Original Article

Proteomic Analysis of Human Pluripotent Stem Cell–Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and MaturationCLINICAL PERSPECTIVE

Ellen Poon, Wendy Keung, Yimin Liang, Rajkumar Ramalingam, Bin Yan, Shaohong Zhang, Anant Chopra, Jennifer Moore, Anthony Herren, Deborah K. Lieu, Hau San Wong, Zhihui Weng, On Tik Wong, Yun Wah Lam, Gordon F. Tomaselli, Christopher Chen, Kenneth R. Boheler, Ronald A. Li
https://doi.org/10.1161/CIRCGENETICS.114.000918
Circulation: Genomic and Precision Medicine. 2015;8:427-436
Originally published March 10, 2015

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Abstract

Background—Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs), but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived VCMs, human fetal and human adult ventricular and atrial CMs.

Methods and Results—Using two-dimensional–differential-in-gel electrophoresis, 121 differentially expressed (>1.5-fold; P<0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator–activated receptor α signaling in cardiac maturation. Consistently, WY-14643, a peroxisome proliferator–activated receptor α agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold, signifying their maturation.

Conclusions—We conclude that the peroxisome proliferator–activated receptor α and thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

Introduction

Human pluripotent stem cells, such as embryonic stem cells (ESC) and induced pluripotent stem cells, self-renew; their differentiation to the cardiac lineage1–6 represents a potentially unlimited source of ventricular cardiomyocytes (VCMs) for therapies and as experimental models to investigate mechanisms involved in human cardiac development and disease progression. Successful application of human ESC (hESC)-ventricular cardiomyocytes (VCMs) to myocardial repair or modeling requires that the cells faithfully recapitulate the phenotype of adult CMs, especially in relation to their electrophysiological, contractile, and metabolic functions.7 Indeed, hESC-CMs are most similar to human embryonic or human fetal (hf) CMs,5,8–11 and with a disorganized sarcomeric arrangement.10,12 Microarray-based experiments have been previously performed to characterize the expression profile of hESC–CMs, purified for ventricular lineage or not, for identifying signaling pathways implicated in their differentiation.13–17 All profiling attempts to date almost exclusively relied on transcriptomic data13–17 or cell surface protein assessments,18 but the global proteome of hESC-VCMs has not been examined. Furthermore, most hESC-VCM studies focus on pathways important for cardiac differentiation, rather than mechanisms implicated in their developmental maturation. In brief, previous results mostly indicate that hESC-CMs are relatively immature compared with fetal and adult hearts, express lower levels of contractile and metabolic genes and show differential expression of specific cardiac ion channels, complementary to known functional data.13–17 For obtaining a better understanding, here we used a combinatorial approach of performing proteomic, bioinformatics, and functional analyses of hESCs, hESC-derived VCMs, hf (18 weeks), and human adult (hA) VCMs to identify novel proteins and pathways important for maturation.

Clinical Perspective on p 436

Methods

Culturing of hESCs, Cardiac Differentiation, and Selection of hESC-VCMs

Undifferentiated hESC (hES2)19 were maintained at 37°C and 5% CO2 on Matrigel (BD Biosciences)-coated wells with mTeSR medium (Stem Cell Technologies). For cardiac differentiation, growth factors were added at specific stages of differentiation.20 The following cytokines were used: days 0 to 1, bone morphogenetic protein 4 (0.5 ng/mL); days 1 to 4, bone morphogenetic protein 4 (10 ng/mL), β fibroblast growth factor (5 ng/mL), and activin A (3 ng/mL); days 4 to 8, Dickkopf 1 (150 ng/mL) and vascular endothelial growth factor (10 ng/mL); after day 8, vascular endothelial growth factor (10 ng/mL), Dickkopf 1 (150 ng/mL), and β fibroblast growth factor (5 ng/mL). Cultures were maintained in a 5% CO2/5% O2/90% N2 environment for the first 10 to 12 days and were then transferred into a 5% CO2/air environment. After ≈12 to 14 days, cardiac derivatives made up ≈50% of the cell population. For profiling experiments, hESC-VCMs were purified using the LV-MLC2V-GFP-T2A-ZEO reporter system to avoid ambiguities because of the presence of contaminating non-VCMs and noncardiac cells. We digested 21-day-old hESC-derived cells, plated them onto matrigel-coated surfaces and transduced with recombinant LV-MLC2V-GFP-T2A-ZEO particles at an multiplicity of infection of 3. Transduced cells were cultured in 5% fetal bovine serum in CM media consisting of DMEM supplemented with 100 μmol/L nonessential amino acids, 2 mmol/L glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin (Invitrogen). Fluorescing GFP positive cells became visible 3 days post-transduction, and zeocin (300 μg/mL) was applied to kill non-VCMs. Please see Figure I in the Data Supplement for characterization of hESC-VCMs.

