Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes

Figures

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  Fig. 1. Effects of human cardiac myofibroblasts on hiPSC-CM electrical function. A Schematic of culture conditions. hiPSC-CMs were cultured with myofibroblasts (MyoFB) in three conditions: with MyoFB conditioned medium, with MyoFBs separated by a semipermeable membrane (noncontact coculture), and with MyoFBs in direct contact (contact coculture). Control: hiPSC-CM cultured with hiPSC-CM conditioned medium. B Spontaneous beating frequency of hiPSC-CM in control, conditioned medium, noncontact and contact coculture (n = 24–28 treated monolayers). C Superimposed representative optical action potentials from hiPSC-CM in control (black), conditioned medium (red), noncontact (blue) and contact coculture (purple) conditions. Cells were electrically stimulated at 1 Hz. D, E Summary of APD50 (C) and APD90 (D) changes for hiPSC-CM cultured with MyoFBs in the three conditions versus control, at 1 Hz pacing rate (n = 21–22 treated monolayers). F Average pacing rate needed to induce loss of 1:1 action potential (AP) capture in hiPSC-CM cultures (n = 11–16 treated monolayers). Bars represent means ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 (one‐way ANOVA with post hoc Tukey’s test)

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  Fig. 2. Effects of human cardiac myofibroblasts on hiPSC-CM calcium handling. A Superimposed representative optical Ca2+ transients from hiPSC-CM in control (black), conditioned medium (red), noncontact (blue) and contact coculture (purple) conditions. Cells were electrically stimulated at 1 Hz. B–E Summary of time to Ca2+ transient peak (B), time to 50% Ca2+ decay (C), time to 80% Ca2+ decay (D), and rate of Ca2+ transient decay (E) changes for hiPSC-CM cultured with MyoFBs in the three conditions versus control, at 1 Hz pacing rate. Bars represent means ± SEM (n = 12–23 treated monolayers). *p < 0.05, **p < 0.01, ****p < 0.0001 (one‐way ANOVA with post hoc Tukey’s test)

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  Fig. 3. Effect of human cardiac myofibroblasts on hiPSC-CM molecular profile. A–F Changes in the abundance of mRNAs associated with AP repolarisation and Ca2+ cycling in hiPSC-CM after culture with MyoFBs conditioned medium (red), with MyoFBs separated by a semipermeable membrane (noncontact coculture, blue), and with MyoFBs in direct contact (contact coculture, purple). Levels of each mRNA were normalised to the control group (hiPSC-CM cultured in the presence of hiPSC-CM-conditioned medium; RQ = 1 for all genes). KCND3 encodes the transient outward potassium channel KV4.3; KCNH2 encodes the potassium voltage-gated channel KV11.1; KCNJ11 encodes the Kir6.2 protein which contributes to IK-ATP; ATP2A2 encodes the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a); SLC8A1 encodes the sodium-calcium exchanger (NCX1); CACNA1C encodes CaV1.2 protein which contributes to ICaL. RT-qPCR performed on mRNA isolated from hiPSC-CM only. Values expressed as means ± SEM (n = 6–7 treated monolayers). $p < 0.05, $$p < 0.01, $$$p < 0.001, $$$$p < 0.0001 versus control. *p < 0.05 (one‐way ANOVA with post hoc Tukey’s test)

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  Fig. 4. Blockade of IL-6 signalling reduces the effects of myofibroblast-conditioned medium and noncontact coculture on hiPSC-CM electrical function. A Spontaneous beating frequency of hiPSC-CM in control (black), conditioned medium (red), noncontact (blue) and contact coculture (purple), in the presence (diagonal pattern) or absence (no pattern) of a neutralising IL-6 antibody. Values expressed as means ± SEM (n = 16–20 treated monolayers). B, C Summary of APD50 (B) and APD90 (C) for hiPSC-CM in control, conditioned medium, noncontact and contact coculture, in the presence (diagonal pattern) or absence (no pattern) of a neutralising IL-6 antibody, at 1 Hz pacing rate. Values expressed as means ± SEM (n = 7–8 treated monolayers). D Average pacing rate needed to induce loss of 1:1 action potential capture in hiPSC-CM cultures. Values expressed as means ± SEM (n = 7–8 treated monolayers). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two‐way ANOVA with post hoc Tukey’s test)

