Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes

Abstract

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na⁺) channels and sarcoplasmic reticulum calcium (Ca²⁺) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na⁺ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

Keywords: cardiomyocyte; dilated cardiomyopathy; disease modeling; engineered heart tissues; induced pluripotent stem cells; long QT syndrome 3; maturation; physiology.

Figure 1.. Influence of MM on Metabolism of iPSC-CMs

(A) Schematic overview of the study. Twenty days after the start of differentiation, hiPSC-CMs were separated into maturation media (MM) or standard RPMI/B27 conditions. (B) Optical AP recordings (VF2.1.Cl dye) revealed functional sodium (Na⁺) channels in MM CMs, with cessation of beating occurring at IC₅₀ ~3.88 μM. Representative optical voltage recording of control (ctr) and 11 μM TTX-treated MM and RPMI/B27 cells. (C) Real-time oxygen consumption rate (OCR) measurements of hiPSC-CMs cultured in RPMI/B27 or MM by Seahorse extracellular flux analyzer. Cells were treated with the ATP synthase inhibitor oligomycin, the respiratory uncoupler FCCP, and the respiratory chain blockers rotenone and antimycin A. MM exhibited greater respiration rate under basal conditions and after mitochondrial uncoupling (n = 3 batches). (D) Real-time ECAR measurements of hiPSC-CMs cultured in RPMI/B27 or MM by Seahorse extracellular flux analyzer. Cells were treated with glucose, ATP synthase inhibitor oligomycin, and glucose analog 2-DG. Maturation conditions induced the glycolytic capacity of cells (n = 3 batches). (E) Flow cytometry analysis of Tom20 expression in RPMI/27- and MM-cultured hiPSC-CMs revealed higher mitochondrial content in maturation culture (n = 4 batches). (F) Representative mitochondria (Tom20) immunostaining in hiPSC-CMs cultured in RPMI/B27 and MM. Pronounced differences were observed in Tom20 distribution between the two culture conditions. Scale bars represent 20 μm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Figure 2.. Metabolism and CM Genes Are Upregulated in MM-Treated iPSC-CMs

(A) Heatmap of the top 1,000 genes with highest variance across the three treatment groups. (B) Top 10 GO terms associated with the differentially expressed genes in the comparison MM versus prolonged RPMI/B27 (top), RPMI/B27 versus baseline differentiation (middle), and MM and baseline differentiation (bottom). The top GO terms associated with maturation are related to metabolism and CM biology. (C–F) Heatmaps of key genes involved in distinct features of CM behavior: (C) fatty acid metabolism, (D) calcium (Ca²⁺) cycling and sarcoplasmic reticulum (SR), (E) sarcomere, and (F) ion channels. RNA-seq experiment was performed on n = 3 iPSC-CM batches.

Figure 3.. MM Increases I_K1, Diastolic Membrane Potential, and Upstroke Velocity of the AP

(A) Large inward rectifier K⁺ current (I_K1) in single hiPSC-CMs cultured in MM. Representative I_K1 traces in ctr and following inhibition with 300 μmol/L Ba²⁺. Outward component of Ba²⁺-sensitive I_K1 and voltage protocol are given in insets. (B) Stimulated APs show highly negative resting membrane potential just prior to the AP (diastolic V_m) and markedly increased AP upstroke velocity (dV/dt_max). Patch-clamp recordings were obtained in single hiPSC-CM cultured in MM and paced at 1 Hz frequency. Data are presented as mean ± SEM.

Figure 4.. Improved Ca²⁺ Handling Properties of iPSC-CMs in MM

(A) Western blot of SR proteins. SR proteins were markedly increased under maturation conditions (n = 3 batches). SERCA2a, sarco/endoplasmic reticulum Ca²⁺-ATPase; CASQ, calsequestrin; JPH2, junctophilin 2; PLN, phospholamban. (B) Representative Fluo-4 traces under both culture conditions either untreated (ctr) or treated with 1 μM thapsigargin (thap). Each trace is an average normalized ΔF/F₀ versus time plot (33 Hz) from multiple peaks in each well (n = 3 peaks). Thapsigargin dose response on hiPSC-CMs highlights the enhanced thapsigargin sensitivity on Ca²⁺ transient duration (CTD) of MM cultured cells (n = 3 batches, n = 3 wells each). (C) siRNA experiment targeting SERCA2a or PLN in hiPSC-CMs. Optical Fluo-4 recordings showed that MM-cultured cells significantly increased CTD after siSERCA2a compared with siControl (siCtr) and RPMI/B27. Conversely, a significantly lowered CTD was recorded in MM after addition of siPLN (n = 3 batches, n = 3 wells each). (D) Representative fura-2 Ca²⁺ traces. The changes in intracellular Ca²⁺ concentrations were measured by ratiometric fura-2 indicator. hiPSC-CMs were paced at 0.5 Hz and exposed to caffeine to measure SR Ca²⁺ content. (E) Quantification of fura-2 recordings. Under pacing conditions, we observed lowered tau, amplitude, and diastolic Ca²⁺ levels in MM cells. After addition of caffeine, MM cells exhibited greater release of SR Ca²⁺ in respect to amplitude (2 batches, n = 39–48 cells each). (F) Traction force microscopy was performed to assess the effect of the culture media on force generation of cells. Comparison of traction stresses (arrows) and intracellular stresses (heatmap) between MM and RPMI/B27 hiPSC-CMs during a contraction cycle. (G) Significantly higher traction stresses were observed in MM culture conditions, with no significant changes to the beat rate (peak-to-peak time). Faster contraction kinetics (contraction and relaxation rate) were established in MM conditions (3 batches, n = 2 wells each). Data are presented as mean ± SEM. Circles (●), squares (■), and triangles (▲) represent experimental batches. For thapsigargin dose-response curve (B), significance was calculated for each dose. For siRNA experiment (C), significance was either calculated compared with respective siControl (†) or with RPMI/B27 conditions (*). † and *p < 0.05, †† and **p < 0.005, ††† and ***p < 0.0005.

