Proteomic Analysis of Human Pluripotent Stem Cell

Article pubs.acs.org/jpr

Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms Sarah A. Konze,†,∥ Sebastian Werneburg,†,∥ Astrid Oberbeck,†,∥ Ruth Olmer,‡,∥ Henning Kempf,‡,∥ Monica Jara-Avaca,‡,∥ Andreas Pich,§ Robert Zweigerdt,‡,∥ and Falk F. R. Buettner*,†,∥ †

Institute of Clinical Biochemistry, Hannover Medical School, 30625 Hannover, Germany Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany § Institute of Toxicology, Core Facility Proteomics, Hannover Medical School, 30625 Hannover, Germany ∥ REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany ‡

S Supporting Information *

ABSTRACT: Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development. KEYWORDS: human pluripotent stem cells, cardiomyocytes, proteomics, SILAC, PDLIM5

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research.8 hPSC-derived cardiomyocytes (hPSC-CMs) can be furthermore used for drug development as well as for the study of cardiogenesis and cardiac diseases.9,10 Differentiation of hPSCs is generally accompanied by alterations of metabolism, which is considered to be functionally connected to mechanisms influencing the cellular differentiation state.11 hESCs have immature and fewer mitochondria compared with their differentiated progenies and primarily rely on glycolysis for ATP production.12,13 In contrast, cardiomyogenic differentiation was shown to be accompanied by the expansion of the mitochondrial network and a shift from glycolysis to oxidative phosphorylation.14 The observation that

INTRODUCTION Human pluripotent stem cells (hPSCs), including embryonic (hESCs) and induced (hiPSCs) pluripotent stem cells, harbor a great potential for the replacement of damaged human tissues or even organs owing to their ability to differentiate into cells of all three germ layers and their respective somatic lineages.1,2 Driven by the shortage of donor organs and the fact that the human heart itself has a very limited regeneration capacity,3−5 one major objective in regenerative medicine is the application of pluripotent stem-cell-derived cardiomyocytes (CMs) or cardiac tissue for treatment of heart failure.6 Transplantation of stem cell-derived CMs upon myocardial infarction has been shown to improve contractile function of mouse and primate hearts. 7,8 However, in the latter case, post-transplant arrhythmias were reported, accentuating the need for further © 2017 American Chemical Society

Received: June 8, 2016 Published: January 31, 2017 1133

DOI: 10.1021/acs.jproteome.6b00534 J. Proteome Res. 2017, 16, 1133−1149

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Journal of Proteome Research

Figure 1. Experimental workflow. Cells were cultivated in SILAC stem cell medium and subsequently differentiated in SILAC medium under chemically defined, serum-free conditions and harvested at the indicated time points (d0, d20, d35).

human system. The work by Van Hoof et al.28 primarily focused on the membrane proteome, leading to the discovery of the CM marker EMILIN2. Poon et al.29 used 2D gel electrophoresis to compare the proteome of hPSC-derived, fetal, and adult ventricular CMs. The focus of this study is more on comparing differentially maturated populations of CMs than CMs versus stem cells, and they identified the peroxisome proliferator-activated receptor α as a factor involved in modulation of metabolism and maturation in stem cell-derived CMs. Furthermore, the applied technique is limited in terms of depth in comparison with shotgun proteomic approaches. In a recent shotgun proteomic comparison between hESCs, cardiac progenitor cells, and hESC-derived CMs by Hofsteen et al.,30 label-free quantification was applied. Hofsteen et al.30 strongly focused on the study of signaling pathways that are changed during differentiation leading to the identification of DAB2 as a WNT signaling antagonist that is relevant for cardiogenesis. In the current study we also applied shotgun proteomics but used metabolic labeling by stable isotope labeling of amino acids in cell culture (SILAC) to quantitatively compare the proteomes of hPSCs and hPSC-derived CMs. hPSCs were differentiated by applying our chemically defined embryoid body (EB)-based differentiation protocol.31 Moreover, genetic selection enabled the generation of highly pure populations of CMs. We observed global alterations in the energy metabolism during cardiomyogenesis and propose that effects caused by differentiation are overlaid by effects of limited oxygen access in differentiating cell aggregates. We furthermore showed strong up-regulation of the Z-disc component PDLIM5, and our study for the first time demonstrates that the transcript variants 2 and 7 of PDLIM5 are expressed and translated into protein (isoforms b and g) in early CMs, concomitantly with the CM differentiation marker NKX2.5.

