Undifferentiated hiPSCs used in this study were obtained from Beijing Cellapy Biotechnology. hiPSCs were cultured in Matrigel (Corning, United States)-coated plates in feeder-free culture conditions with fresh PGM1 PSC culture medium (Cellapy, China), and the cells were passaged at 80–90% confluence using a dissociation solution (Cellapy, China). The schematic shown in Supplementary Figure S1A illustrates the cell culture, differentiation, and dissociation timeline. The undifferentiated hiPSCs were induced to differentiate into cardiomyocytes by transient activation/inhibition of the Wnt signaling pathway. Cardiac differentiation methods were adapted from previously published articles (Lian et al., 2012; Sharma et al., 2015). Cells at 90% confluence were used for differentiation (day 0), and the medium was replaced with RPMI 1640 (Sigma, United States) with B27 supplement minus insulin (Thermo Fisher Scientific, United States), which was considered basal medium. Day 0 cells were incubated with 6 μmol of CHIR99021 (GSK-3 inhibitor) (Selleck, United States) for 48 h (day 2). Day 2 cells were cultured in the basal medium for 24 h (day 3). Day 3 cells were treated with 5 μmol of IWP2 (WNT inhibitor) (Selleck, United States) for 48 h (day 5). Then, day 5 differentiated cells were cultured in the basal medium for 48 h (until day 7). On day 7, the medium was replaced by RPMI 1640 with B27 supplement with insulin (Thermo Fisher Scientific, United States). Approximately 8 days after differentiation, some cells began to beat (Supplementary Video 1). Subsequently, cells were maintained in the medium containing RPMI 1640 plus B27 supplement with insulin for 7 days followed by maintenance in the cardiac enrichment medium [RPMI 1640 medium (Thermo Fisher Scientific, United States) supplemented with 4 mM sodium L-lactate (Sigma-Aldrich, United States)]. Cells were maintained in the cardiac enrichment medium for 3 days. After this enrichment phase, the medium was changed to RPMI 1640 with knockout serum replacement (KSR) (Thermo Fisher Scientific, United States). On day 20, spontaneously beating cardiomyocytes were dissociated with TrypLE Express enzyme (Thermo Fisher Scientific, United States), centrifuged, resuspended, and replated onto Matrigel-coated plates for follow-up experiments. hiPSC-derived cardiomyocytes were treated with 0.5 mM AICAR (Selleck, United States) or vehicle (DMSO) for 7 days from day 23 to day 30 (Supplementary Video 2). All cultures were grown at 37°C in 5% O₂ and 5% CO₂.

Total RNA preparations were extracted with TRIzol reagent (TaKaRa, Japan) from the cells and quantified using a NanoDrop (Thermo Fisher Scientific, United States). One microgram of total RNA was reverse transcribed to cDNA using a PrimeScript RT reagent kit (TaKaRa, Japan). Quantitative real-time PCR was performed using amplified cDNA, gene-specific primers, and a SYBR Green dye kit (TaKaRa, Japan) using QuantStudio 3 (Thermo Fisher Scientific, United States). GAPDH was used as the housekeeping gene. The relative gene expression was calculated by the 2^{–Δ Δ Ct} method. Mitochondrial DNA content was quantified as described previously (Ulmer et al., 2018). Genomic and mitochondrial DNA was extracted with a genomic DNA extraction kit (BioFlux, China). The relative content of mtDNA was detected by quantitative real-time PCR using mitochondria-encoded NADH dehydrogenase (mt-ND1 or mt-ND2) primers and normalized to β-globin. All primers were purchased from TSINGKE Biological, and the sequences of the primers are listed in Supplementary Table S1.

Cells were lysed with a solution containing lysis buffer (KeyGEN, China), protease inhibitors, phosphatase inhibitors, and PMSF (Beyotime, China) at 4°C for 30 min on a rocker. A BCA protein assay kit (KeyGEN, China) was used to quantify total protein. Protein samples were mixed with 5× buffer and boiled for 10 min before loading onto a 10% SDS-PAGE gel (Epizyme, China). After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States). Based on the position of the markers, the membranes were cut into pieces, blocked with QuickBlock^TM blocking buffer (Beyotime, China) for 1 h, and incubated with primary antibodies (Supplementary Table S2) overnight at 4°C. The membranes were washed with TBST and then incubated with the corresponding secondary antibody (Supplementary Table S2) at room temperature for 1 h. The membranes were developed by ECL (Millipore, United States) using a ChemiDoc^TM Touch imaging system (Bio-Rad, United States). Intensity of the Western blot bands was quantified using Quantity One (United States).

