In vitro model of ischemic heart failure using human induced pluripotent stem cell-derived cardiomyocytes

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here, our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated into functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the nonmyocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures that had more than 94% cTnT+ cells. After purification, hiPSC-CMs were replated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. The phenotype of metabolic stress-selected hiPSC-CM monolayers recapitulated many of the functional and structural hallmarks of ischemic CMs, including elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy-induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate, and abnormal response to β1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generated cell populations with phenotype similar to what is well known to occur in the setting of ischemic heart failure and thus provide a opportunity for study of human ischemic heart disease.

Keywords: Cardiology; Cardiovascular disease; Cell Biology; Heart failure.

Figure 1. hiPSC-CM differentiation protocol and purification approaches.

(A) Timeline of small molecule–based cardiac-directed differentiation and purification approaches. (B) Phase-contrast images showing the impact of metabolic selection on monolayer confluence (19-1-11 hiPSC line, day 24). (C) hiPSC-CM spontaneous calcium transient recordings in 19-9-11 hiPSC-CMs without any selection (black, n = 6 monolayers) and with metabolic selection (red, n = 5 monolayers). Spontaneous calcium transient alternans were induced by metabolic selection media (red traces and bars). *Denotes significant difference of amplitude between even and odd numbered beats. Quantification in C shows the relative amplitude of each beat after beat 1, normalized to beat 1 amplitude. Without selection, the amplitude did not vary beat to beat (beat 2 = 0.97 ± 0.02, beat 3 = 0.98 ± 0.01; n = 6). However, in lactate selection media (CDML3), the beat amplitude varied on a beat-to-beat basis (beat 2 = 0.84 ± 0.11, beat 3 = 0.99 ± 0.01; n = 6; paired t test, P < 0.05), indicating calcium transient alternans. hiPSC-CMs, human induced pluripotent stem cell–derived cardiomyocytes.

Figure 2. hiPSC-CMs generated using 2 distinct purification approaches.

(A) Each purification approach is equally effective to enrich hiPSC-CM population analyzed by flow cytometry with cTnT labeling. (B) Connexin43 (Cx43) mislocalization was apparent in metabolic stress–selected monolayers. (C) Metabolic stress–selected hiPSC-CMs had shorter sarcomere length (MACS = 1.85 ± 0.17 μm; n = 35 vs. metabolic stress selection = 1.77 ± 0.13 μm; n = 40; unpaired t test, *P = 0.009). Sarcomere length was quantified using the repeating fluorescence pattern of the α-actinin staining. (D) Phalloidin staining was used to examine the actin cytoskeleton. Stress fibers rather than sarcomeres were identified in the metabolic stress–selected hiPSC-CMs. hiPSC-CMs, human induced pluripotent stem cell–derived cardiomyocytes; cTnT, cardiac troponin I; MACS, magnetic-activated cell sorting.

Figure 3. hiPSC-CM purification approach effects on CM intracellular calcium flux.

(A) Metabolic stress selection significantly alters baseline calcium flux of hiPSC-CMs. (B) SERCA2a expression levels were reduced in metabolic stress–selected hiPSC-CMs. (C) MACS-purified CMs (black traces, symbols) responded as expected to isoproterenol; however, metabolic stress–selected hiPSC-CMs responded only with positive chronotropy. (D) cTnI phosphorylation state was elevated at baseline in metabolic stress–selected hiPSC-CMs (0.19 ± 0.05, n = 3 vs. 0.03 ± 0.02 AU, n = 3). cTnI phosphorylation increased after isoproterenol stimulation (metabolic stress = 0.75 ± 0.29, n = 3 and MACS = 0.99 ± 0.30 AU, n = 3). (E) PLN phosphorylation levels (PLN-P) were similarly low at baseline in each group, and only in MACS-sorted hiPSC-CMs was PLN-P detected at significant levels after isoproterenol treatment (metabolic stress = 0.05 ± 0.06 AU, n = 3 vs. MACS = 0.77 ± 0.35 AU, n = 3; 1-way ANOVA, *P = 0.02). hiPSC-CMs, human induced pluripotent stem cell–derived cardiomyocytes; cTnI, cardiac troponin I; PLN, phospholamban; MACS, magnetic-activated cell sorting.

Figure 4. hiPSC-CM purification approach impacts electrophysiology phenotypes.

(A) Microelectrode recordings show MDP was depolarized in metabolic stress–selected hiPSC-CMs APA was greater in MACS-selected hiPSC-CM monolayers (MACS = 103.5 ± 12.5 mV; n = 11 vs. metabolic stress selection = 81.5 ± 17.9 mV; n = 9; unpaired t test, *P < 0.001). (B) Metabolic stress–selected monolayers had significantly greater spontaneous beating frequency. (C) Metabolic stress selection monolayers had significantly longer APD80 than MACS-selected monolayers over a range of pacing frequencies (1 Hz: metabolic = 357.12 ± 6.85 ms; 1.5 Hz: metabolic = 312.24 ± 7.03 ms; 2.0 Hz: metabolic = 279.38 ± 6.21 ms; 2.5 Hz metabolic = 250.29 ± 4.20 ms; n = 9-12 monolayers) vs. (1 Hz: MACS = 309.56 ± 6.85 ms;1.5 Hz: MACS = 279.16 ± 3.83 ms; 2.0 Hz: MACS = 248.60 ± 6.31 ms; 2.5 Hz: MACS = 216.93 ± 4.20 ms; n = 12 monolayers, mean ± SEM). (D) Arrhythmia testing was done by abrupt slowing of pacing from 2.5 to 1.0 Hz with continuous recording. MACS-purified monolayers could follow the pacing change with 1:1 capture; however, 72.2% of metabolic stress–selected monolayers displayed arrhythmias. hiPSC-CM, human induced pluripotent stem cell–derived cardiomyocyte; MDP, maximal diastolic potential; APA, action potential amplitude; MACS, magnetic-activated cell sorting.

