Graphical abstract

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Highlights

     * •
       MAPK/PI3K/AKT signaling decreases across time in human ventricular
       heart tissue
     * •
       A 5 day treatment of hiPSC-CMs with MAPK/PI3K/AKT inhibitors
       advances maturation
     * •
       CM maturation reached with inhibitors is superior to untreated
       older cultures
     __________________________________________________________________

   In this article, Garay and colleagues show that the MAPK and PI3K/AKT
   signaling pathways are downregulated in the adult ventricular heart
   tissue. Inhibition of these signaling pathways in vitro, for only
   5 days, can enhance the maturation status of human iPSC-derived
   cardiomyocytes across multiple domains. This short protocol opens the
   possibility for synergistic use with other known inducers of cardiac
   maturation to potentially reach adult-level maturation.

Introduction

   Cardiovascular disease is the leading cause of morbidity and mortality
   worldwide ([61]Roth et al., 2020). Human induced pluripotent stem cell
   (hiPSC)-derived cardiomyocytes (hiPSC-CMs) hold great promise for drug
   discovery and heart disease modeling. Unfortunately, traditional
   methods of CM differentiation produce cells that show an early fetal
   transcriptional, epigenetic, and metabolic profile, thus limiting their
   application for diseases of the adult heart.

   CM maturation is a complex and finely tuned process from prenatal to
   postnatal stages. During the embryonic and fetal periods of heart
   development, the expression of many sarcomeric genes becomes temporally
   and topographically restricted. For example, expression of the
   ventricular isoform of myosin light chain (MLC-2v) is detected in the
   primitive heart several days after expression of the atrial isoform
   (MLC-2a) ([62]Franco et al., 1998) but becomes restricted to the
   ventricular chambers in the adult heart ([63]Iorga et al., 2017). As
   such, the MLC-2v/MLC-2a ratio is commonly used as a maturation
   benchmark of ventricular hiPSC-CMs. Similarly, the transition from the
   fetal to neonatal and adult heart can be traced by the stoichiometric
   molecular switch from ssTnI (slow skeletal troponin I), present in the
   fetal heart; to the neonatal heart, which expresses both ssTnI and cTnI
   (cardiac troponin I); to the adult heart, which expresses only cTnI
   ([64]Bedada et al., 2014). Postnatally, human CMs show an increase in
   cell volume (hypertrophy), multinucleation, and a decrease in
   proliferation ([65]Bergmann et al., 2015). Structurally, the sarcomere
   length (SL) during diastole increases to an average of 2.25 μm
   ([66]Sonnenblick et al., 1967). These structural changes are intimately
   coupled to a series of physiologic changes that adapt the heart to work
   under increasing pressure, including improved calcium (Ca^2+) handling
   ([67]Høydal et al., 2018) and a metabolic switch from glycolysis to
   oxidative phosphorylation (OXPHOS) ([68]Stanley et al., 2005).

   To date, numerous protocols have been developed to improve the
   maturation status of hiPSC-CMs, ranging widely in the type of
   intervention and ease of implementation. Early studies showed that
   increased culture time ([69]Lundy et al., 2013) greatly improved
   several morphological features, such as SL, multinucleation, and cell
   surface area. Others have shown that electrical pacing ([70]Chan
   et al., 2013), 3-dimmensional tissues ([71]Ronaldson-Bouchard et al.,
   2018), and soluble factors ([72]Yoshida et al., 2018) can enhance CM
   maturation. Unfortunately, most of these methods require sophisticated
   platforms, equipment, and expertise that are not as readily affordable
   or accessible, making their wide application more limited.

   In this study, we curate multiomic datasets from both hiPSC-CMs and
   human cardiac tissue to gain insights into signaling pathways that
   regulate CM maturation. We show that inhibition of the
   mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase
   (PI3K)-AKT pathways on hiPSC-CMs, for only 5 days, results in enhanced
   maturity in many domains: transcription, structure, metabolism and
   electrophysiology. Therefore, these results represent a short and
   robust protocol to reach day 60 maturation levels in day 30 hiPSC-CMs.