Isolation of Fetal and Adult CMs

Human fetal and hA CMs were isolated and experimented according to protocols approved by the UC Davis International Union of Pure and Applied Chemistry (IUPAC) and institutional review board (IRB; Protocol No. 200614787-1 and No. 200614594-1; Table I in the Data Supplement).

Two-Dimensional–Differential-In-Gel Electrophoresis

Cells were lysed in 2D lysis buffer. Each sample was labeled with CyDye-3 or CyDye-5 (GE Healthcare/Amersham). As an internal standard, equal amounts of all cell lysates were mixed and labeled with CyDye-2. CyDye-labeled protein samples were mixed and 250 μL was loaded per isoelectric focusing strip (GE Healthcare/Amersham). Isoelectric focusing was then done for a total of 25 000 Vh with standard conditions using Ettan IPGPhore II. After the isoelectric focusing, electrophoresis was performed at 16°C. The resulting 2D gel was scanned using a Typhoon Trio scanner (GE Healthcare/Amersham). Analyses were performed using the Profenesis Samespot v2.3 (Nonlinear Dynamics). Selected protein spots were excised from the gel using Ettan spot picker (GE Healthcare/Amersham), subjected to in-gel digestion and protein IDs were determined by mass spectrometry/mass spectrometry analysis using a matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometer, AB SCIEX-4800 (AB SCIEX).

For more details, please see Data Supplement.

Data Analysis

For hierarchical clustering, we used average linkage based on Euclidean distance to examine proteins with fold changes >1.5 (P<0.05). Principal component analysis of the processed proteomic data was performed using the samespot software (Nonlinear Dynamics). Functional annotation was performed based on gene ontology classifications (www.geneontology.org). For differential protein abundance, proteins satisfying expression fold difference of at least 1.5 and P value (t test) <0.05 were considered to be differentially expressed.

Pathway Analysis of Differentially Expressed Proteins

Differentially expressed proteins were imported into Ingenuity Pathway Analysis 2013 (http://www.ingenuity.com/) to evaluate their association with corresponding canonical pathway and upstream regulators. The enrichment analysis was performed by Fisher right-tailed exact tests, where P<0.05 based on false discovery rate <5%.

Quantitative Real-Time Polymerase Chain Reaction

CDNA was prepared using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time polymerase chain reaction was performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems) and gene expressions were quantified using StepOnePlusTM Real-Time PCR system (Applied Biosystems). Primer sequences are available on request. Gene expression was normalized to GAPDH and is presented as fold changes relative to dimethyl sulfoxide–treated control (mean±SEM). A Student t test was used to determine statistical significance. P<0.05 were considered as statistically significant.

Measurement of Mitochondrial Membrane Potential

Mitochondrial membrane potential (Δψm) was measured using the well-established JC-1 dye (Invitrogen). JC-1 exhibits Δψm-dependent accumulation. At high Δψm, JC-1 forms complexes with intense red fluorescence (≈585 nm). At low Δψm, JC-1 remains in the monomeric form, which shows only green fluorescence (≈530 nm). Therefore, the ratio of red/green signal is an indication of Δψm. hESC-VCMs were incubated with 0.5 μmol/L of JC-1 dye at 37°C for 30 minutes in CM media. Cells were imaged at green and red channels and signal intensities were analyzed using ImageJ software to calculate Δψm as the ratio of red/green fluorescent signal.