embryonic stem cells rely on fermentation is consistent with the demonstration that mammalian embryos develop under reduced oxygen conditions15 and that hypoxia is supportive for the maintenance of hESC pluripotency16 and for somatic cell reprogramming.17 Lactic acid fermentation, which occurs under oxygen starvation, is a mechanism to replenish the NAD+ storage under hypoxic conditions by converting glycolysisderived pyruvate to lactate. In contrast with aerobic respiration, in which glycolysis is followed by the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, lactic acid fermentation produces 13 times fewer ATP molecules per unit glucose. However, CMs as well as other somatic cells can, at least transiently, survive on glycolysis/fermentation.18 Besides adjustment of metabolism, cardiomyogenic differentiation is associated with vast structural changes of the cytoskeleton.19 One component of the forming sarcomeres is the Z-disc, which includes the protein enigma homologue (ENH), also known as PDLIM5. PDLIM5 is a member of the enigma subfamily of PDZ and LIM domain-containing proteins, comprising, among others, enigma and cypher/ ZASP.20 Through their PDZ domain these proteins bind to the cytoskeleton while the LIM domains are making contacts with signaling proteins or transcription factors.20 Various transcript variants of PDLIM5 coding for different isoforms are known in men21 and mice,22 and the expression pattern of these splice variants has been shown to change during postnatal heart development of rat.23 Induction of heart hypertrophy in a mouse model affects alternative splicing of PDLIM5, and different splice variants were shown to act antagonistically on the expression of hypertrophy markers in rat, with ENH1 promoting and ENH4 preventing hypertrophy.23 Opposing functions of different splice variants were also reported for ENH1 and ENH3 in neonatal rat CMs. In this model, the ENH1 interacts with and activates the transcription factor cAMP-responsive element binding protein (CREB), whereas the LIM-less splice variant ENH3 inhibited CREB.24 A complete knockout of Enh (Pdlim5) function in mice causes an adult onset of dilated cardiomyopathy.22 To globally describe changes in the proteome of differentiating cells, mass spectrometry is an efficient tool. Several proteomic studies comparing stem-cell-derived CMs and their parental pluripotent precursors have been reported for the murine system,14,25−27 but only a few studies exist for the

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MATERIALS AND METHODS

Cultivation of hPSCs

Unless stated otherwise, all reagents used for tissue culture were purchased from Life Technologies (Carlsbad, CA) and all cell culture vessels were purchased from Greiner (Greiner Bio-One, Frickenhausen, Germany). In general, hPSCs were maintained at 85% relative humidity, 37 °C, and 5% CO2. The following hPSC lines were used in this study: (i) the genetically modified human embryonic stem cell line hES3_αMHCneoPGKhy1134


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Journal of Proteome Research gro_1,32,33 carrying a neomycin resistance under the control of the CM-specific myosin heavy chain 6 (MYH6/α-MHC) promoter, which is in the following referred to as H3M, (ii) the human embryonic stem cell line ES03 (ES Cell International, National Stem Cell Bank Wisconsin, WI), in the following referred to as H3, and (iii) the human induced pluripotent stem cell line hCBiPSC2 (human cord blood derived induced pluripotent stem cell clone 2, reprogrammed from cord blood derived endothelial cells, LEBAO, MHH),34 in the following referred to as I2. hPSCs were cultured on feeder cells (γ-irradiated human foreskin fibroblasts, CCD919, ATCC, Manassas, VA) as previously described.35 In brief, feeder cells were seeded in six-well plates at a density of 2.5 × 104 cells/cm2 in stem-cell medium consisting of KnockOut DMEM, 20% (v/ v) KnockOut Serum Replacement, 1% (v/v) MEM NonEssential Amino Acids, 0.5% (v/v) GlutaMAX, and 0.1 mM 2mercaptoethanol supplemented with 50 ng/mL (H3M, H3) or 10 ng/mL (I2) basic fibroblast growth factor (bFGF, Institute of Technical Chemistry, Leibniz University Hannover, Hannover, Germany). Cells were passaged every 6 to 7 days (H3M, H3) or every 3−5 days (I2) by collagenase IV treatment.

