Functional expression and subcellular localization of f-channels in native human 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, it is localized into membrane caveolae, where the interaction with caveolin-3 (Cav3) regulates current properties. HCN4 is abundant in undifferentiated human embryonic stem cells (hESC) and immature hESC-derived CM (hESC-CM). To date, no information is available on 1) developmental changes of HCN4 channel localization and function in human CM 2) the relationship with HCN channel expression/function in adult CM. Confocal microscopyshowed that HCN4 and Cav3 colocalize in native adult human CM. In the same cells, f-current was consistently recorded (70% cells), with a voltage of half maximal activation (Vh) of -102 and -101 mV in atrial and ventricular CM, respectively. 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 d30 hESC-CMs, but decreased in d60 and d110 hESC-CMs. In the d110 cells, HCN4 appeared to be associated with cav3. Activation properties of f-current recorded from d110 hESC-CMs, resembled those measured in native atrial and ventricular CMs. Current activation occurred at more positive potentials in d60 hESC-CMs and native fetal CMs. In native atrial CMs disruption of caveolae shifted If activation curve to more positive potentials. In conclusion, our data shows for the first time a HCN4/cav3 association in native human CMs and hESC- derived CM. Expression of cav3 and its association with ionic channels likely represents a crucial step of cardiac maturation, which may result in changes of cellular electrophysiological properties and modulation by endogenous signals.