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

Abstract

Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) is accompanied by changes in ion channel expression, with relevant electrophysiological consequences. In rodent CM, the properties of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, a major f-channel isoform, depends on the 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 real time PCR and immunocyto/histochemistry. F-current was measured in patch-clamped single cells. HCN4 and Cav3 colocalize in adult human atrial and ventricular CM, but not in fetal CM. Proteins and mRNA for Cav3 were not detected in undifferentiated hESC, but expression increased during hESC-CM maturation. At 110 days, HCN4 appeared to be colocalized with Cav3. Voltage-dependent activation of the f-current was significantly more positive in fetal CM and 60-day hESC-CM (midpoint activation, V1/2, ∼ -82 mV) than in 110-day hESC-CM or adult CM (V1/2∼-100 mV). In the latter cells, caveolae disruption reversed voltage dependence toward a more positive or an immature phenotype, with V1/2 at -75 mV, 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.

FIG. 1.

Expression of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4 and Caveolin-3 (Cav3) in human adult and fetal cardiomyocytes (CM). Human adult ventricular (hAV) (A) and atrial (hAA) (B) CM expressing HCN4 (green) and Cav3 (red). Colocalization is observed upon merging (yellow). (C) Human fetal (hF) CM expressing HCN4 (green) and Cav3 (red). No colocalization is observed upon merging. Color images available online at www.liebertpub.com/scd

FIG. 2.

Expression of HCN4 and Cav3 in undifferentiated human embryonic stem cell (hESC). (A) Undifferentiated hESC expressing HCN4 (green), but not Cav3. (B) 3D reconstruction of a stack of confocal images showing HCN4 expression (red) in a single undifferentiated hES cell, showing clearly the cell surface (sarcolemmal) localization of the ion channel. Scale bar=10 μm. Color images available online at www.liebertpub.com/scd

FIG. 3.

Expression of Cav3, HCN4, and α-actinin in d30, d60, and d110 hESC-CM. (A) Cav3 and HCN4 double staining (d30: white arrow=low density; yellow arrow=high density) (B) α-actinin and HCN4 double staining to verify expression of HCN4 in cardiac-specific cells. Scale bar=10 μm. Color images available online at www.liebertpub.com/scd

FIG. 3.

Expression of Cav3, HCN4, and α-actinin in d30, d60, and d110 hESC-CM. (A) Cav3 and HCN4 double staining (d30: white arrow=low density; yellow arrow=high density) (B) α-actinin and HCN4 double staining to verify expression of HCN4 in cardiac-specific cells. Scale bar=10 μm. Color images available online at www.liebertpub.com/scd

FIG. 4.

Immunohistochemical (IHC) staining of whole hESC-CM clusters. IHC staining of hESC-CM at d60 and d110 with α-actinin, Cav3, and HCN4. Color images available online at www.liebertpub.com/scd

FIG. 5.

Transcriptional regulation of HCN4 and Cav3 in undifferentiated hESC and hESC-CM. Real time PCR data for the expression of HCN4 and Cav3 in hESC-CM over time (*P<0.05).

FIG. 6.

Cardiomyocyte electrophysiological recordings. (A–E) Representative I_f current traces in hESC-CM (60 and 100 days), hF-CM and hAA- and hAV-CM. (F) Activation curves of I_f current for each cell type (*P<0.05). (G) Effects of membrane cholesterol depletion on activation properties of f-channels in hAA-CM (n=3) and hF-CM (n=6). (H) Bar graph showing mean potential (±SEM) of half maximal activation of f-current (I_f V_1/2) in hAA-CM and hF-CM, treated or not with methyl-β-cyclodextrin (MβCD). ***P<0.001.