CHIR99021 was removed after 24 h; the medium was changed to RPMI/B27 w/o insulin (day 1). On day 3, 5 μM IWP-4 (Stemgent, Cambridge, MA) was added. On day 7, the medium was changed to RPMI 1640 with B-27 Supplement with insulin. Spontaneously beating areas in the monolayer were visible from days 7−10 onward. SILAC Labeling of hPSCs

For SILAC labeling, hPSCs on feeder cells were cultured for at least five passages in SILAC stem-cell medium, in which the Knockout DMEM component was replaced by DMEM-F12 for SILAC (Thermo Fisher Scientific, Waltham, MA) supplemented with 7 × 10−4 mol/L of the respective arginine (Arg) isotopologue (Arg-6 = L-[13C6] Arg-HCl or Arg-10 = L[13C615N4] Arg-HCl) and 5 × 10−4 mol/L of the respective lysine (Lys) isotopologue (Lys-4 = L-[2H4] Lys-HCl or Lys-8 = L-[13C615N2] Lys-HCl,) (Silantes, München, Germany). The combination of Arg-6 and Lys-4 is referred to as “medium”; the combination of Arg-10 and Lys-8 is termed “heavy”. Subsequent EB-based cardiomyogenic differentiation was conducted in SILAC differentiation medium for 20 or 35 days, in which the DMEM component was replaced by DMEM for SILAC (Thermo Fisher Scientific) supplemented with 4 × 10−4 mol/L Arg-6 and 8 × 10−4 mol/L Lys-4 as “medium” or 4 × 10−4 mol/L Arg-10 and 8 × 10−4 mol/L Lys-8 as “heavy” condition. A table showing the SILAC labeling pattern of different cell lines and biological replicates is provided as Supporting Information Table S1.

EB-Based Cardiomyogenic Differentiation

hPSCs (day (d)0) were differentiated in a chemically defined, EB-based approach31 for 20 (H3M, H3, I2) or 35 days (H3M, Figure 1). In brief, to induce EB formation, colonies of H3M cultured on feeder cells for 4 to 5 days in stem-cell medium were detached and transferred to six-well suspension culture plates in basic serum-free (bSF) differentiation medium composed of high glucose DMEM, 1% MEM Non-Essential Amino Acids, 1% GlutaMAX, 0.1 mM 2-mercaptoethanol, 5.5 μg/mL transferrin, and 6.7 ng/mL sodium selenite, supplemented with 5 μM of the p38 mitogen-activated protein kinase inhibitor SB203580 (Jena Bioscience, Jena, Germany).31 Subsequently, this medium was changed every 3 to 4 days. Beating foci became visible from day 10 onward and were counted on day 20. For antibiotic selection of CMs from H3M differentiations, 200 μM G418 (Calbiochem, Darmstadt, Germany) was added to the medium starting on day 12, and cells were cultured until d35. The differentiation outcome on d35 is referred to as human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). H3 and I2 were differentiated equally except for the first day, when cells were placed in SF-medium composed of high glucose DMEM, 1% MEM Non-Essential Amino Acids, 1% GlutaMAX, 0.1 mM 2-mercaptoethanol, and 1% InsulinTransferrin-Selenium (ITS) supplement.

Sample Preparation for Protein Determination and MS

For harvest of samples on day 0 (hPSCs) and day 20 (aggregates), cells were scraped off and pelleted by centrifugation. On day 35, beating aggregates (cardiac bodies) of purified CMs from H3M cells were manually separated from cellular debris before centrifugation. hPSCs, differentiated cells, or feeder cells were lysed by sonification in RIPA buffer (50 mM Tris-HCl, pH 8.0 with 1% NP-40 (Roche, Basel, Switzerland), 0.5% sodium deoxycholate (Sigma-Aldrich, St. Louis, MO), 0.1% sodium dodecyl sulfate (SDS, SigmaAldrich) and 150 mM NaCl) containing 1 mM PMSF (Roche, Mannheim, Germany) and 1% HALT protease inhibitor cocktail (Thermo Fisher Scientific). Sonification was carried out in a cup horn for two cycles of 1 min and 30 s, respectively, using a Branson Sonifier 450 with settings for output control at 5 and duty cycle at 50%. Determination of protein concentrations of cell lysates was carried out with the Pierce 660 nm protein assay reagent (Thermo Fisher Scientific). Equal protein amounts of two differently labeled SILAC samples were pooled and precipitated with acetone at −20 °C overnight (o/n). Precipitates were dissolved in Laemmli buffer (35 mM Tris-HCl pH 6.8, 7% (v/v) glycerol, 2.8% (w/v) SDS, and 0.005% (w/v) bromophenol blue), heated to 95 °C for 5 min, and separated at a length of 7 cm on 10% SDS polyacrylamide gels. Gels were stained with Roti-Blue (Roth, Karlsruhe, Germany); then, the sample lanes were manually sliced and cut into small pieces of ∼1 mm3 that were subjected to in-gel digestion according to the method described by Shevchenko et al.37 In brief, gel pieces were destained in 25% methanol (J.T. Baker, Deventer, Netherlands) and dehydrated in LiChrosolv liquid-chromatography-grade acetonitrile (ACN, Merck, NJ). Reduction of disulfide bonds and subsequent carbamidomethylation was performed by incubation with 10 mM DTT and 100 mM iodoacetamide in 100 mM NH4HCO3 (AmBic, all from Sigma-Aldrich) for 30 min each.

