Atrial fibrillation-associated electrical remodelling in human induced pluripotent stem cell-derived atrial cardiomyocytes: a novel pathway for antiarrhythmic therapy development

Figures

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  Figure 1. Cellular electrophysiology of atrial (iPSC-aCM) and ventricular (iPSC-vCM) induced pluripotent stem cell-derived cardiomyocytes. (A) Schematic of the iPSC-CM differentiation protocols used in this study. Application of 1 µmol/L retinoic acid (RA) from day 3 (d3) to day 6 induces an atrial specific subtype. (B) Representative optical action potentials (AP) elicited at 1 Hz in intact single iPSC-aCM (A, left) and iPSC-vCM (V, right) before (baseline) and after application of the M-receptor agonist carbachol (CCh, 2 µmol/L). (C) AP duration at 50 and 90% repolarization (APD50, APD90; atrial: n = 74/4, ventricular: n = 89/3). (D) Percentage change of APD90 following CCh application (atrial: n = 11/2, ventricular: n = 10/2). (E) Representative voltage-clamp recordings of inward-rectifier K+ current current in single iPSC-aCM (left) and iPSC-vCM (right) before (baseline, IK1) and after CCh application, revealing the acetylcholine-activated inward-rectifier K+ current (IK,ACh) in iPSC-aCM. (F), IK1 measured at −100 mV (atrial: n = 77/6, ventricular: n = 31/4). (G) Concentration-response curves for CCh-mediated activation of IK,ACh defined as CCh-dependent increase of…

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  Figure 2. Electrophysiology of atrial (aEHM) and ventricular (vEHM) engineered human myocardium. (A) Schematic of the EHM culture procedure used in this study. (B) Representative action potentials (AP) elicited at 1 Hz in aEHM (A, left) and vEHM (V, right) before (baseline) and after application of the M-receptor agonist carbachol (CCh, 10 µmol/L). (C) AP duration at 20, 50, and 90% repolarization (APD20, APD50 and APD90, top left), AP amplitude (APA, top right), upstroke velocity (dV/dtmax, bottom left), and resting membrane potential (RMP, bottom right; atrial: n = 13/3, ventricular: n = 5/2). (D), Percentage change of APD90 following CCh application. (E), Percentage change of RMP following CCh application (atrial: n = 6/2, ventricular: n = 5/2). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. aEHM using unpaired Student’s t-test or Welch’s t-test (E). n/N = number of recordings/EHM.

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  Figure 3. Twenty-four hour TP-induced remodelling of cellular electrophysiology in atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-aCM) and atrial engineered human myocardium (aEHM). (A) Representative optical action potentials (AP) elicited at 1 Hz in intact single iPSC-aCM after 24 h electrical pacing at 1 Hz (left) or 3 Hz (right). (B) AP duration at 50 and 90% repolarization (APD50, APD90, 1 Hz: n = 23/3, 3 Hz: n = 28/3) (C), Voltage-clamp protocol (0.5 Hz, top) and representative membrane current (IM) trace (bottom) of L-type Ca2+ current (ICa,L) in iPSC-aCM after 24 h electrical pacing at 1 Hz (left) or 3 Hz (right). (D), Peak ICa,L (left), integrated ICa,L expressed as estimated cytosolic Ca2+ influx (right; 1 Hz: n = 23/4, 3 Hz: n = 28/4). (E), Voltage-clamp protocol (0.5 Hz, top) and representative membrane current (IM) trace (bottom) of ICa, L in 24 h 3 Hz paced iPSC-aCM before and after application of type 1 and type 2A phosphatase inhibitor okadaic acid (OA, 1 µmol/L). (F), Change in peak ICa,L following OA application (1 Hz: n = 11/1, 3 Hz: n = 22/1). (G) Representative AP were elicited at 1 Hz in aEHM after 24 h optical pacing at 1 Hz (left) or 3 Hz (right).…