Quantification of Contractile Force in Human Ventricular Cardiac Microtissues

Human ventricular cardiac microtissues (hvCMTs) were prepared as previously described.21 In brief, hESC-VCMs were dissociated after trypsin digestion. A cooled suspension of ≈106 cells within reconstitution mixture, consisting of 1.5 mg/mL liquid neutralized collagen I (BD Biosciences) and 0.5 mg/mL fibrinogen (Sigma–Aldrich), was added to the substrate and the entire assembly was centrifuged to drive the cells into the micropatterned wells, where hESC-VCMs self-assembled into microtissues within 24 hours. For quantifying microtissue forces, brightfield and fluorescence images were taken at 100 Hz with a fast CCD camera (Allied Vision), and an A-Plan ×10 objective on a Nikon Eclipse Ti (Nikon Instruments, Inc) equipped with a live cell incubator. Only tissues that were uniformly anchored to the tips of the cantilevers were included in the analysis. The displacement of fluorescent microbeads at the top of the cantilevers was then tracked with using the SpotTracker plug-in in ImageJ (National Institutes of Health). Microtissues were treated with the thyroid hormone triiodothyronine (T3; 100 nM) at the time of seeding for 6 consecutive days. Measurements were made on day 6 of T3 treatment.

Measurements of Action Potential

Action potentials in hESC-VCMs treated with or without T3 (100 nM) for 6 days were analyzed by loading hESC-VCMs with the voltage sensitive dye Di-8-ANEPPS (5 μmol/L; Invitrogen) for 30 minutes at 37°C in DMEM/F12, followed by imaging with a CMOS-based camera (MiCAM ULTIMA, SciMedia USA Ltd, CA) in Tyrode solution containing (in mmol/L): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH adjusted to 7.4 with NaOH. Electric stimulation was applied to evoke action potentials.

Results

Proteomic profiling was performed using 2D-differential-in-gel electrophoresis on protein extracts from undifferentiated hESCs, hESC-VCMs, hF-VCMs, and hA-VCMs. This technique was chosen such that protein abundance in multiple samples can be compared and accurately quantitated. For comparison, human atrial samples (ie, hF-atrial cardiomyocytes [ACM] and hA-ACMs) were also studied. Protein extracts were separated by 2D gels to detect individual protein spots. As an example, Figure 1A shows the separation of proteins present in undifferentiated hESCs and hESC-VCMs. Superimposition of the 2 images enabled the visualization of the differences in protein expression: green and red protein spots corresponded to proteins that were overexpressed in hESC and hESC-VCM cells, respectively. Yellow spots represented protein spots that were equally expressed in both the samples.

Figure 1.
Figure 1.

Two-dimensional gels scanned for (A) hES2 (ie, Cy3) and human embryonic stem cell (hESC)-ventricular cardiomyocyte (VCM; ie, Cy5), (B) human fetal (hF)-atrial cardiomyocytes (ACM) and hF-VCM, and (C) human adult (hA)-ACM and hA-VCM. Overlay of the gels where Cy5 and Cy3 are red and green, respectively. Proteins that were equally expressed in both the samples are yellow.

Figure 1B and 1C shows the separation of proteins present in hF-ACMs, hF-VCMs, hA-ACMs, and hA-VCMs. Overall, a total of 554 individual protein spots were detected and matched to all gels (Figure 2A). Three hundred sixteen spots were differentially expressed between any 2 samples (>1.5 fold; P<0.05). One hundred sixty-one unambiguous IDs were obtained by mass spectroscopy, corresponding to 121 unique gene products. Cross analyses enabled us to examine the protein expression profile across these samples. As an example (Figure 2B and 2C), the expression of MLC2V (MYL2) was most robust in hA-VCMs and hF-VCMs but present at lower levels in hESC-VCMs and the atrial samples (hF-ACMs and hA-ACMs), and was virtually absent in undifferentiated hESCs. These data were entirely consistent with trends reported previously using other techniques,22–25 validating the use of this approach for identifying novel proteomic differences.

