A detailed characterization of the hyperpolarization-activated “funny” current (If) in human-induced pluripotent stem cell (iPSC)–derived cardiomyocytes with pacemaker activity

Fig. 1. Action potential properties and If density and kinetic in pCM. a (Top) Representative spontaneous action potentials recorded from d15, 30, and 60 pCMs, as indicated; (Bottom) Summary table of the AP parameters: APA, action potential amplitude; MDP, maximum disatolic potential; APD, action potential duration at 50% (APD50) or 90% (APD90) of repolarization; APD90c, rate corrected APD90. b Representative traces of the If current density recorded in the range −35/ −115 mV (20 mV increment), at the three different differentiation stages (d15, triangles; d30, circles; d60, squares throughout the figure). c Plot of mean If current density–voltage relations obtained at the different time points. Mean values at −115 mV were as follows: − 3.75 ± 0.29 pA/pF (n = 19), − 4.19 ± 0.45 pA/pF (n = 21), and − 4.64 ± 0.90 pA/pF (n = 13). d Plot of the mean If activation curves; V1/2 and inverse-slope factors were as follows: − 76.5 ± 0.71* mV and 4.8 ± 0.36 (n = 19), − 79.0 ± 0.8 mV and 5.6 ± 0.36 (n = 21), − 81.2 ± 1.37 mV and 6.4 ± 0.47 (n = 13) at days 15, 30, and 60, respectively. Asterisk indicates P = 0.041 day 15 vs 30 and P = 0.0011 day 15 vs 60. e Plots of If activation time constant (τ)…

Fig. 2. hiPSC-CMs express HCN isoforms and auxiliary proteins. a Box-plot showing qRT-PCR analysis of HCN1-4 genes in iPSC-CMs at d15, 30, and 60, as indicated; Troponin T expression was used as reference gene in each sample to normalize for cardiomyocyte yield in different differentiations. b qRT-PCR analysis of genes known to be f-channels auxiliary subunits (caveolin-3-CAV3; Alpha-1,2-Glucosyltransferase-KCR1; Discs large homolog 1-SAP97; and Potassium channel βsubunit-MIRP1) in beating cultures at the various time points. c WB analysis of HCN4 and HCN1 in three hiPS-CM cultures at the various time points. cTnT expression was used for estimating content in cardiomyocytes. d Confocal microscopy image of hiPSC-CM showing co-expression of HCN4 (green) and caveolin-3 (red); nuclei were counterstained with DAPI (blue)

Fig. 3. Characterization of the ionic nature of If current. a Representative normalized If traces recorded at day 30 of differentiation elicited by the protocol used to obtain the fully activated I-V. b If traces recorded at −125 mV before (CTRL) and during the superfusion of 2 mM Caesium (Cs) at day 30 of differentiation. c Mean fully activated I-V relations obtained without (empty circles, n = 27) or with 2 mM Cs (black circles, n = 20). d Mean fully activated I-V relations in the presence of 30 mM external potassium (filled triangles, n = 13) or 30 mM external sodium (empty triangles, n = 14). The inset shows a blow up of the x-axis to appreciate changes in the Erev

Fig. 4. If current in pacemaker pCMs is modulated by sympathetic and parasympathetic agonists. a Time course of If current elicited by voltage steps at − 80 mV at d30 during perfusion of 1 μM isoproterenol (Iso). b Overlapped If traces in Tyrode (Tyr), during iso perfusion and after wash-out (WO). c Dose–response curve for isoproterenol. d Time course of If current elicited by voltage steps at −95 mV at d30 during perfusion of 100 nM carbachol (Cch). e Representative If current traces recorded before (Tyr), during (CCh), and after carbachol wash-out (WO). f Plot of dose–response curve for CCh. Continuous lines in panels c and f represent the best fitting to the Hill equation. g Time course of If current elicited by voltage steps at − 95 mV at d30 during perfusion of 100 nM iso alone and Iso + Cch 100 nM. h Overlapped If traces in Tyrode (Tyr), during superfusion of Iso (Iso), and during Iso+CCh.. i Dot plot of the shifts of the If activation curve caused by 100 nM CCh with or without pre-stimulation with 100 nM Iso, as indicated. *P = 0.0096

Fig. 5. Spontaneous rate of pCMs is modulated by sympathetic and parasympathetic agonists. a Representative time course of the action potential rate in clusters of pCMs at 30 days of differentiation before, during 1 µM isoproterenol perfusion, and after washout, as indicated. b Representative time course of the action potential rate before, during, and after washout of 100 nM acetylcholine (ACh) perfusion. c, d Representative action potential traces recorded before (solid line) and during (dashed line) isoproterenol (c) and Ach (d) stimulation; traces correspond to points 1 and 2 in of the respective time courses. e, f Dot plot of the percentage change in firing rate during 1 µM Iso (e) and 100 nM Ach (f). Mean ± SEM value is reported as filled square and whiskers