ABSTRACT
Title
Functional expression and subcellular localization of f-channels in native human and hESC-derived cardiomyocytes
Authors
M. Del Lungo1, L. Sartiani1, V. Spinelli1, A. Bosman2, A. Mugelli1, M. Jaconi2, E. Cerbai1.
1Center of Molecular Medicine University of Florence, Florence, Italy, 2Department of Pathology and Immunology, Geneve, Switzerland.
1Center of Molecular Medicine University of Florence, Florence, Italy, 2Department of Pathology and Immunology, Geneve, Switzerland.
Abstract
The Hyperpolarization-activated Cyclic-Nucleotide gated (HCN) genes encode for the alpha subunit of f-channel present in the heart. HCN4 is the predominant isoform in the sinoatrial node cells and, in the rabbit, it is localized into membrane caveolae, where the interaction with caveolin-3 (cav3) regulates f-current properties (Barbuti, A. et al., 2007). HCN4 is abundant in undifferentiated human embryonic stem cells (hESC) and immature hESC-derived cardiomyocytes (hESC-CMs). Maturation is associated with modifications of f-channel functional properties. To date, no information is available on i) the subcellular localization of HCN4 and cav3 in hESC- and native human CMs ii) the functional consequences of their association on f-current properties. Confocal microscopy showed that HCN4 and cav3 colocalize in native human adult CMs. In the same cells, f-current was consistently recorded (70% cells), with a voltage of half maximal activation of -102 and -101 mV in atrial and ventricular CMs, respectively. Protein and mRNA for cav3 were not detected in undifferentiated hESC, but expression increased during maturation of hESC-CMs. 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 human fetal CMs. In native atrial CMs disruption of caveolae shifted f-current activation curve to more positive potentials.
In conclusion, our data shows for the first time that HCN4 and cav3 associate in native human and hESC-derived CMs. 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.
In conclusion, our data shows for the first time that HCN4 and cav3 associate in native human and hESC-derived CMs. 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.