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  Fig. 5. Blockade of IL-6 signalling reduces the effects of myofibroblast-conditioned medium and noncontact coculture on hiPSC-CM calcium handling. A–E Time to Ca2+ transient peak (A), time to 50% Ca2+ decay (B), time to 80% Ca2+ decay (C), time from 50 to 80% Ca2+ decay (D) and rate of Ca2+ transient decay (E) for hiPSC-CM in control (black), conditioned medium (red), noncontact (blue) and contact coculture (purple), in the presence (diagonal pattern) or absence (no pattern) of a neutralising IL-6 antibody, at 1 Hz pacing rate. Values expressed as means ± SEM (n = 8–12 treated monolayers). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two‐way ANOVA with post hoc Tukey's test)

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  Fig. 6. Effect of Cx43 silencing in myofibroblasts on hiPSC-CM electrical function following contact coculture. A Quantification of changes in Cx43 mRNA abundance in MyoFBs following transfection with a negative control siRNA, a GAPDH-specific siRNA, and a Cx43-specific siRNA (Ambion Silencer™ Select siRNA s5759) by RT-qPCR. RNA was collected at two timepoints: immediately following transfection (24-h transfection time; 1DT), and 48 h after transfection was completed (cells washed post-transfection and then cultured in basal DMEM; 1DT + 2). Values expressed as percentage relative to the negative control ± SEM (n = 3 independent experiments). **p < 0.01, ****p < 0.0001 (one‐way ANOVA with post hoc Tukey's test). B Superimposed representative optical AP from hiPSC-CM cultured in direct contact with MyoFBs treated with a non-targeting negative control siRNA (HiPSC-CM + Control KD MyoFBs, light blue) or in contact with MyoFBs treated with a Cx43-specific siRNA (HiPSC-CM + Cx43-KD MyoFBs, orange). Pure hiPSC-CM cultures (HiPSC-CM Only, black): hiPSC-CM cultured with basal DMEM media. Cells were electrically stimulated at 1 Hz. C, D APD50 (C) and APD90 (D) changes for hiPSC-CM cultured in…

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  Fig. 7. Effect of Cx43 silencing in myofibroblasts on hiPSC-CM calcium handling following contact coculture. A Superimposed representative optical Ca2+ transients from hiPSC-CM cultured in direct contact with MyoFBs treated with a non-targeting negative control siRNA (HiPSC-CM + Control KD MyoFBs, light blue) or in contact with MyoFBs treated with a Cx43-specific siRNA (HiPSC-CM + Cx43-KD MyoFBs, orange). Pure hiPSC-CM cultures (HiPSC-CM Only, black): hiPSC-CM cultured with basal DMEM media. Cells were electrically stimulated at 1 Hz. B–E Summary of time to Ca2+ transient peak (B), time to 50% Ca2+ decay (C), time to 80% Ca2+ decay (D) and rate of Ca2+ transient decay (E) changes for hiPSC-CM cultured in contact with control KD MyoFBs or Cx43-KD MyoFBs versus hiPSC-CM alone, at 1 Hz pacing rate. Bars represent means ± SEM (n = 9 treated monolayers). **p < 0.01, ****p < 0.0001 (one‐way ANOVA with post hoc Tukey's test)

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  Fig. 8. Effect of myofibroblast Cx43 silencing and IL-6 signalling blockade on hiPSC-CM electrical function and calcium handling following contact coculture. A–F APD50 (A), APD90 (B), time to Ca2+ transient peak (C), time to 50% Ca2+ decay (D), time to 80% Ca2+ decay (E) and rate of Ca2+ transient decay (F) for hiPSC-CM cultured in contact with control KD MyoFBs or Cx43-KD MyoFBs versus hiPSC-CM alone, in the presence (diagonal pattern) or absence (no pattern) of a neutralising IL-6 antibody, at 1 Hz pacing rate. Values expressed as means ± SEM (n = 3–9 treated monolayers). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two‐way ANOVA with post hoc Tukey’s test)