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  Figure 4. Twenty-four hour TP-induced remodelling of inward-rectifier K+ currents in atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-aCM) and atrial engineered human myocardium (aEHM). (A), Representative optical action potentials (AP) elicited at 1 Hz in intact single iPSC-aCM after 24 h electrical pacing at 1 Hz (left) or 3 Hz (right) before (baseline) and after application of the M-receptor agonist carbachol (CCh, 2 µmol/L). (B), Percentage change of AP duration at 90% repolarization (APD90) following CCh application (1 Hz: 26/4, 3 Hz: n = 16/3). (C), Representative voltage-clamp recordings of inward-rectifier K+ current in single iPSC-aCM after 24 h electrical pacing at 1 Hz (left) or 3 Hz (right) before (baseline, IK1) and after CCh application, revealing the acetylcholine-activated inward-rectifier K+ current (IK,ACh). (D), Peak IK1 measured at −100 mV. E, Peak IK,ACh measured at −100 mV (D, E: 1 Hz: n = 22/2, 3 Hz: n = 34/2). (F), Representative voltage-clamp recordings of basal inward-rectifier K+ current in single iPSC-aCM after 24 h electrical pacing at 1 Hz (left) or 3 Hz (right) before (baseline) and after application of selective IK,ACh blocker tertiapin…

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  Figure 5. Gene expression of ionic channels and receptors in atrial engineered human myocardium (aEHM) subjected to 24-h optical TP. (A), mRNA levels of ion channels CACNA1C (ICa,L, top) and KCNJ2 (IK1, bottom). (B), mRNA levels of IK,ACh ion channel subunits KCNJ3 (top) and KCNJ5 (bottom). (C), mRNA levels of receptors CHRM2 (M2 receptor, top) and ADORA1 (A1 receptor, bottom). All values are plotted as single values against the corresponding action potential duration at 90% repolarization (APD90). Data are mean ± SEM. **P < 0.01 vs. 1 Hz using the Mann–Whitney U test.

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  Figure 6. Prolonged (7-day) TP-induced remodelling of cellular electrophysiology in atrial engineered human myocardium (aEHM) and atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-aCM). (A), Representative action potentials (AP) elicited at 1 Hz in aEHM after 7 days of optical pacing at 1 Hz (left) or 3 Hz (right). (B), AP duration at 20, 50, and 90% repolarization (APD20, APD50 and APD90, left), resting membrane potential (RMP, middle left), upstroke velocity (dV/dtmax, middle right), AP amplitude (APA, right; 1 Hz: n = 45/8, 3 Hz: n = 23/9). (C) Representative voltage-clamp recordings of basal inward-rectifier K+ current (IK1) current in iPSC-aCM after 7 day of optical pacing at 1 Hz (left) or 3 Hz (right). (D) Peak IK1 measured at −100 mV (1 Hz: n = 49/1, 3 Hz: n = 64/1). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 1 Hz using paired Student’s t-test. n/N = number of iPSC-CM/differentiation or number of recordings/EHM.

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  Figure 7. Prolonged (7-day) TP-induced contractile dysfunction in atrial human engineered myocardium (aEHM). (A) Representative partial trace of aEHM contraction as it is continuously optically stimulated at 3 Hz for 7 days. Contractile measurements were taken every 24 h at a window of 1 Hz pacing, of which a representative example is shown. (B) Representative average contractile signals from aEHM during 7 day 1 Hz optical pacing on day 0 and day 7 (left), representative partial traces of aEHM contraction on day 0 (top right) and day 7 (bottom right) of 1 Hz pacing. (C) Representative average contractile signals from aEHM tissue during 7 day 3 Hz optical pacing on day 0 and day 7 (left), representative partial traces of aEHM contraction on day 0 (top right) and day 7 (bottom right) of 3 Hz pacing. (D) Time course of contractile function of aEHM optically paced for 7 d at 1 Hz (n = 3). (E) Change in contractile force after 7 day of optical pacing at 1 Hz. Single point data extracted from (D). (F) Time course of contractile function of aEHM optically paced for 7 day at 3 Hz (n = 6). (G) Change in contractile force after 7 day of optical pacing at 3 Hz. Single point data extracted from (F).…