Figure 2.
Figure 2.

A, Image of preparatory gel. Differentially expressed spots identified are marked. B, Topographical and spot images of MLC2V (MYL2) in human embryonic stem cell (hESC), hESC-ventricular cardiomyocyte (VCM), human fetal (hF)-atrial cardiomyocytes (ACM), hF-VCM, human adult (hA)-ACM, and hA-VCM. C, Normalized spot intensities of MLC2V (MYL2) in the 6 CM populations.

Global Proteomic Expression Profiling

We next performed hierarchical clustering and principal component analyses to examine the proteomic landscape of our samples. Figure 3 shows that biologically independent replicates clustered closely together, consistent with a high experimental reproducibility. Although replicates of samples generally clustered according to their developmental stages and chamber-specificities, the close distances between hF-VCMs and hF-ACMs, and between hA-VCMs and hA-ACMs suggested that the proteomes of VCMs and ACMs from the same developmental stages were similar. HESC-VCMs were preferentially grouped together with hF-VCMs and hF-ACMs, indicating that the global proteome of hESC-VCMs was fetal-like, mirroring their comparable global transcriptomes.26

Figure 3.
Figure 3.

A, Hierarchical clustering showing that biological replicates cluster together. Relative gene expression is color-coded: red indicates upregulation and green indicates downregulation. B, Principal component analysis showing that human embryonic stem cell (hESC)-ventricular cardiomyocytes (VCMs) are grouped more closely with fetal CMs. hA indicates human adult; and hF, human fetal.

Identification of Proteomic Changes in Cardiac Contraction and Metabolism

To reveal specific protein changes associated with different stages of ventricular maturation, we focused on the differential protein expression among hESC-VCM, hF-VCMs, and hA-VCMs. Among the proteins identified, 37 and 33 were significantly increased and decreased in hA-VCMs compared with hF-VCMs, respectively, whereas 24 and 19 were significantly increased and decreased in hF-VCMs compared with hESC-VCMs, respectively. Expression changes in individual proteins are shown in Table II in the Data Supplement. Table 1 summarizes the general trends in protein expression among different functional groups in the 3 CM populations. Figure 4 shows the spot intensities and topographical images of select key proteins for contraction, metabolism, cell survival, ion transport, and regulation of mRNA and protein expression of CMs.

View this table:
Table 1.

Summary of Differential Protein Expression Changes in Different Functional Groups

Figure 4.
Figure 4.

Differential expression of selected proteins in human embryonic stem cell (hESC), hESC-ventricular cardiomyocyte (VCM), human fetal (hF)-VCM, and human adult (hA)-VCM. Topographical images are shown. The graphs show normalized spot intensities.

For contraction, as anticipated from their level of mechanical activities, hESC-VCMs had the lowest levels of sarcomeric proteins such as β-myosin heavy chain (MYH7) and α-actin (ACTA1), followed by hF-VCMs, and then hA-VCMs, potentially underlying the weaker developed twitch force of engineered ventricular tissue strip that we recently reported.27 For insights into cardiac metabolism, we next examined the abundances of proteins involved in FA oxidation (eg, very long-chain–specific acyl-coenzyme A dehydrogenase, ACADVL) and glycolysis (eg, cytosolic malate dehydrogenase, MDH1). These were significantly increased and decreased, respectively, in hA-VCMs relative to hESC-VCMs and hF-VCMs whose levels were comparable. Nine of 33 known proteins involved in energy generation showed significant 1.6- to 14.0-fold expression differences between hESC-VCMs and hF-VCMs. Indeed, glycogen phosphorylase (PYGB), a rate-limiting step for glycogen breakdown, displayed the highest difference by being >14-fold enriched in hF-VCMs compared with hESC-VCMs. In addition, we also observed higher levels of proteins involved in energy transfer from the mitochondria to the contractile apparatus and this includes four-and-a-half-LIM domain protein 2 (FHL2; Figure 4), creatine kinase (CKM) and mitochondrial CK (CKMT2) in hF-VCMs relative to hESC-VCMs. CKMT2 protein level was increased further in hA-VCMs relative to hF-VCMs, consistent with increased mitochondrial maturation.