Results

Signaling pathways downregulated in the adult heart

   To better understand the signaling pathways that regulate cardiac
   maturation, we retrieved transcriptomic and epigenomic datasets from
   hiPSC-CMs and human heart tissue across the lifespan ([73]Figure 1A;
   [74]Table S1). We focused on protein-coding genes and loci of human
   ventricular tissue to facilitate comparative analysis with ventricular
   hiPSC-CMs generated via Wnt modulation ([75]Lian et al., 2012).
   Principal-component (PC) analysis showed distinct separation between
   hiPSC-CMs and prenatal and postnatal human heart samples
   ([76]Figures 1B and [77]S1A), with in vitro CMs clustering closer to
   the fetal samples than to the adult ones ([78]Figures S1B–S1D), in
   agreement with previous observations ([79]Uosaki et al., 2015). Of
   note, the 3-year-old child samples clustered together with the adult
   samples ([80]Figures S1A–S1D). Pairwise comparisons identified 10,597
   and 11,571 differentially expressed genes (DEGs) between adult versus
   fetal, and adult versus in vitro, respectively, with 7,736 DEGs in
   common ([81]Figure 1C). Unsupervised hierarchical clustering of the top
   1,000 DEGs shared among all groups revealed three distinct clusters
   ([82]Figures 1D and [83]S1E). Gene Ontology (GO) analysis of these
   clusters showed enriched genes related to canonical Wnt pathway
   (cluster 1), lipid transport (cluster 2), and OXPHOS (cluster 3).
   Similarly, gene set enrichment (GSE) analysis of adult versus in vitro
   CMs revealed that the Wnt/Hippo signaling pathways and cell cycle are
   significantly downregulated in the adult, while OXPHOS, peroxisome
   proliferator-activated receptor (PPAR) signaling pathway, and fatty
   acid degradation are significantly upregulated in the adult
   ([84]Figure S1F).

Figure 1.

   [85]Figure 1
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   Signaling pathways downregulated in the postnatal heart

   (A) Scheme for comparative analyses of RNA-seq and H3K27ac ChIP-seq.

   (B) Principal-component analysis for adult (orange), fetal (purple),
   and in vitro (green) samples.

   (C) Venn diagram of RNA-seq pairwise comparisons showing DEGs.

   (D) RNA-seq hierarchical clustering of the top 1,000 DEGs shared among
   all samples. Clusters are indicated on left margin. C, 3-year-old
   child.

   (E) Volcano plot for coding genes.

   (F) ChIP-seq hierarchical clustering for 25,149 loci with differential
   H3K27ac deposition. Clusters are indicated on left margin. E, embryo
   (CS22).

   (G) ChIP-seq tracks for M protein (MYOM2, cluster 2) and p38α (MAPK14,
   cluster 3) in pooled biological replicates.

   (H) Venn diagram for DEGs shared among ChIP-seq, clusters 1 and 3, and
   RNA-seq.

   (I) Gene-network plot of KEGG pathway enrichment analysis of the most
   downregulated genes in the adult heart, with cluster size scale at the
   bottom right.

   Globally, cell cycle-related genes, such as CDK1 and AURKB, are
   downregulated in the adult, while structural and metabolic genes, such
   as MYOM2 and CD36, are upregulated in the adult compared with fetal and
   in vitro CMs ([87]Figure 1E). Similarly, several MAPK genes, such as
   MAP2K6 and MAP3K1, or upstream activators of the pathway, such as FGF13
   ([88]Lin et al., 2019) and IGF2BP3 ([89]Suvasini et al., 2011), show
   significant downregulation postnatally. Meanwhile, negative regulators,
   such as the dual-specificity phosphatases DUSP1 and DUSP6 ([90]Owens
   and Keyse, 2007), are significantly upregulated in the adult compared
   with fetal and in vitro CMs ([91]Figure 1E).