Figure 5.. Matured CMs Showed Improved Ability to Recapitulate Contractile and Electrophysiological Dysfunction of DCM and LQT Mutations

(A) Schematic overview highlighting the implication of splicing defect caused by RBM20-R634Q in dilated cardiomyopathy (DCM). Two known mis-spliced targets of RBM20-R6434Q are sarcomeric protein Titin (TTN) and Ca²⁺ regulator ryanodine receptor 2 (RyR2). (B) Detection of RyR2 isoform expression in RBM20-WT and mutant cell lines under both culture conditions with PCR products on agarose gel. RBM20-R634Q exclusively expressed RyR2 isoform containing 24 bp insert, while RBM20-WT cells also expressed RyR2 without the insert. (C) qRT-PCR performed on MM and RPMI/B27 samples to establish expression levels of different TTN isoforms in WT or R634Q iPSC-CMs. Significant increased level of fetal TTN was observed in R634Q mutant line with no differences between culture conditions (n = 3 batches). (D) Contractility performance (KIC, contractility assay) of R634Q iPSC-CMs was significantly impacted by maturation conditions. No significant differences were established in RPMI/B27 media R634Q, but mutation led to profound changes in physiology in MM (RPMI 2 batches, n = 3 wells each; MM 3 batches, n = 6 wells each). (E) The SCN5A F1473C mutation causes congenital long QT syndrome variant 3 (LQT3) by perturbation of channel inactivation. (F) Schematic overview on the effect of mexiletine. Mexiletine blocks the voltage-gated Na⁺ channel to reduce the late Na⁺ current (I_NaL) and shorten action potential duration (APD) in LQT3 CMs. (G) Optical recordings with voltage-sensitive dye VF2.1Cl on MM healthy donor (HD) or F1473C hiPSC-CMs grown in either RPMI/B27 or MM. Each trace is an average normalized ΔF/F₀ versus time plot (33 Hz) from multiple peaks in each well (n = 3–5 peaks). (H) Dose-effect plots of mexiletine on APD (APD₇₅). No APD modulation was noticed in MM-grown HD lines or RPMI/B27-F1473C hiPSC-CMs across mexiletine dose range. A clear dose-dependent APD shortening was observed for MM-F1473C hiPSC-CMs. (I) Quantification of baseline (no drug treatment) APD (APD₇₅) of individual iPSC-CM lines. MM F1473C hiPSC-CMs exhibited a significantly prolonged APD compared with HD and RPMI/B27-F1473C hiPSC-CMs (2 batches, n = 3–6 wells each). (J) The effect of mexiletine on iPSC-CMs as shown by the dose with the maximum APD₇₅ shortening effect in each culture condition. APD₇₅ shortening times (ms) are represented as change from their respective ctr wells. Mexiletine significantly shortened APD in MM F1473C compared with HD and RPMI/B27-F1473C hiPSC-CMs (2 batches, n = 3–6 wells each). Data are presented as mean ± SEM. For mexiletine dose-response curve (H), significance was calculated as change from respective untreated ctr. Circles (●), squares (■), and triangles (▲) represent experimental batches. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Figure 6.. MM Improves the Stability and Functional and Structural Properties of EHTs

(A) Representative images of EHTs prior to switching media (day 17) and after 6 weeks of culture (day 59) in ctr or MM, and schematic overview of the metrics used to quantify EHT integrity and morphology. (B) Impact of culture conditions on EHT width (plotted on left y axis) and length (plotted on right y axis, dotted lines). Length and width were normalized to baseline (randomization), depicting significantly less thinning of EHTs cultured in MM (n = 18–24, 3 batches). (C) Average contractile force normalized to the mean force of ctr of the respective batch and spontaneous beat rate (BPM) in the respective medium on the last day of culture (n = 14–18, 3 batches). (D) Average normalized contraction peaks under electrical stimulation in 1.8 mM Ca²⁺ Tyrode’s solution. Note higher time to peak (TTP; 80%) in MM, but similar relaxation time (RT; 80%; n = 9, 3 batches). (E) Representative MLC2a and MLC2v staining in EHT after 30-day culture in MM or ctr media, depicting mostly MLC2v-positive CMs with a clear longitudinal orientation in both groups. (F) Representative fluorescent images of α-actinin staining in EHT grown in ctr or MM (scale bar corresponds to 10 μm). (G) Sarcomeric length as determined by α-actinin staining. Longer sarcomeric length was observed in MM conditions (n = 100 sarcomeres, 2 batches). (H) Representative electron microscopy images from ctr and MM EHT. Ultrastructures in EHT cultured in MM appeared more defined. Data are presented as mean ± SEM. Circles (●), squares (■), and triangles (▲) represent experimental batches. *p < 0.05, **p < 0.005, ***p < 0.0005.