Cellular ATP content was measured by an ATP assay kit (Beyotime, China) according to the manufacturer’s instructions. Cell lysates were centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was used for subsequent determination. ATP detection working solution was added into the detection wells; sample or standard was added and quickly mixed with a pipettor, and the chemiluminescence signal (RLU) was measured with a chemiluminescence instrument (BioTek, United States) after at least a 2 s interval. Concentrations of ATP in the samples were calculated according to the standard curve; total protein concentration in the sample was determined by a BCA protein assay kit, and ATP concentration was converted into nmol/mg protein.

hiPSC-CMs were seeded into 12-well plates at a density of 1 × 10⁶ cells and treated with 0.5 mM AICAR (Selleck, United States) or vehicle (DMSO) for 7 days from day 23 to day 30. Cellular HK activities and lactate production were measured by HK assay kit (Solarbio^® BC0745, China) and LA assay kit (Solarbi^® oBC2230, China) according to the manufacturer’s instructions, respectively. All experiments were normalized by the cell numbers.

Cells were dissociated from 12-well plates and replated into Matrigel-coated coverslips. Cells were washed with PBS and fixed in 4% paraformaldehyde or 75% ice-cold ethanol for 20 min. Then, the cells were permeabilized with 0.5% Triton X-100 for 15 min, blocked with 5% BSA for 1 h, and incubated with primary antibodies (Supplementary Table S2) overnight at 4°C. After washing with PBS, the cells were incubated with secondary antibodies (Supplementary Table S2) for 1 h at room temperature in the dark and then stained using Hoechst 33342 (Beyotime, China) for 10 min. Imaging was performed using a fluorescence microscope (BX51; Olympus) and quantified using NIS-Elements software. Cells were analyzed for cell area, perimeter, circularity index, and sarcomere length with ImageJ software (United States). The circularity index equals 4Π^∗Area/Perimeter², and the sarcomere length was measured in two adjacent α-actinin bands (Liu et al., 2020).

The cells were fixed with 4% glutaraldehyde solution for 2 h and postfixed for 2 h with 1% osmium tetroxide at 4°C. The cells were dehydrated with ethanol and methanol gradients and then embedded in epoxy resin. The samples were cut into thin (60 nm) sections and stained with citrate and uranyl acetate. TEM images were acquired at random locations throughout the samples and captured using a transmission electron microscope (TEM; H-7500).

The OCR was detected using a Seahorse XF24 extracellular flux analyzer (Agilent Technologies, United States). hiPSCs were cultured in a Matrigel-coated Seahorse XF-24 cell plate before analysis. On day 23, hiPSC-CMs were dissociated and subsequently plated onto a Seahorse XF-24 cell plate at 2.5 × 10⁵ cells/well. hiPSC-CMs were treated with 0.5 mM AICAR (Selleck, United States) or vehicle (DMSO) for 7 days. Mitochondrial function was analyzed using an XF Cell Mito Stress kit (Agilent Technologies, United States). Cells were washed with XF assay medium (unbuffered DMEM + 10 mM glucose + 2 mM L-glutamine + 1 mM sodium pyruvate) 1 h before the assay and incubated in 500 μL of the base medium in a non-CO₂ incubator at 37°C. OCRs were analyzed by sequential automatic injections of mitochondrial inhibitors, including 2.5 μM oligomycin, 2 μM FCCP, 0.5 μM rotenone, and 0.5 μM antimycin A. OCR (pmol/min) was measured according to the manufacturer’s instructions (Agilent Technologies, United States). The results were used to calculate basal respiration, maximal respiration, proton leakage, ATP production, and non-mitochondrial respiration. The results were normalized to 1 μg of protein determined by a BCA protein assay kit.

Fatty acid oxidation (FAO) was assessed by an Agilent Seahorse XF Substrate Oxidation Stress Test Kit (Agilent Technologies, United States), same with mitochondrial stress test. Except for OCR assessment, there are four reagents Etomoxir (Eto, 4 μM), oligomycin [1.5 μM), FCCP (2 μM), and antimycin A/rotenone (0.5 μM)] were added to the system, other operation of the experiment is similar to Mito Stress Test. Basal respiration, proton leakage, maximal respiration, ATP product and spare respiratory capacity were measured in a XF24 analyzer.