   To further understand these developmentally determined transcriptional
   profiles, we looked at the loci with differential expression of histone
   3 lysine 27 acetylation (H3K27ac) marks, thereby suggesting epigenetic
   activation or repression of such genes. We found 3,998 loci with
   significantly reduced H3K27ac sites in the adult compared with the
   prenatal samples (clusters 1 + 3; [92]Figures 1F and 1H). GO analysis
   of these clusters revealed enriched genes related to Wnt, Hippo, and
   BMP signaling pathways (cluster 1) and heart morphogenesis (cluster 3;
   [93]Figure S2A). A closer inspection of the chromatin
   immunoprecipitation sequencing (ChIP-seq) tracks revealed that some
   MAPK genes, such as MAPK14 encoding p38α, have increased H3K27ac signal
   early in development, but they are decreased in the postnatal period
   ([94]Figures 1G and [95]S2B). On the other hand, hiPSC-CMs do not show
   a decrease in H3K27ac at the MAPK14 locus even after 80 days in
   culture. In contrast, structural genes, such as MYOM2, turn on only in
   adulthood ([96]Figure 1G).

   After combining both RNA sequencing (RNA-seq) and ChIP-seq datasets, we
   found a narrow list of 1,452 genes that were shared ([97]Figure 1H).
   Over-representation analysis of these genes using Kyoto Encyclopedia of
   Genes and Genomes (KEGG) shows that the MAPK pathway (q = 0.0137),
   along with the Hippo (q < 0.0001) and Wnt (q = 0.0002) pathways are
   significantly enriched ([98]Figure 1I). These findings suggest that
   these pathways are downregulated in the adult. Upon closer inspection,
   39% of the genes annotated to the MAPK pathway were also shared with
   the PI3K-AKT pathway ([99]Figure S2C). The MAPK and PI3K-AKT pathways
   have been shown to regulate cardiac hypertrophy postnatally, but it
   remains unknown whether MAPK and PI3K-AKT signaling is more broadly
   implicated in regulation of CM maturation.

Sarcomeric protein expression switch upon MAPK and PI3K-AKT inhibition

   To investigate whether inhibition of the MAPK/PI3K/AKT pathways could
   promote hiPSC-CM maturation, we generated ventricular CMs from three
   independent hiPSC lines, using the Wnt modulation protocol ([100]Lian
   et al., 2012). After enrichment with DL-lactate ([101]Burridge et al.,
   2014), we determined if MAPK/PI3K/AKT inhibition could enhance MLC-2v^+
   expression as a marker of maturation. We tested the dose response to
   SB203580, a p38 and phosphoinositide-dependent protein kinase-1 (PDK1)
   inhibitor ([102]Kumar et al., 1997; [103]Lali et al., 2000) (referred
   to hereafter as S), and PD0325901, a MEK1/2 ([104]Allen et al., 2003)
   (referred to hereafter as P), separately, on MLC-2v^+ expression from
   days 25–30 ([105]Figure S3A). Here, we found that 5 μM S
   ([106]Figures S3B and S3D) and 10 μM P ([107]Figures S3C and S3E) were
   optimal for increasing MLC-2v^+ expression. We confirmed that these
   concentrations resulted in inhibition of the signaling pathways
   ([108]Figures S3F and S3G). We then compared the effects of these small
   molecules alone (S or P) or in combination (SP) at their optimized
   concentration ([109]Figures 2A–2D and [110]S3H). Although the
   expression of cardiac troponin T (cTnT) was similarly high across all
   three hiPSC-CM preparations, we found that the treatment with SP
   yielded CMs with even greater cTnT^+MLC-2v^+ expression than the single
   treatments ([111]Figure 2C). More important, treatment with SP was
   found to be optimal for enhancement of the MLC-2v^+/MLC-2a^+ expression
   ratio ([112]Figure 2D), a feature of ventricular CM maturation.
   Independently, we then confirmed that days 25–30 were indeed the ideal
   time window and duration for this isoform switch ([113]Figures S3I and
   S3J). Taken together, these data suggest that treatment with SP
   accelerated the switch in myosin light chain isoform expression to the
   more mature MLC-2v isoform.

Figure 2.

   [114]Figure 2
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   Inhibition of MAPK/PI3K/AKT improves the expression of adult isoform
   sarcomeric proteins in day 30 hiPSC-CMs to day 60 levels

   (A) Scheme for ventricular CM differentiation and treatment with
   MAPK/PI3K/AKT inhibitors.