Cardiomyogenic Differentiation from Monolayer Cultures

Cardiomyogenic differentiation from monolayers of hPSCs was performed essentially as developed by Lian et al.36 In brief, hPSCs were cultivated on Matrigel (BD Biosciences, Bedford, MA) in mTeSR1 medium (STEMCELL Technologies, Grenoble, France). For cardiomyogenic differentiation, hPSCs were singularized with TrypLE Select and seeded in mTeSR1 with 5 μM Rho-associated coiled-coil kinase (ROCK) inhibitor Y27632 (RI, Institute of Technical Chemistry, Leibniz University Hannover) (day −4). The medium was changed to mTeSR1 without RI on day −2. On day 0, the medium was changed to RPMI 1640 incl. GlutaMAX with B-27 Supplement w/o insulin (RPMI/B27 w/o insulin) containing 20 μM (H3M, H3) or 8 μM (I2) of the glycogen synthase kinase 3 beta inhibitor CHIR99021 (Selleckchem, Houston, TX). 1135


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allowed for quantification. Two missed cleavages of trypsin (cleavage C-terminal of K, R) were allowed. The mass tolerance for precursor ions and fragment ions was set to 20 ppm and 0.5 Da, respectively. Known contaminants were excluded from the protein lists. Proteins were stated as identified if at least two peptides per protein with a minimum peptide length of six amino acids were identified applying a false discovery rate of 0.01 on protein and peptide level and allowing the requantification mode. Protein ratios were calculated from razor and unique peptides for d20 or d35 cardiomyogenic differentiations versus hPSCs and log2-transformed. An unpaired, two-sided, heteroscedastic Student’s t test was applied to compare ratios with a p value 1.5-fold up-regulated and 230 were significantly (p < 0.05) >1.5-fold down-regulated in the d35 samples (Supporting Information Table S6). Again, the hierarchical clustering revealed a similar regulation pattern among the three replicates (Figure 2B). Regulation of Proteins during Cardiomyogenic Differentiation

Regulation ratios of d20 or d35 versus d0 were plotted against the significance level (Volcano plot, Figure 2C,D). As expected, among the most strongly and most significantly down-regulated proteins in the d20/d0 samples were known stem cell-specific proteins43 like DNMT3B, octamer-binding protein 3/4 (OCT3/4), E-cadherin (CDH1), Sal-like protein 4 (SALL4), undifferentiated embryonic cell transcription factor 1 (UTF1), ES cell-associated protein (ECAT11), and developmental pluripotency-associated protein 4 (DPPA4) (Figure 2C). Although slightly failing the significance threshold, a similar down-regulation was observed for ECAT11, UTF1, DNMT3B, and CDH1 in the d35/d0 comparison. The higher significances for these proteins obtained in the d20/d0 versus the d35/d0 comparison might be caused by the fact that four biological replicates were analyzed for d20/d0 but only three for d35/d0.

Figure 2. Bioinformatics analysis of proteomic data. (A) Heatmap generated by hierarchical clustering of proteins identified in at least two of the four replicates of the d20/d0 comparison upon log2 transformation of d20/d0 ratios for four replicates of H3M. Proteins down-regulated on d20 are presented in green, and proteins upregulated are depicted in red. (B) Heatmap generated as described for panel A applying three biological repeats of the H3M cell line for the d35/d0 comparison. (C) Plotting log2 of mean d20/d0 ratios against the negative decadic logarithm of the p value (obtained by Student’s t test). Mean ratio was calculated for all proteins found in >2 samples of the d20/d0 comparison. (D) Same presentation as in panel C but for the d35/d0 comparison. 1138


