histochemistry. F-current was measured in patch-clamped single cells. HCN4 and Cav3 co-localize in adult human atrial and ventricular CM, but not in fetal CM. Protein and mRNA for Cav3 were not detected in undifferentiated hESC, but expression increased during hESC-CM maturation. At 110 days, HCN4 appeared to be co-localized with Cav3. Voltage-dependent activation of the f-current was significantly more positive in fetal CM and 60-day hESC-CM (midpoint activation, V_1/2, ~ -82mV) than in 110-day hESC-CM or adult CM (V_1/2 ~ -100mV). In the latter cells, caveolae disruption reversed voltage-dependence toward a more positive, or immature phenotype, with V_1/2 at -75mV, while in fetal CM voltage-dependence was not affected. Our data show, for the first time, a developmental change in HCN4-Cav3 association in hESC-CM. Cav3 expression and its association with ionic channels likely represent a crucial step of cardiac maturation.