   (B) Representative flow cytometry plots of day 30 hiPSC-CMs show cTnT^+
   (upper panel) and the cTnT^+ MLC-2v^+ (lower panel) population.

   (C) Bar graph shows summary of fluorescence-activated cell sorting
   (FACS) analysis for cTnT^+MLC-2v^+ hiPSC-CMs per treatment at day 30.

   (D) Graph shows MLC-2v^+/MLC-2a^+ ratio by FACS on day 30 hiPSC-CMs.

   (E) Representative FACS plot for cTnT^+MLC-2v^+ expression per
   condition.

   (F) Bar graph shows the frequency of cTnT^+MLC-2v^+ in day 30 hiPSC-CMs
   treated with SP or DMSO, compared with untreated day 60 CMs as a
   reference.

   (G and H) Western blot for cTnI, ssTnI, and α-tubulin (G) and
   (H) quantification of stoichiometric relationship in day 30 DMSO- or
   SP-treated hiPSC-CMs.

   Data are presented as mean ± SEM. For (C) and (D), n = 3 (PLZ/19004) or
   5 (MYL2) independent experiments per condition using all 3 cell lines;
   for (F), n = 3 independent experiments per condition using all 3 cell
   lines; for (G), n = 3 (PLZ/MYL2) independent experiments per condition.
   ns, p > 0.05; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

   To better contextualize these changes in isoform expression after
   treatment with SP, we carried out a new round of experiments in which
   we compared the day 30 SP-treated hiPSC-CMs with their untreated day 60
   counterparts ([116]Figures 2E, 2F and [117]S3K). Flow cytometry
   analysis of the cTnT^+MLC-2v^+ population showed no statistical
   difference between day 30 SP-treated and day 60 hiPSC-CMs from the same
   cell preparation ([118]Figure 2F), while both groups were found to have
   a higher percentage of cTnT^+MLC-2v^+ hiPSC-CMs compared with day 30
   DMSO control. We confirmed these results with immunofluorescence (IF)
   studies ([119]Figures 3A and [120]S3L–S3O). Indeed, the frequency of
   the cTnT^+MLC-2a^+MLC-2v^− population significantly decreased in the
   day 30 SP-treated group to levels similar to day 60 compared with day
   30 DMSO controls, whereas the cTnT^+MLC-2a^−MLC-2v^+ population
   significantly increased to similar levels in both the SP-treated day 30
   and day 60 hiPSC-CMs ([121]Figure S3L). No differences were observed in
   the intermediary cTnT^+MLC-2a^+MLC-2v^+ population among any of the
   treatment groups ([122]Figure S3L). Taken together, these results
   indicate that the increase in MLC-2v expression after a 5 day treatment
   with SP at day 30 is equivalent to the expression levels reached after
   60 days in culture.

Figure 3.

   [123]Figure 3
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   Morphological improvements in day 30 hiPSC-CMs upon MAPK/PI3K/AKT
   inhibition

   (A) Representative IF images for α-actinin-2, MLC-2v, MLC-2a, DAPI, and
   merge for DMSO- and SP-treated day 30 hiPSC-CMs as well as untreated
   day 60 CMs. Inset indicates representative mean for SL. Scale bar,
   20 μm.

   (B) Violin plot of SL with mean values represented by black line.

   (C–F) Bar graphs show quantification data for cell surface area (C),
   cell perimeter (D), length-to-width ratio (E), and nucleation levels
   (F).

   (G) Representative 3D volume rendering of T-tubules in day 30 DMSO- and
   SP-treated hiPSC-CMs.

   (H and I) Quantification of T-tubule density (H) and (I) CM volume.