Cardiac Metabolism and Maturation of hESC-VCMs

To seek further insights, we performed the complementary approach of combining clustering, Ingenuity pathway, and upstream regulator analyses of differentially expressed proteins, to identify pathways or regulators important for CM maturation (Figure 5A to 5C; Table IIIA and IIIB in the Data Supplement). Ingenuity Pathway Analysis result shows that proteins differentially expressed among hESC-VCMs, hF-VCMs to hA-VCMs were most significantly associated with metabolic processes (such as mitochondrial dysfunction), raising the intriguing possibility that metabolism and CM developmental maturation are related. Ingenuity Pathway Analysis also provides upstream regulator analysis to identify regulators that may be important for affecting gene/protein expression based on statistical analysis. The identified regulators can be used to predict whether the regulatory cascades or target proteins are involved in the biological activities. As shown in Figure 5C, we detected peroxisome proliferator–activated receptor γ coactivator 1-α (PPARGC1A)/estrogen-related receptor α/peroxisome proliferator–activated receptor α (PPARA) as a group of transcription factors that regulates metabolic protein expression, particularly those related to FA metabolism (eg, ACADM and ACADVL), in hESC-VCMs. Of the 11 proteins targeted by PPARGC1A, the transcripts of 9 were indeed significantly upregulated in hA-VCMs or hF-VCMs relative to hESC-VCMs (Figure 5E).

Figure 5.
Figure 5.

Bioinformatic analysis of proteins upregulated during cardiac maturation. A, Cluster analysis of differentially expressed proteins. Maturation proteins (as indicated) were subject to Ingenuity Pathway Analysis. B, Summary of pathway analysis results. The P value indicates the likelihood that the association between the maturation proteins and a given process or pathway is because of random chance. The smaller the P value, the more significant the association. C, Summary of upstream regulator analysis. D, Schematic of upstream regulation. Solid arrows indicate regulatory relationships between upstream regulator and targets identified by Ingenuity Pathway Analysis. Dotted line indicates interaction between upstream regulators reported in literature. Dotted arrow indicates directional regulation between upstream regulators, as reported in literature. E, Predicted targets of peroxisome proliferator–activated receptor γ coactivator 1-α (PPARGC1A). Black/Red indicates proteins, which were/were not significantly enriched on the mRNA level in human adult (hA)-ventricular cardiomyocytes (VCMs) or human fetal (hF)-VCMs relative to human embryonic stem cell (hESC)-VCMs (Table IIIA–IIIC in the Data Supplement). ILK, integrin-linked kinase