Mitochondrial morphology was visualized using Mito-Tracker^®, green (Beyotime, China) according to the manufacturer’s instructions. Cells were incubated with 25 nM MitoTracker green for 30 min at 37°C. Then, the cells were washed with PBS and imaged utilizing a confocal microscope (Nikon, Japan). The average fluorescence intensity was analyzed by NIS-Elements Viewer software (Japan).

Mitochondrial membrane potential (ΔΨm) was measured utilizing a mitochondrial membrane potential assay kit with JC-1 (Beyotime, China) according to the manufacturer’s instructions. Cells were incubated with JC-1 working solution for 30 min at 37°C. Then, the nuclei were stained with Hoechst 33342 (Beyotime, China) for 10 min at 37°C. Then, the cells were washed with PBS and imaged utilizing a confocal microscope (Nikon, Japan). At low ΔΨm, JC-1 is a green fluorescent monomer. At higher ΔΨm, JC-1 forms red fluorescent aggregates. The average fluorescence intensity was analyzed by NIS-Elements Viewer software. Data are shown as a ratio of red fluorescence intensity to green fluorescence intensity.

hiPSCs were cultured in a 96-well plate and then treated with various concentrations of AICAR for 7 days. CCK-8 solution (Solarbio, China) was added to each medium, and the conditions were maintained for 3 h at 37°C. Optical density was measured by a plate reader (BioTek, United States) at a wavelength of 450 nm. Every measurement was performed at least three times.

hiPSC-CMs were dissociated into single cells, washed with sterile DPBS and pelleted at 200 × g for 5 min. Following fixation in 4% paraformaldehyde, the cells were washed twice with DPBS, pelleted and resuspended in 200 μl of DPBS. Fixed hiPSCs and hiPSC-CMs were incubated with fluorescently labeled anti-cTnT antibodies (see Supplementary Table S1) for 1 h at room temperature, respectively. The cells were then washed three times. Analysis was performed on BD FACSCanto II cytometer, and the results were analyzed and plotted using FlowJo v10 software.

The data are expressed as the mean and standard error of the mean (SEM). Each measurement was performed at least three times. P-values were evaluated by unpaired t-test or one-way ANOVA using GraphPad Prism software (United States). For all analyses, a value of P < 0.05 was considered significant.

hiPSCs cultured on Matrigel-coated plates formed colonies with tight arrangement and clear boundaries according to microscopy (Supplementary Figure S1B). hiPSCs consistently stained positive for the pluripotent stem cell-specific markers Nanog and Sox2 (Supplementary Figure S1D). Then, hiPSCs were differentiated into cardiomyocytes using a monolayer protocol according to the cardiac differentiation method (Supplementary Figure S1A). hiPSC-CMs began spontaneous contraction after 8 days of differentiation. hiPSC-CMs were purified on day 14, digested, and replated on day 20. The cells grew in a monolayer and showed a clear cell shape on Matrigel-coated plates (Supplementary Figure S1D). The flow analysis indicated that metabolic purification led to a purified population containing more than 92% cTnT-positive cells by day 20 (Supplementary Figure S1C). Immunostaining demonstrated that hiPSC-derived CMs expressed CM-specific markers, sarcomeric α-actinin, Cx43, and cTnT (Supplementary Figure S1E). Immunofluorescence detection of MLC2v, and MLC2a (Supplementary Figure S1F) showed that hiPSC-CMs mainly consist of ventricular CMs and a few other atrial CMs.

The ultrastructural changes in mitochondria in hiPSCs and hiPSC-derived CMs were analyzed by transmission electron microscopy. Images of hiPSCs suggest that the mitochondria were granular with sparse cristae (Figure 1A). We found that the mitochondria were characterized by the presence of a few more mature, elongated, and large mitochondria with denser intramitochondrial cristae and a compacted matrix in hiPSC-CMs compared to those in hiPSCs. We also observed that hiPSC-CMs contain Z-bands and regular arrangement of myofibrils (Figure 1A). These findings are consistent with the data of previous studies on myocardial structure during cardiomyocyte differentiation (Hoque et al., 2018). The density of mitochondria in cardiomyocytes is high, which contributes to ATP production (Piquereau et al., 2013). To determine the metabolic status of hiPSCs and hiPSC-CMs, we measured the OCR of the cells by an XF24 extracellular flux analyzer, which reflects the cellular respiration capacity. The detection diagram is shown in Figure 1B. We observed that hiPSC-CMs had significantly higher OCRs associated with basal respiration, ATP production, maximal respiration, and proton leakage compared to those in hiPSCs (Figures 1C,D). An increase in these mitochondrial oxidation parameters indicated the maturation or enhancement of mitochondrial oxidative capacity.

