Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system

Figures

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  Fig. 1. General cell culture and human-induced pluripotent stem cell (hiPSC)-derived pacemaker differentiation. a SFS.1CVB3∆VP0 hiPSC in culture. Left: Phase contrast image of SFS.1CVB3∆VP0 cells. Right: Fluorescence microscopy picture of doxycycline-induced SFS.1CVB3∆VP0 cells. The green VENUS signal indicates sufficient CVB3 expression. b Differentiated hiPSC cells in culture. Left up: Differentiated ventricular-like cells in an autonomously contracting network. Left down: Zoom into the ventricular-like cell network structure. Right up: Differentiated pacemaker-like cells. Right down: Zoom into the pacemaker-like cell network structure. c FACS analysis of differentiated ventricular-like cells and pacemaker-like cells showing expression of connexins 40, 43 and 45 (n = 3). Connexin 45 indicates sinoatrial node (SAN)-like differentiation and was only found in hiPSC-derived pacemaker-like cells. Background signal of non-stained cells of the same sample is indicated with the red line. Bottom panel: Statistical analysis shows significant increase of SAN-specific connexin 45 in hiPSC-derived pacemaker-like cells and significantly more cardiac connexin 43 (*p < 0.05). d Immunofluorescent…

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  Fig. 2. Immunofluorescence staining and analysis under basal conditions (control) and after 5 days of CVB3 expression (CVB3) in pacemaker-like cells. a Immunofluorescence staining of pacemaker-like cells with antibodies against N-Cadherin (membrane marker) and either anti-HCN4 (top panel), anti-Nav1.5 (middle panel) and anti-Cav1.2 (bottom panel). The distributions were visualized via linescan. b Quantitative distribution analysis of N-Cadherin (n = 30), HCN4 (n = 30), Nav1.5 (n = 5) and Cav1.2 (n = 5). The localization of all targets in the membrane and the cytoplasmatic fraction was monitored and put into a ratiometric dependence (***p < 0.001). c Quantitative expression analysis of N-Cadherin (n = 30), HCN4 (n = 30), Nav1.5 (n = 5) and Cav1.2 (n = 5). The whole fluorescence intensity of all targets in the cells was monitored and put into a percental dependence (**p < 0.01)

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  Fig. 3. Electrophysiological analysis of differentiated pacemaker-like cells. a Multielectrode array (MEA) measurements of young (14 days old) pacemaker-like cells. Cells were first monitored under basal conditions (left) and then under the influence of 10 μM ivabradine (middle) for HCN4 block. The signal events were averaged (10 signals) for detailed signal analysis (right). b Multielectrode array of aged (40 days old) pacemaker-like cells. Cells were first monitored under basal conditions (left) and then under the influence of 10 μM ivabradine (middle) for HCN4 block. The signal events were averaged (10 signals) for detailed signal analysis (right). c Multielectrode array of aged (40 days old) pacemaker-like cells with additional 5 days of CVB3 induction. Cells were first monitored under basal conditions (left) and then under the influence of 10 μM ivabradine (middle) for HCN4 block. The signal events were averaged (10 signals) for detailed signal analysis (right). d The number of electrochemical events in the MEA measurements of young, aged and aged + CVB3 -induced pacemaker-like cells was counted under basal conditions and under the influence of 10 μM ivabradine (***p < 0.001). e…

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  Fig. 4. Influence of CVB3 on the channel functionality of the HCN4 Channel. Representative current traces and a normalized current–voltage (I–V) curves b of the HCN4 channel without (n = 32) and with coexpression of CVB3 (n = 35) after 5 days of channel expression. The injection of cRNA was performed separately and in each case two days before the electrophysiological recordings. The currents were activated by applying xx-s hyperpolarizing test potentials between − 140 and − 30 mV, starting from a holding potential of 0 mV. The current amplitudes of the HCN4 channel under CVB3 influence were normalized to the control current at − 140 mV of the HCN4 channel without CVB3 influence (***p < 0.001). Non-injected cells and cells with only CVB3 expression did not resulted in HCN4 currents. c The normalized mean initial tail currents (± SEM) of the HCN4 channel without (n = 32) and with coexpression with CVB3 (n = 35)