Using the above pathway insights as a guide, the relationship between cardiac metabolism and maturation of hESC-VCMs was further investigated. To seek functional evidence, we next tested the pharmacological effect of WY-14643, a well-characterized PPARA agonist,28 on Δψm of hESC-VCMs using the JC-1 dye. Δψm is an index of cellular metabolic output and mitochondrial functionality and arises from the asymmetrical distribution of charges between the inner and outer sides of the inner mitochondrial membrane as a result of the electron transport chain and its ATP production.29 WY-14643 treatment significantly hyperpolarized Δψm by 34±16% in hESC-VCMs in the presence of oleic acid (P<0.05; Figure 6A); conversely, WY-14643-treated hESC-VCMs cultured without oleic acid supplement had an Δψm similar to control (P>0.05). Along this line, WY-14643 (50 μmol/L) significantly increased the transcript levels of genes involved in FA metabolism, such as medium-/long-/very long-chain–specific acyl-coenzyme A dehydrogenases (ACADM, ACADL, and ACADVL; Figure 6B). Although hESC-VCMs had significantly lower levels of both ACADM (1.9±0.1-fold) and ACADVL (3.8±0.6-fold) compared with hA-VCMs, WY-14643 treatment raised ACADVL expression to adult levels. CPT1B, important for FA transport, was also increased by 2.2±0.2-fold; 100 μmol/L WY-14643 produced similar effects. Collectively, these observations were in accordance with a WY-14643-induced FA oxidation, which provides substrates for the electron transport chain. As for the mitochondrial morphology, control hESC-VCMs had disorganized punctate and perinuclear COXIV staining (Figure 6C). By contrast, WY-14643-treated hESC-VCMs showed stronger COXIV staining. Furthermore, mitochondria became elongated and formed filamentous networks between myofibril filaments, suggestive of a more developmentally mature phenotype.30 We also examined the staining pattern of four-and-a-half-LIM domain protein 2, an anchoring protein that mediates energy transfer between the mitochondria and the contractile apparatus.31 Immunostaining of hESC-VCMs indicated that WY-14643 improved the organization and alignment of FHL2, which colocalized with α-actinin with a clearer-striated staining pattern (Figure 6C). In addition, the mitochondrial elongation factor GFM1 was also increased in WY-14643-treated cells relative to control (Figure 6C).

Figure 6.
Figure 6.

Effect of peroxisome proliferator–activated receptor α/peroxisome proliferator–activated receptor γ coactivator 1-α activation on human embryonic stem cell (hESC)-ventricular cardiomyocytes (VCMs). A, Effect of WY-14643 (50 μmol/L) on mitochondrial membrane potential with and without oleic acid supplement (0.2 mmol/L); *P<0.05. B, Effect of WY-14643 on the expression of FA metabolic genes. WY-14643 (100 and 50 μmol/L) was applied to hESC-VCMs for 4 days. Dimethyl sulfoxide (DMSO)–treated and untreated hESC-VCMs acted as control. Data are presented as fold changes relative to DMSO-treated control cells (mean±SEM). Untreated cells were also assessed and gene expression was not significantly different from DMSO-treated cells; n=3, *P<0.05, ** P<0.01. C, Effect of WY-14643 on proteins important for mitochondrial morphology and energy transfer. COXIV, FHL2, and GFM1 staining are shown in red. Samples were also costained with α-actinin antibody, in green.

T3 Treatment Increases the Contractile Forces of Engineered Human Ventricular Microtissues

Because PPARA/PPARGC1A is known to be regulated by or interact with the thyroid hormone pathway,32–35 which promotes the maturation of fetal VCMs and ESC-CMs,36,37 we then examined the functional consequences of T3 treatment on contractile forces. Although shortening of single hESC-CMs or their clusters has been measured as an surrogate index for contractile forces,38,39 a multicellular 3D hvCMT system has been developed, where true dynamic tension developed by the tissue in real time can be measured.21 Figure 7A shows that hvCMT engineered from ≈1000 hESC-VCMs each of ≈0.5 mm in length allowed continuous measurement of their spontaneous, as well as stimulated dynamic twitch tension after T3 (100 nM) treatment for 6 days. For time-matched hvCMTs treated with T3, the developed twitch tension increased significantly (by 3-fold; P<0.05; n=6). However, the spontaneous twitch frequencies did not reach statistical differences after T3 treatment (P>0.05; Figure 7B). High-resolution optical mapping of T3-treated single hESC-VCMs showed that their action potential parameters were not different from those of time-matched untreated controls (Figure 7C and 7D).

Figure 7.
Figure 7.

Effect of T3 treatment on human embryonic stem cell (hESC)-ventricular cardiomyocytes (VCMs). Effect of T3 treatment on (A) dynamic tension and (B) spontaneous contraction frequencies in human ventricular cardiac microtissue (hvCMTs). hvCMTs were treated with T3 (100 nM) for 6 days after which spontaneous dynamic tensions were measured. Data represent mean±SEM; n=6, **P<0.05. C, Representative tracing of action potential in control and T3-treated (100 nM; 6 days) hESC-CMs at 1 Hz and measured with Di-8-ANEPPS and a high-resolution optical mapping system. D, Effect of T3 treatment (100 nM; 6 days) on action potential upstroke time and 50% decay time (APD50). Data represent mean±SEM. n=6 to 8.

