title: 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.
authors: Department of Pathology and Immunology, Faculty of Medicine, Geneva University, Geneva, Switzerland.
authors: Alexis
authors: A
authors: Bosman
authors: Laura
authors: L
authors: Sartiani
authors: Valentina
authors: V
authors: Spinelli
authors: Martina
authors: M
authors: Del Lungo
authors: Francesca
authors: F
authors: Stillitano
authors: Daniele
authors: D
authors: Nosi
authors: Alessandro
authors: A
authors: Mugelli
authors: Elisabetta
authors: E
authors: Cerbai
authors: Marisa
authors: M
authors: Jaconi
mesh_terms: D021941:Caveolae / Q000378:metabolism; D051244:Caveolin 3 / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism; D055724:Electrophysiological Phenomena; D053595:Embryonic Stem Cells / Q000502:physiology*; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism*; D012508:Sarcolemma
chemical_list: C417736:CAV3 protein, human; D051244:Caveolin 3; D054815:Cyclic Nucleotide-Gated Cation Channels; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D009124:Muscle Proteins; D015221:Potassium Channels