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  Fig. 5. Impact of non-structural CVB3 proteins on the HCN4 channel function. a and b Normalized (−V) curves (± SEM) and the mean normalized currents at − 140 mV (***p < 0.001; ****p < 0.0001) of the HCN4 WT channel alone (n = 41) and with coexpression of the non-structural proteins 2A (A, n = 14), 2B (B, n = 27), 2C (C, n = 35), 2BC (D, n = 31), 3A (E, n = 35), 3B (F, n = 30), 3C (G, n = 29), 3D (H, n = 32), 3AB (I, n = 30), 3CD (J, n = 36) after 5-day expression of the channel. Injection of cRNA of viral proteins was performed separately and 2 days or in case of 2A 1 day before the electrophysiological measurements were performed. The currents were activated by applying xx-s hyperpolarizing test potentials between − 140 mV and − 30 mV, starting from a holding potential of 0 mV. The current amplitudes of the HCN4 channels expressed alone or in coexpression with the respective non-structural protein was analyzed at − 140 mV. c Genome and proteins of Coxsackievirus B3 and enteroviral polyprotein processing

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  Fig. 6. CVB3 expression leads to enhanced autophagy and autophagy-related HCN4 internalization in hiPSC-derived pacemaker-like cells. a Immunofluorescence staining of hiPSC-derived pacemaker-like cells without CVB3 expression and after 5 days of CVB3 expression. The cells were stained against HCN4 and the autophagy markers LC3, Beclin-1 or p62. b The cellular fluorescence intensities of the fluorescence markers LC3 (n = 13), Beclin-1 (n = 13) and p62 (n = 16) were analyzed in CVB3-expressing, hiPSC-derived pacemaker-like cells and normalized to CVB3-negative control cells (*p < 0.05; **p < 0.01; ***p < 0.001). c The immunostained hiPSC-derived pacemaker-like cells were analyzed for colocalization of HCN4 and the autophagy markers LC3 (n = 7), Beclin-1 (n = 11) and p62 (n = 15) with JAcoP in ImageJ (*p < 0.05; **p < 0.01; ***p < 0.001)

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  Fig. 7. Interplay between HCN4, Rab7 and LC3 under coexpression of CVB3-2C or CVB3-3A. a Colocalization of HCN4, Rab7 and LC3 in HeLa cells only under HCN4 expression (n = 15) or under the coexpression of either CVB3-2C (n = 17) or CVB3-3A (n = 21). The HeLa cells were fixed and immunostained 12 h after transfection and colocalization of HCN4, Rab7 and LC3 was analyzed with JAcoP in ImageJ (*p < 0.05; **p < 0.01; ***p < 0.001). b Pharmacological treatment of CVB3-2C- or CVB3-3A-expressing HeLa cells for the potential reversion of the malignant, cytoplasmatic HCN4 accumulation. The treatment with either N6-Benzyladenosine, GW5074 or CID 106770 was done for 8 h, 12 h or 24 h after transfection with a CVB3-2C- or CVB3-3A-construct (n = 10). The cells were imaged and membranous (Im) versus cytoplasmatic (Ii) HCN4-GFP intensity was compared (*p < 0.05; **p < 0.01; ***p < 0.001)

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  Fig. 8. a Differentiated hiPSC-derived pacemaker cells express the SAN channel HCN4 and the cardiac channels Nav1.5 and Cav1.2. b Immunofluorescence staining of hiPSC-derived ventricular-like cells and hiPSC-derived pacemaker-like cells against connexin 43 (magenta), connexin 40 (red) and connexin 45 (green). Single channel pictures. c Schematic interaction between CVB3 and hiPSC-derived pacemaker cells concerning HCN4 transport and function as it was observed in this study. Not only is CVB3 a player in general HCN4 expression but mainly disturbs HCN4 recycling via Rab7-dependent autophagy combined with elevated autophagosome formation, leading to the loss of HCN4 function