   Data are presented as mean ± SEM of 3 independent experiments using all
   three cell lines (PLZ/19004/MYL2). For (G–I), n = 4–10 cells per
   experiment, 3 independent experiments in MYL2 line. ^∗p < 0.05,
   ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

   Given the observed isoform switch from MLC-2a to MLC-2v, we
   investigated whether treatment with SP could also have an impact on the
   molecular switch of TnI isoforms, a different maturation marker. Here,
   we found that SP-treated hiPSC-CMs had higher expression of the adult
   TnI isoform and lower expression of the fetal TnI isoform compared with
   DMSO-treated controls ([125]Figures 2G, 2H, and [126]S3P). Altogether,
   these results indicate that treatment of hiPSC-CMs with SP from days
   25–30 can engage the molecular switch driving adult isoform replacement
   of sarcomeric proteins.

Structural features of hiPSC-CMs upon MAPK/PI3K/AKT inhibition

   To elucidate the effects of MAPK/PI3K/AKT inhibition beyond sarcomeric
   protein expression, we conducted morphological studies, which revealed
   that changes in SL were accompanied by hypertrophy. Assessment of SL,
   across all the cell lines, showed changes from a baseline of 1.64 ±
   0.01 μm in day 30 DMSO-treated controls to 2.02 ± 0.01 μm in day 30
   SP-treated hiPSC-CMs ([127]Figures 3A, 3B, and [128]S4A). Surprisingly,
   these enhancements in SL were also higher than those observed on the
   day 60 hiPSC-CMs ([129]Figure 3B).

   We observed a similar pattern in other key structural parameters
   relevant to CM maturation. Here, we observed an increase in the cell
   surface area, perimeter, and cell length-to-width ratios in both day 30
   SP-treated and day 60 hiPSC-CMs relative to day 30 DMSO-treated
   controls ([130]Figures 3C–3E and [131]S4B–S4D). As observed with SL,
   the changes detected in surface area were greater in day 30 SP
   treatment than day 60 hiPSC-CMs ([132]Figures 3C and [133]S4B).

   When we looked at the number of nuclei per CM, another parameter of
   maturation status, we observed a significant increase in
   multinucleation proportional to the decrease in mononucleation. Similar
   to day 60 hiPSC-CMs, the percentage of mononucleated CMs decreased in
   day 30 SP-treated hiPSC-CMs relative to day 30 DMSO-treated controls by
   ∼10% ([134]Figures 3F and [135]S4E). In contrast, the percentage of
   binucleation and trinucleation of day 30 SP-treated hiPSC-CMs increased
   by ∼9 and ∼1%, respectively, similar to day 60 hiPSC-CMs
   ([136]Figure 3F). Of note, in all instances, there were no
   statistically significant differences between day 30 SP-treated and day
   60 hiPSC-CMs, but there was a trend toward greater improvement in the
   day 30 SP-treated group compared with day 60.

   Finally, a hallmark of structural changes associated with maturation is
   the development of transverse tubules (T-tubules), which regulate the
   excitation-contraction coupling and Ca^2+ handling under physiological
   conditions. To our surprise, we found that even day 30 DMSO-treated
   controls had well-defined T-tubules. Their density increased
   significantly upon treatment with SP from 0.15 ± 0.01 to 0.24 ±
   0.01 μm^2/μm^3 ([137]Figures 3G and 3H). This increase in T-tubule
   density was also accompanied by a doubling of CM volume
   ([138]Figure 3I). Altogether, these data suggest that despite a similar
   expression pattern of sarcomeric proteins between day 30 SP-treated and
   day 60 hiPSC-CMs, there are structural changes that go beyond day 60
   during the 5 day treatment period with SP.

Transcriptional profile of hiPSC-CMs upon MAPK/PI3K/AKT inhibition

   To more comprehensively understand the effects of MAPK/PI3K/AKT
   inhibition on the maturation status of hiPSC-CMs, we conducted bulk
   RNA-seq analysis on day 30 SP-treated hiPSC-CMs and compared these with
   their day 30 DMSO-treated controls as well as day 90 hiPSC-CMs. We used
   day 90 as a more stringent reference of maturation considering that
   some maturation parameters improved beyond day 60 levels after
   treatment with SP. As shown in [139]Figures 4A and 4B, GSE analysis of
   KEGG pathways of day 30 DMSO- versus SP-treated hiPSC-CMs revealed
   significant upregulation of the cholesterol metabolism pathway
   (normalized enrichment score [NES] = +1.63), with CD36 and APOA1 being
   highly represented. In contrast, we observed significant downregulation
   of (1) NF-κB (NES = −1.47) in genes including PTGS2 and VCAM1, (2)
   PI3K-AKT (NES = −1.44) in genes including THBS1 and EPHA2, and (3)
   TGF-β (NES = −1.54) in genes including TGFB2 and LEFTY2. These data
   suggest that the effects observed upon inhibition of the MAPK/PI3K/AKT
   pathways could be a result of concomitant downregulation of key
   interacting signaling pathways such as NF-κB and TGF-β.