AMPK is an energy-sensing protein kinase that plays a key role in the regulation of cellular energy metabolism (Herzig and Shaw, 2018); however, the role of AMPK in the maturation of hiPSC-CMs has not been defined. We examined the expression profile of AMPK during cardiac maturation of hiPSCs. The phosphorylation of AMPK was time-dependently increased in hiPSC-CMs (Figure 2A). Simultaneously, the protein levels of the downstream genes PGC-1α and CPT1α, which regulate energy metabolism, were dramatically increased (Figure 2A). The protein level of α-actinin was upregulated during cardiac differentiation of hiPSCs (Figure 2A). Additionally, the expression of PGC-1α (a crucial regulator of oxidative metabolism in cardiac development) and CPT1α (a regulator of fatty acid transport in the mitochondrial membrane) mRNAs was significantly time-dependently upregulated in the hiPSC-CM culture (Figures 2B,C). The mRNA expression levels of FABP3, FAT-CD36, and SLC27a6 were significantly increased during hiPSC-CM maturation to promote fatty acid transport and oxidation (Figures 2D–F). Hence, these data demonstrate that enhanced energy metabolism of hiPSC-CMs is related to AMPK phosphorylation and that activation of AMPK may be an effective approach to promote hiPSC-CM maturation.

To determine whether AMPK activation promotes energy metabolism and maturation of hiPSC-CMs, we treated hiPSC-CMs after replating for 7 days with AICAR (AMPK activator) at various concentrations (0, 0.1, 0.5, and 1 mM). Western blot was used to detect the protein expression of AMPK and phospho-AMPK, and CCK-8 assay was used to assess cell viability. As shown in Figures 3A,B, the activation effect of AICAR on AMPK was the strongest at a concentration of 0.5 mM and did not show significant cytotoxicity. This treatment condition was used in the following experiments. AMPK activation was reported to modulate glucose and fatty acid metabolism and mitochondrial function (Qi and Young, 2015). Therefore, hiPSC-CMs (day 23) were treated with DMSO (control) or AICAR at a concentration of 0.5 mM for 7 days and then subjected to biological and functional analysis on day 30. As shown in Figure 3C, AICAR increased the expression of phosphorylated AMPK and did not change the total AMPK protein levels. With regard to the effects of AMPK on the expression of the downstream metabolism-related targets, we found that AICAR increased the protein and transcript levels of PGC-1α, which improved mitochondrial biogenesis and metabolic maturation (Figures 3C,D; Liu et al., 2020). We observed significantly increased PPARα protein and mRNA levels in AICAR-treated cells compared to those in the control cells suggesting that AICAR treatment increased fatty acid β-oxidation (Figures 3C,D; Colasante et al., 2015). Additionally, AICAR treatment significantly increased the expression of ERRα (Figures 3C,D), which plays a key role in mitochondrial fatty acid oxidation (Alaynick, 2008). Western blot analysis revealed that the expression of fatty acid β-oxidation (CPT-1α, CPT-1β, and SLC24a6) and mitochondrial OxPhos markers (COX5b, COXIV, Cyt-c, C-S, and MPC1) was significantly upregulated in hiPSC-CMs following AICAR treatment compared to those in the control (Figures 4A,C). AICAR treatment significantly increased the mRNA expression of fatty acid β-oxidation (CPT-1α, CPT-1β, FABP3, FAT-CD36, SLC27a6, SLC25a20, LCAD, and MCAD) and mitochondrial OxPhos markers (COX5b, ATP5a, and Cyt-c) (Figures 4B,D).

We then used the same hexokinase assay to compare the glycolysis rates of hiPSC-CMs treated with AICAR or control. hiPSC-CMs treated with AICAR had significantly lower hexokinase activities compared with control group (Figure 4E). Glycolysis results in the conversion of glucose into pyruvate, which is subsequently reduced into lactate. To further validate that the increase in hexokinase activity is because of an increase in glycolysis, we compared the total cellular lactate levels in cardiomyocytes using a commercial lactate assay kit (Figure 4F). hiPSC-CMs treated with AICAR had significantly lower lactate levels compared with control group. hiPSC-CMs treated with AICAR had reduced glycolysis rates as would be expected during myocardial maturation.

