hERG1 channel subunit composition mediates proton inhibition of rapid delayed rectifier potassium current (IKr) in cardiomyocytes derived from hiPSCs

Figures

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  Figure 1. hiPSC-CM maturation with PDMS hyperpolarizes the AP and increases IKrdensity.A, AP recordings from cells cultured on glass (blue) and PDMS (green). B and C, AP parameters. Cells plated on PDMS demonstrated more hyperpolarized resting membrane potential and greater AP amplitude than cells plated on glass. D, representative IKr traces elicited by the protocol at bottom. E, steady-state IKr measured at the end of the step pulse, recorded from immatured and matured hiPSC-CMs. F, steady-state current densities at −10 mV. G, tail IKr in immatured and matured hiPSC-CMs. H, tail current densities at −10 mV. hiPSC-CMs plated on PDMS had larger ERG currents than hiPSC-CMs plated on glass. I, there was no significant difference in the voltage dependence (V1/2) of IKr activation recorded from immatured hiPSC-CMs versus matured hiPSC-CMs. J, there was no statistically significant difference in the cellular capacitance between immatured and matured hiPSC-CMs. Data were compared using a two-way ANOVA test and a two-tailed Mann–Whitney test where appropriate. Errors bars represent mean ± SD. N value = 3, n value ≥8. ∗∗∗∗p < 0.0001, ∗∗∗p = 0.0002, and ∗p < 0.05. AP, action potential; hiPSC-CM,…

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  Figure 2. Effect of cell maturation on IKrdeactivation.A, tail IKr traces recorded from immatured (blue) and matured (green) hiPSC-CMs at −40 mV. B, corresponding deactivation time constants for IKr recorded from hiPSC-CMs cultured on glass (immatured) versus PDMS (matured). C, representative IKr traces recorded from immatured (blue) and matured (green) hiPSC-CMs using a ventricular AP clamp protocol. D, repolarizing charge normalized to the cell capacitance in matured and immatured hiPSC-CMs. E, repolarizing charge normalized to the peak-tail IKr of the same cell recorded from matured and immatured hiPSC-CMs. Two-tailed Mann–Whitney test. Errors bars represent mean ± SD. N value = 3, n value ≥10. AP, action potential; hiPSC-CM, human-induced pluripotent stem cell–derived cardiomyocyte; IKr, rectifier potassium current; PDMS, polydimethylsiloxane.

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  Figure 3. Proton sensitivity of native IKrcorresponds with hiPSC-CM maturation.A, representative IKr traces elicited by the protocol below from matured and immatured hiPSC-CMs at pH 7.4 and 6.3. B, steady-state IKr density in immatured and matured hiPSC-CMs at pH 7.4 and 6.3. C, normalized steady-state current densities at −10 mV. D, peak-tail IKr density in immatured and matured hiPSC-CMs in control and acidic environment. E, normalized peak-tail IKr densities at −10 mV. The symbols ∗ (black and red) and # represent the statistical significance of normalized peak tail and steady-state IKr at pH 7.4 versus pH 6.3 in immatured and matured hiPSC-CMs, respectively. The symbol ∗ (red only) denotes significant difference between immatured and matured hiPSC-CMs at pH 6.3. F, voltage dependence of activation (V1/2) for IKr from immatured and matured hiPSC-CMs in control and acidic environment. G, time constants of IKr deactivation recorded from immatured and matured hiPSC-CMs at pH 7.4 and acidic pH 6.3. Data were compared using a two-way ANOVA and a two-tailed Mann–Whitney test. Errors bars represent mean ± SD. N value = 3, n value ≥9. ∗∗∗∗p < 0.0001, ∗∗∗p = 0.0008, ∗∗p = 0.0024, and ∗p < 0.05.…

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  Figure 4. hERG1 subunit abundance in matured and immatured hiPSC-CMs.A, representative immunostainings for hERG1a and F-actin. B, quantification of mean hERG1a immunofluorescence from matured and immatured hiPSC-CMs. C, representative immunostainings for hERG1b and F-actin. D, quantification of mean hERG1b immunofluorescence from matured and immatured hiPSC-CMs. E, hERG1a and hERG1b mRNA levels in matured and immatured hiPSC-CMs. Data were compared using a two-tailed Mann–Whitney test. Errors bars represent mean ± SD. N value = 3, n value ≥8. ∗∗∗p = 0.0002 and ∗p < 0.05. Scale bars indicate 25 μM. hiPSC-CM, human-induced pluripotent stem cell–derived cardiomyocyte.

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  Figure 5. PAS domain expression diminishes IKrproton sensitivity in immatured hiPSC-CMs.A, representative IKr traces elicited by the protocol below from PAS (orange) and GFP (blue)-transduced hiPSC-CMs at pH 7.4 and 6.3. B and C, steady-state I–V relationships normalized to the maximum peak tail IKr from immatured hiPSC-CMs expressing either GFP or PAS, at pH 7.4 and 6.3, respectively. D, steady-state I–V relationships normalized to the maximum peak tail IKr recorded from the same cell for matured hiPSC-CMs at pH 7.4 (solid symbols) and 6.3 (open symbols), respectively. E, steady-state IKr at pH 7.4 and 6.3 in immatured hiPSC-CMs overexpressing PAS or GFP. F, normalized steady-state current densities at −10 mV. G, peak-tail IKr levels measured at pH 7.4 and 6.3 in immatured hiPSC-CMs overexpressing PAS (orange) or GFP (blue). H, normalized tail current densities at −10 mV. I, deactivation kinetics from GFP and PAS-expressing hiPSC-CMs. J, voltage dependence of activation (V1/2) in immatured and matured hiPSC-CMs at pH 7.4 and 6.3. The symbols ∗ (black and red) and # represent the statistical significance of normalized peak tail and steady-state IKr at pH 7.4 versus pH 6.3 in GFP and…

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  Figure 6. Cryo-EM structure of the hERG1a channel with predicted locations of residues identified as proton sensors. Predicted pronatable amino acids modeled on the hERG1a cryo-EM structure (Protein Data Bank ID: 5VA1) using the Research Collaboratory for Structural Bioinformatics Protein Data Bank (rcsb.org) (33, 34, 41, 69, 70, 99). Eight residues are highlighted: E575, H578, and H587 in the pore turret, D509 in the S3 helix, D456 and D460 in the S2 helix, H562 in the S5 helix, and H674 in the S6 helix–CNBHD linker. Black boxes depict expanded view of each residue position. The CNBHD and PAS are highlighted in green and yellow, respectively. Dashed blue lines represent regions unresolved in the cryo-EM structure. Inset depicts a top–down view of the tetrameric subunit arrangement of the hERG1a channel cryo-EM structure. cyclic nucleotide–binding homology domain; PAS, Per–Arnt–Sim.