Figure 5. Metabolic selection induces mitochondrial dysfunction in hiPSC-CM monolayers.

(A) JC-1 staining indicates more polarized mitochondria in MACS-purified monolayers. Numeric values are in the main text. (B) MitoTracker Red CMX Ros staining supports the JC-1 results. MitoTracker signal, MACS = 1369.11 ± 355.66 AU vs. metabolic stress selection = 635.45 ± 302.12, t test, *P < 0.001. hiPSC-CM, human induced pluripotent stem cell–derived cardiomyocyte; MACS, magnetic-activated cell sorting.

Figure 6. Metabolic stress–selected hiPSC-CM monolayers have increased sensitivity to chemotherapy-induced cardiotoxicity (DOX-TOX).

(A) Annexin V staining indicates greater extent of apoptosis in metabolic-purified hiPSC-CMs treated with 1000 nM doxorubicin (DOX). (B) Calcium transient optical mapping using rhod2 example traces show greater sensitivity of electrophysiological function of metabolic stress selection hiPSC-CMs (red traces). (C) Metabolic stress–selected hiPSC-CM monolayers show arrhythmias in response to DOX-TOX with greater sensitivity than MACS-purified hiPSC-CM monolayers. hiPSC-CMs, human induced pluripotent stem cell–derived cardiomyocytes. hiPSC-CM, human induced pluripotent stem cell–derived cardiomyocyte; MACS, magnetic-activated cell sorting.

Figure 7. SERCA2a calcium pump gene therapy reverses hiPSC-CM heart failure phenotype.

(A) Viral transduction was verified by mCherry live-cell expression in hiPSC-CM monolayers. Further, SERCA2a protein expression was determined by Western Blotting. Quantification indicates that SERCA2a protein expression was reduced in metabolic stress–treated hiPSC-CMs at baseline (MACS = 2.11 ± 0.22, n = 3 vs. metabolic stress = 1.27 ± 0.06, n = 3). AdSERCA2a treatment restored SERCA2a levels to control levels (metabolic stress plus Ad SERCA2a = 2.15 ± 0.17, n = 3; ANOVA, *P < 0.05). (B) Calcium transient measurements indicate that metabolic stress selection media prolongs the cardiac action potential and spontaneous arrhythmias were observed in the metabolic stress selection purification approach. hiPSC-CM monolayers treated with AdSERCA2a did not develop arrhythmias. (C) CaTD50 was prolonged in metabolic stress media–treated cells at baseline and was corrected to control values with AdSERCA2a gene therapy (CaTD50s: MACS baseline = 475.5 ± 18.1 ms; metabolic stress baseline = 577.37 ± 17.7 ms; MACS plus AdSERCA2a = 396.6 ± 11.2 ms; metabolic stress plus Ad SERCA2a = 493.67 ± 19.9 ms; ANOVA, **P = 0.002, ns). CaTD, calcium transient duration; hiPSC-CMs, human induced pluripotent stem cell–derived cardiomyocytes; MACS, magnetic-activated cell sorting.

Figure 8. CDML3 metabolic selection media induces arrhythmia phenotypes in MACS-purified 19-9-11 hiPSC-CM monolayers and antibiotic resistance–purified hiPSC-CMs.

(A) Action potential traces (fluovolt) of hiPSC-CMs maintained in RPMI plus B27 maintenance media. (B and C) CDML3 induced arrhythmia phenotypes observed using voltage sensitive dye. (D) APD30, (E) APD50, and (F) action potential triangulation are greater in CDML3-treated hiPSC-CMs. RPMI plus B27, APD30 = 284.7 ± 27.8 ms; CDML3 APD30 = 309.7 ± 29.0 ms; unpaired t tests, *P < 0.001, n = 94 and n = 95. RPMI APD90 = 823.2 ± 366.1 ms; CDML3 APD90 = 1266.7 ± 499.9 ms; unpaired t test, *P < 0.001. RPMI triangulation = 0.60 ± 0.13; CDML3 triangulation = 0.72 ± 0.12; unpaired t test, *P < 0.001. (G) None of the 94 wells showed spontaneous arrhythmias in RPMI plus B27 monolayers, whereas 24 of 95 monolayers presented with spontaneous arrhythmias in the CDML3-treated group. (H–J) Electrophysiology and calcium flux of iCell² CMs is affected by maintenance in CDML3 media. APD50 and CaTD50 are prolonged in the CDML3 media–treated monolayers. Calcium transient amplitude of CDML3 media–treated monolayers plated in 96-well plates is reduced compared with RPMI-B27 maintenance media. (K) Seahorse measurement of OCR shows reduced maximal OCR (L) and reduced reserve respiratory capacity (M) in CDML3-treated hiPSC-CM monolayers. hiPSC-CMs, human induced pluripotent stem cell–derived cardiomyocytes; MACS, magnetic-activated cell sorting; OCR, oxygen consumption rate. Unpaired t test, **P < 0.05.