To explore the effect of AICAR treatment on the morphology and gene expression of hiPSC-CMs, the cells with antibodies to α-actinin. In the AICAR treatment and control groups, the results of α-actinin immunostaining showed a clear sarcomere structure (Figure 5A). Additionally, AICAR-treated hiPSC-CMs had longer sarcomeres and lower circularity index than those in the control group (Figures 5B–E). Immunostaining demonstrated that In the AICAR treatment and control groups both expressed CM-specific markers TNNI3, and MYH7 (Supplementary Figures S2B,C).

We found that the protein levels of α-actinin, TNNI3, and MYH7 were significantly increased in AICAR-treated cells compared with that in the control cells (Figure 5F). We also analyzed the effect of AICAR treatment on the gene expression profiles of hiPSC-CMs. RT-PCR results showed that most CM-related sarcomere protein-encoding genes (TNNI3, TNNT2, MYH7, MYL3, MYL4, MYBPC3, and CX43) were significantly upregulated in AICAR-treated hiPSC-CMs (Figure 5G). Additionally, AICAR treatment induced the expression of calcium voltage-gated channel subunit (CACNA1C), potassium voltage-gated channel subfamily genes (KCND2, KCNE1, KCNJ2, KCNQ1, and KCNH2), and sodium voltage-gated channel subunit gene (SCN5A) (Figure 5H). These data suggest that AICAR treatment promotes the maturation process of hiPSC-CMs in multiple aspects, including cell morphology, sarcomeric structure, ultrastructural change, and gene expression.

The mitochondrion is an energy center of cardiomyocytes that controls cardiomyocyte metabolism and survival by constantly providing ATP. In the process of development, mitochondria have evolved in morphology and function (Yang et al., 2014b). We used JC-1 dye as an indicator of mitochondrial membrane potential (ΔΨm) to validate the changes in mitochondrial function. Results of confocal microscopy showed that AICAR treatment induced an overall increase in ΔΨm as compared to that in the control (Figure 6A). To characterize mitochondrial energy metabolism, we investigated the effect of AICAR treatment on mitochondrial respiration by the addition of oligomycin, FCCP, rotenone, and antimycin A (Figure 6C). AICAR treatment significantly increased the OCRs associated with basal respiration, maximal respiration, spare respiratory capacity, and ATP production (Figure 6D). Additionally, ATP production was increased by AICAR treatment of hiPSC-CMs (Figure 6B). In the Substrate Oxidation Stress Test, Etomoxir, a specific inhibitor of carnitine palmitoyl transferase 1α (CPT1α), was used to specifically inhibit mitochondrial FAO. Treatment with ETO abolished OCR increases in AICAR-treatment hiPSC-CMs. The increased fatty acid oxidation energy was shown by the decrease in OCR upon incubation with etomoxir in hiPSC-CMs treated with AICAR (Figure 6E). The maximum respiration rate, and the production of ATP were lower in the AICAR-treated hiPSC-CMs than that in the control group (Figure 6F). Thus, these data suggest that activation of AMPK may enhance mitochondrial oxidative capacity and promote metabolic maturation of hiPSC-CMs.

Then, hiPSC-CMs treated with AICAR or control were imaged using transmission electron microscopy to visualize the cellular ultrastructure and mitochondrial morphology. We observed that the number of mitochondria was increased in AICAR-treated hiPSC-CMs and that AICAR-treated hiPSC-CMs contained more mitochondrial crista than those in the control (Figure 7A). In control and AICAR-treated hiPSC-CMs, the myofibrils were plentiful and well-organized, and the Z-lines of the muscle fibers were visible (Figure 7A). Imaging of the cellular distribution of mitochondria using MitoTracker revealed disparate mitochondrial morphologies between control and AICAR-treated hiPSC-CMs; the former contained isolated mitochondria with fragments, and the latter consisting of an extensively interconnected filamentous network (Figure 7B). The mean fluorescence intensity of MitoTracker in AICAR-treated hiPSC-CMs was increased compared to that in the control (Figure 7B). Moreover, western blot analysis showed that mitochondrial fusion proteins MFN1 and MFN2 were upregulated, and a fission protein was downregulated compared with those in the control (Figure 7C). To determine whether AICAR treatment influenced the mitochondrial DNA content, we detected the mtDNA copy number by qPCR. AICAR treatment induced upregulation of the mitochondrial DNA to nuclear DNA ratio (Figures 7D,E). Overall, these data indicate that activation of AMPK increases mitochondrial fusion events leading to mitochondrial network expansion.