Post-Transcriptional Regulation is Crucial for CM Maturation

We find that normalized transcript abundance relative to protein abundance during the developmental continuum of CM differentiation (hESCs to hESC-VCMs) and maturation (hESC-VCMs and hF-VCMs, hF-VCMs and hA-VCMs) is not always linear. Of the 121 proteins detected here, transcripts for 116 could be detected by microarray.26 Among proteins that showed differential abundance between hESC-VCMs and hF-VCMs, 37% had significant transcriptomic changes in the same direction. Only 19% of proteins differentially expressed between hF-VCMs and hA-VCMs showed the same trend. Pearson correlation analysis confirmed that the correlation between mRNA and protein expression changes during maturation was lower than that during differentiation (Table IV in the Data Supplement). Thus, a majority of the protein changes identified here during stages of development/differentiation could not be accounted for by transcriptional regulation.

Discussion

This study was the first to investigate cardiac maturation by profiling the proteome of CMs isolated from fetal and adult ventricles and atria, and comparing to that of hESC-VCMs. Our global profiling analysis indicates that the proteome of hESC-VCMs are grossly similar to those of hF-VCMs and hF-ACMs. Previous global profiling attempts primarily involved microarray-based transcriptomic data.13–17 A study by Van Hoof et al18 focused exclusively on plasma membrane proteins in CMs derived from hESCs and hF hearts, and identified elastin microfibril interfacer 2 as a surface marker of CMs. By examining the global proteomes, here we report novel differences and similarities in contractile and metabolic enzymes among our CM populations. In combination with bioinformatics and functional analyses, our experiments further enable us to reveal the role of PPARA/PPARGC1A and related signaling pathways in hESC-VCM maturation.

Metabolism is crucial for supplying energy for contraction and mutations in key metabolic genes such as ACADVL can result in cardiomyopathy.40 Chung et al41 have shown that mitochondrial oxidative metabolism is crucial for the cardiac differentiation of mouse ESCs. It is, therefore, possible that potential differences in the mitochondrial capacities of hESC-VCMs and hA-VCMs underlie the process of maturation. Specifically, we show that the protein levels of important FA metabolic enzymes are low in hESC-VCMs relative to hA-VCMs and that an established PPARA/PPARGC1A agonist WY-14643 significantly increases the mRNA levels of FA metabolic genes, elevates Δψm and improves the organization of the mitochondria and the phosphocreatine system of hESC-VCMs. Although a hyperpolarized Δψm drives ATP production and is an indication of enhanced mitochondrial/metabolic output, a promoted alignment of mitochondria to the contractile apparatus and improved organization of components of the phosphocreatine system further enables a more efficient transfer of energy between the 2 units. These results in human cells are reminiscent of those that take place during mouse embryonic development,30 and are also consistent with those of Birket et al42 who showed that repression of PPARGC1A decreased mitochondrial content, and thereby compromised the capacity for coping with energetic stress. Indeed, our recent transcriptomic profiling experiment also identifies PPARGC1A as a member of a cardiac transcription factor network, which regulates genes important for heart development and function26; similarly, Xu et al14 report that PPARGC1A/PPARA signaling regulate genes enriched in CMs relative to undifferentiated hESCs. Most interestingly, the thyroid hormone T3 significantly augments the contractile force without affecting the electric properties as gauged by their action potential parameters. In neonatal rat cardiomyocytes, T3 has a biphasic effect on PPARGC1A level dependent on the treatment duration.32 In other systems such as hepatocarcinoma HepG2 cells, PPARA inhibits the transcriptional activity of the thyroid hormone receptor by competing for the retinoid X receptor.33 How the different pathways interact and the intricate cause-and-effect relationships among their components deserve additional investigations.