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Figure 3. Enrichment analyses. Enrichment analyses searching for enriched KEGG pathways in the lists of proteins that were significantly (p < 0.05) regulated. (A) Proteins >1.5-fold down-regulated on d20 (irrespective of their regulation ratio on d35). (B) Proteins >1.5-fold up-regulated on d20 (irrespective of their regulation ratio on d35). (C) Proteins >1.5-fold up-regulated on day 20 and >4-fold up-regulated on d35. (D) Proteins >1.5fold up-regulated on day 20 and remaining up-regulated on d35. (E) Proteins >1.5-fold down-regulated on day 20 but not down-regulated on d35. 1139


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Journal of Proteome Research Figure 3. continued

(F) Proteins >1.5-fold up-regulated on day 20 but not up-regulated on d35. (A−F) The 12 most significantly up-regulated pathways are shown. The bars present the number of proteins found for each pathway, and the red dots display the respective p value. The numbers in parentheses behind the KEGG pathways describe the entry code of the respective KEGG pathway map. Pathways of interest belonging to central metabolism or heart function are highlighted in red or blue, respectively.

Figure 4. Regulation of enzymes belonging to glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), and electron transport chain (ETC) in H3M for the d20/d0 comparison (upper panel) and for the d35/d0 comparison (lower panel). Significantly >1.5-fold regulated proteins are depicted with black letters on dark green (down-regulation) or red (up-regulation) boxes. Significantly regulated proteins with a regulation ratio 1.5-fold, but with a p value above 0.05 are depicted in green or red boxes with white letters. Enzymes that were identified but that were not regulated are presented as white boxes with black letters.

N-cadherin (CDH2) and the myosin components embryonic myosin light chain 1 (MYL4), myosin light chain 3 (MYL3), myosin heavy chain 6 (MYH6/α-MHC) and myosin heavy chain 7 (MYH7/β−MHC), were among the most highly and significantly up-regulated proteins in both the d20/d0 and d35/ d0 data sets. Moreover, other constituents of the cardiac muscle, particularly of the Z-disc,45 are among the up-regulated

Additionally, the stem-cell-specific proteins SALL4 and DPPA4 as well as podocalyxin-like protein 1 (PODXL), which is known to be highly expressed in hPSCs, were strongly and significantly down-regulated in the d35/d0 comparisons (Figure 2D). Known CM-specific proteins,44 for example, creatine kinase M chain (CKMM), cardiac α-actin (ACTC), α-actinin-2 (ACTN2), the troponins I and T type 2 (TNNI, TNNT2), 1140


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Figure 5. Analysis of PDLIM5 expression during cardiomyogenesis. (A) Regulation ratios of PDLIM5 protein levels in the individual replicates of H3M cells for the d20/d0 (squares) and d35/d0 comparisons (triangles) showing up-regulation of PDLIM5 in differentiated cells. Horizontal bars represent mean regulation rate and error bars show standard deviation of replicates. (B) qPCR analysis of PDLIM5 expression comparing d0, d20, and d35 of H3M, H3, I2, and commercially available Cytiva cardiomyocytes. PDLIM5 expression of H3 on d0 was set to one. Significance testing was performed on ΔCT values applying a Student’s t test. (C) Western blot analysis using antibodies against PDLIM5 (upper panel) and actin as loading control (lower panel). Ten μg protein of each hPSCs (I2, H3, H3M), human fibroblasts (huF), d20 aggregates (H3, H3M), and d35 hPSCCMs (H3M) was loaded. The ∼65 kDa form of PDLIM5 was ubiquitously expressed but increased from d0 to d20 differentiation. Additionally, shortened PDLIM5 variants (predominantly at ∼55, but also at ∼40 and ∼30 kDa) became visible after differentiation. (D−F) Western blot analysis against PDLIM5, NKX2.5, and actin (loading control). A monolayer differentiation of H3M (D), H3 (E), and I2 (F) according to Lian et al.36 was performed, and cells were harvested in a time-course series during cardiomyogenic differentiation. Thirty μg protein of whole cell lysates was loaded in each lane. NKX2.5 showed synchronized expression to PDLIM5 short isoforms appearing around d7. Quantitative proteomic data revealed that expression of actin was not significantly different between d0, d20, and d35 and actin was therefore applied as loading control.