Figure 4.

   [140]Figure 4
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   Transcriptional signatures of hiPSC-CMs upon MAPK/PI3K/AKT inhibition

   (A) GSE analysis with upregulated and downregulated KEGG pathways
   (arrows).

   (B) Gene-network plot for differentially expressed KEGG pathways.

   (C) Venn diagram of pairwise comparison DEGs.

   (D) Principal-component analysis of adult, 3-year-old child, fetal, and
   day 30 DMSO- and SP-treated hiPSC-CMs with 95% confidence interval.

   (E) Hierarchical clustering of 308 shared DEGs; clusters on left
   margin.

   (F) Cluster 1 GO analysis.

   (G) Hierarchical clustering of selected genes annotated to cell
   division/proliferation biological processes.

   (H) Bar plot with log-transformed counts per million values for MKI67,
   each dot is an independent cell line.

   (I) Summary of myosin heavy chain (MHC^+) and Ki-67^+ FACS data; n = 3
   independent experiments per condition for each cell line
   (PLZ/19004/MYL2).

   (J) Representative IF images of cTnT and phospho-histone H3 (pHH3)
   staining. Scale bar, 500 μm.

   (K) Summary dot-plot quantification of cTnT^+ pHH3^+ nuclei. n = 30
   technical replicates, 4 independent experiments per condition from a
   representative cell line (PLZ).

   Data are presented as mean ± SEM; n = 1–3 independent experiments per
   cell line unless specified. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and
   ^∗∗∗∗p < 0.0001.

   Pairwise comparisons identified 713 and 2,402 DEGs between day 30 DMSO-
   versus SP-treated and day 30 DMSO-treated versus day 90 hiPSC-CMs,
   respectively, with 308 DEGs in common ([142]Figure 4C). Using this
   defined list of DEGs, we conducted principal-component analysis and
   found the greatest axis of variation to be that separating fetal CMs
   from child/adult. Notably, day 30 DMSO-treated controls clustered with
   fetal samples, while day 30 SP-treated hiPSC-CMs clearly shifted to the
   right on PC1, the maturation axis, demonstrating a shift away from
   fetal state in the direction of the adult state ([143]Figure 4D).
   Unsupervised hierarchical clustering of this core gene list revealed
   four main clusters of DEGs where the two largest clusters (1 and 3)
   have a similar expression pattern between day 30 SP-treated and day 90
   hiPSC-CMs ([144]Figure 4E). GO analysis of cluster 1 showed significant
   enrichment of genes related to cell division and ERK1/2 cascade
   ([145]Figure 4F). Inspection of the genes annotated to the largest and
   most significant category of biological process revealed significant
   downregulation of AURKB and MKI67, which are involved in cell
   proliferation ([146]Figures 4G and 4H).