Here, we show that transcriptomic and proteomic changes among CM samples are largely discordant. This finding suggests that post-transcriptional mechanisms, such as RNA processing, stability or transport, translation and post-translational pathways, or protein stability must account for at least some of these differences. This also demonstrates the importance of a proteomic approach. In comparison to the results of Van Hoof et al,18 who examined the plasma membrane proteome of hESC-CM cultures and hF-CMs. The small overlap maybe attributed to the precipitation of plasma membrane proteins during 2D-differential-in-gel electrophoresis experiments and are, therefore, mostly excluded from our analysis43 Gundry et al44 examined published proteomic data on pluripotent stem cells and showed that overlap among different proteomic studies are generally small and this is consistent with what we observed. Of the ones that were found in both the studies, only 31% showed concordant changes between hESC-CMs and hF-VCMs, which could result from the unsorted and MLC2v-purified populations used in Van Hoof et al’s18 and our studies.

Our laboratory has previously reported several ion channels,8 Ca2+-handling proteins,45 microRNAs,46 and epigenetic components47 that are determinants of the maturation of hESC-CMs. Led by these data and other clues, we found that induced contractions via electric stimulation increase the expression of contractile genes and improve the organization of myofilaments.8 Induced contractions place an increased demand on energy, which may be met by driven metabolic maturation. Taken collectively, our data raise the intriguing possibility that additive synergistic effects of such promaturation mechanisms such as electric conditioning,8 epigenetics priming,47 T3 treatments, etc, exist for facilitated maturation of hESC/induced pluripotent stem cells-VCMs.

Conclusions

Our present proteomic study identified new candidate proteins and pathways that are less abundant in hESC-VCMs compared with their fetal and adult CMs. Specifically, we have implicated a role for PPARA/PPARGC1A in hESC-CM metabolic and mitochondrial maturation with T3 treatment significantly enhancing the contractile force. The successful use of hESC-CMs as human heart disease models and cardiotoxicity screening tools depends on their ability to recapitulate the properties of their adult counterparts. The results may provide new insight for devising mechanism-based in vitro maturation strategies.

Sources of Funding

This work was supported by the Research Grant Council of Hong Kong (T13-706/11, HKU772913, and HKU17113514) and the Faculty Core of the University of Hong Kong.

Disclosures

None.

  • Received May 9, 2013.
  • Accepted February 18, 2015.

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CLINICAL PERSPECTIVE

Cardiac differentiation of human pluripotent stem cells such as human embryonic stem cells represents a potentially unlimited source of ventricular cardiomyocytes. However, human pluripotent stem cell-ventricular cardiomyocytes are developmentally immature, limiting their applications. Mechanisms that control ventricular cardiomyocyte maturation are unclear. In this study, we systematically compared the proteomes of human embryonic stem cells, human embryonic stem cell–derived ventricular cardiomyocytes, human fetal and human adult ventricular and atrial CMs for identifying differentially expressed proteins, followed by assessing their functional roles in cardiac maturation. Using the peroxisome proliferator–activated receptor (PPAR) signaling pathway as a guide, we demonstrated that treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in an engineered human ventricular cardiac microtissue system, signifying their facilitated maturation. These results may lead to mechanism-based methods for deriving mature chamber-specific human pluripotent stem cell-CMs for applications in drug discovery, disease modeling, and regenerative medicine.

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  • Proteomic Analysis of Human Pluripotent Stem Cell–Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and MaturationCLINICAL PERSPECTIVE
    Ellen Poon, Wendy Keung, Yimin Liang, Rajkumar Ramalingam, Bin Yan, Shaohong Zhang, Anant Chopra, Jennifer Moore, Anthony Herren, Deborah K. Lieu, Hau San Wong, Zhihui Weng, On Tik Wong, Yun Wah Lam, Gordon F. Tomaselli, Christopher Chen, Kenneth R. Boheler and Ronald A. Li
    Circulation: Genomic and Precision Medicine. 2015;8:427-436, originally published March 10, 2015
    https://doi.org/10.1161/CIRCGENETICS.114.000918
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