Functional expression and subcellular localization of f-channels in human ventricular and hESC-derived cardiomyocytes.

Caveolae are specialized lipid rafts in the plasma membrane responsible for the interaction, sublocalization and function of proteins and ion channels. The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) genes encode for the alpha subunit of f-channel present in cardiac pacemaker cells, in the adult and developing cardiac myocytes (CM). HCN4 is the predominant isoform in the sinoatrial node and, in the rabbit, experimental evidence indicate that it localizes into membrane caveolae, where caveolin-3 (Cav3)-channel interaction regulates current properties and autonomic modulation. HCN4 is abundant in undifferentiated human embryonic stem cells (hESC) and immature hESC-derived CM (hESC-CM). To date, no information is available on i) developmental changes of HCN4 channel localization and function in human CM ii) the relationship with HCN channel expression/function in adult CM. Confocal analysis showed that HCN4 and Cav3 colocalize in adult human ventricular CM. In the same cells, f-current was consistently recorded upon hyperpolarization (70% cells), with a voltage of half maximal activation (Vh) of -94 mV. Protein and mRNA for Cav3 were not detected in undifferentiated hESC, but expression increased during maturation of hESC-CM. Oppositely, HCN4 was highly expressed in hESC and early hESC-CM, but a 5-fold decrease in mRNA levels occurred in late hESC-CM. In these cells, HCN4 appeared to be associated with Cav3. Activation properties of f-current recorded in late hESC-CM resembled those measured in adult ventricular CM (AV-CM) (Vh=-93 mV). Current activation was faster and occurred at more negative potentials in hESC and early CM. In conclusion, cardiac maturation is associated with the recruitment HCN4 channel and CAV3 into membrane lipid rafts, suggesting that sub-cellular localization of f-channel in lipid rafts is a fundamental step in cardiac maturation.