Proliferation status of hiPSC-CMs upon MAPK/PI3K/AKT inhibition

   Given that the cell proliferation status is another key CM maturation
   marker and we observed transcriptional signatures in this domain, we
   conducted experiments to determine whether there was any change in
   proliferation upon MAPK/PI3K/AKT inhibition. We first assessed our
   enriched population of CMs for Ki-67 expression, a proliferation marker
   present in all stages of the cell cycle. We observed a significant
   reduction in Ki-67^+ expression from 19.1% ± 1.3% to 7.0% ± 1.2% upon
   treatment with SP ([147]Figure 4I, right panel; [148]Figures S4G–S4I)
   across all three cell lines. Here, we also observed no statistically
   significant difference between our day 30 SP-treated and day 60
   untreated reference ([149]Figure 4I, right panel; [150]Figure S4H).
   These results were further validated with IF studies of phospho-histone
   H3 (pHH3) expression, an M-phase marker, which revealed a significant
   decrease in the percentage of cTnT^+ pHH3^+ nuclei in day 30 SP-treated
   hiPSC-CMs relative to DMSO controls ([151]Figures 4J and 4K), and this
   decrease was also found to be greater than day 60 untreated CMs
   ([152]Figure 4K). These results suggest that there is a decrease in CM
   proliferation upon inhibition of MAPK/PI3K/AKT signaling, consistent
   with enhanced maturation of CMs.

Force profile of hiPSC-CMs upon inhibition of MAPK/PI3K/AKT

   We next investigated whether there were any changes in contractile
   force generation, a physiological marker of maturation, upon treatment
   with SP. Toward this end, we conducted single-cell force measurements
   using traction force microscopy on micropatterned CMs ([153]Figures 5A,
   5B, and [154]S5A) in day 30 DMSO- and SP-treated CMs as well as day 60
   untreated reference. Here, we found no significant differences in force
   generation between DMSO- and SP-treated CMs across two different hiPSC
   lines ([155]Figures 5C and [156]S5B). Similarly, we found no
   significant increase in force generation in the day 60 untreated cohort
   ([157]Figures 5C and [158]S5B). These data suggest that under our
   minimal culture conditions, there is no increase in force generation
   after 60 days in culture and that the MAPK/PI3K/AKT pathways do not
   contribute to force generation despite a robust increase in contractile
   sarcomeric proteins.

Figure 5.

   [159]Figure 5
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   Traction force profile and metabolic shift upon MAPK/PI3K/AKT
   inhibition

   (A) Bright-field image of micropatterned hiPSC-CM (cell outlined by a
   white dashed line) and heatmaps of the cell-induced substrate surface
   tractions.

   (B) Total traction force exerted by the representative CM in the
   direction of its long and short axes over 3 s.

   (C) Normalized contraction force in day 30 DMSO- and SP-treated groups
   compared with day 60 untreated CMs.

   (D–F) Mean kinetic profile of OCR and ECAR from day 30 DMSO- and
   SP-treated and day 60 untreated hiPSC-CMs.

   (G–L) Dot-plot quantification of (G) basal, (H) non-mitochondrial,
   (I) proton leak, (J) ATP-linked, (K) percentage spare capacity OCR, and
   (L) basal ECAR.

   (M and N) Normalized gene expression of (M) HK1 and (N) PKM.

   (O) Representative transmission electron micrograph of mitochondria on
   hiPSC-CMs; scale bar, 0.6 μm.

   (P) Quantification of mitochondrial cross-sectional area.

   (Q and R) Normalized gene expression of (Q) ESRRG and (R) PPARGC1B.

   (S and T) Representative FACS plot for ROS measurement (S) and
   (T) quantification of mean fluorescence intensity (MFI).

   Data are presented as mean ± SEM. For (C), n = 6–14 cells per
   experiment, 3 independent experiments in PLZ/MYL2; for (D)–(L), n =
   8–48 technical replicates per experiment per condition per line, 3
   independent experiments in PLZ/19004/MYL2; for (M), (N), (Q), and (R),
   n = 3 independent experiments per condition in PLZ/19004/MYL2; for (P),
   n = 20–40 mitochondria per cell, 4 or 5 cells per condition, 1
   independent experiment in MYL2; for (T), n = 3 independent experiments
   in PLZ. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Metabolic profile upon MAPK/PI3K/AKT inhibition

   We next wanted to interrogate the metabolic status of hiPSC-CMs in the
   context of MAPK/PI3K/AKT inhibition. We conducted Seahorse
   mitochondrial stress test studies to look at oxygen consumption rate
   (OCR), an indicator of aerobic respiration, with simultaneous
   measurements of the extracellular acidification rate (ECAR), an
   indicator of glycolytic flux, in our day 30 DMSO- and SP-treated CMs
   with their day 60 untreated references. Here, we found that there was a