proteins in the d20 and d35 samples including obscurin (OBSCN), palladin (PALLD), nexin (SNX1), and the PDZ and LIM domain containing proteins PDLIM5 and LIM domain-binding protein 3 (LDB3, alternatively cypher or ZASP; Figure 2C,D and Supporting Information Table S5 and S6). Additionally, among the 30 most strongly up-regulated proteins of the d35/d0 comparison, 10 were well known constituents of the cardiac muscle, namely, the cardiac myosin binding protein C (MYBPC3), cardiomyopathy-associated protein 1 (CMYA1), myophosphorylase (PYGM), and myosin

heavy chain 3 (MYH3) as well as the already mentioned OBCSN, CKMM, ACTC, ACTN2, MYL4, TNNT2, MYH6, and MYH7 (Figure 2D, Supporting Information Table S6). Housekeeping proteins like beta-actin (ACTB), vinculin (VCL), laminin B1 (LAMB1), or cofilin (CFL1) did not change significantly between d0, d20, and d35 (Supporting Information Tables S5 and S6). In summary, the clear finding that known stem-cell-specific proteins were down-regulated and known CM-specific proteins were up-regulated in H3M-CMs (best visible after G418 1141


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compared d20 aggregates as well as enriched CMs (d35) against hPSCs (d0). On d20 we observed up-regulation of proteins belonging to glycolysis and pentose phosphate pathway (PPP), whereas proteins of the electron transport chain (ETC), involved in oxidative phosphorylation, were mostly down-regulated (Figure 4 upper panel). In a population of >90% CMs on d35, enzymes of central glucose metabolism (glycolysis, PPP, and TCA cycle) were rather consistently upregulated in comparison with hPSCs (d0). Levels of ETC proteins were partially elevated on d35 compared with d0 (Figure 4 lower panel) but considerably increased related to aggregates on d20. To exclude that these observations are exclusive to the cell line H3M, we repeated the proteomic comparison of d20 CMs against d0 hPSCs with the two cell lines H3 and I2 (Supporting Information Figure S8). A combined analysis of H3M, H3, and I2 confirmed the previously seen effects on central metabolism (Supporting Information Figure S9).

selection in the d35 samples) confirmed our proteomics approach and established a solid basis for further analyses of the obtained data set. To evaluate to which extent the regulation ratios of the two time point comparisons correlate, log2 values of the regulation ratios of proteins identified in both the d20/d0 and the d35/d0 analysis were plotted against each other. This type of analysis revealed broad conformity for both experimental setups with cardiac muscle proteins up-regulated and pluripotency-related proteins down-regulated upon cardiomyogenic differentiation (Supporting Information Figure S6). From the d20/d0 and d35/d0 ratios we calculated d35/d20 values that showed considerable overlap between the different replicates (Supporting Information Figure S7A). Of 2736 proteins, 122 were significantly down- and 106 were significantly up-regulated by a factor of ≥1.5 each (Supporting Information Figure S7B and Table S7). Most strikingly, proteins belonging to TCA cycle and oxidative phosphorylation were significantly up-regulated comparing d35 versus d20 (highlighted in Supporting Information Figure S7B), but we did not see obvious further up-regulation of CM-specific proteins comparing these two time points.

Expression of the PDLIM5 Transcript Variants 2 and 7 and of NKX2.5 Started at a Similar Time during Cardiomyogenic Differentiation

By searching the proteins that were strongly up-regulated (“up/ further-up”) over the course of differentiation in the KEGG database, we identified a number of heart-related KEGG pathways being enriched, that is, “hypertrophic cardiomyopathy”, “dilated cardiomyopathy”, “cardiac muscle contraction”, and “arrhythmogenic right ventricular cardiomyopathy” (Figure 3C). These contained a number of structural proteins (highlighted in Figure 2C,D) including PDLIM5, which not only is a structural component of the Z-disc in striated muscle46 but also has been implicated in signaling processes during heart development.23,24 Our proteomics data identified an upregulation of PDLIM5 in differentiating H3M isolated at d20 and d35 (Figure 5A and Supporting Information Tables S5 and S6). The up-regulation was confirmed by qPCR for H3M, H3, and I2 but was only significant for H3M (Figure 5B). Using a polyclonal antibody made against the epitope comprising amino acids 131 to 262 of PDLIM5, we observed a different pattern of PDLIM5 size variants in undifferentiated (d0) and differentiated (d20 and d35) samples. Several smaller forms of PDLIM5, predominantly at ∼55 kDa, but also weak bands at ∼40 and ∼30 kDa became essentially visible in the differentiated samples of H3M in addition to the major form of PDLIM5 at ∼65 kDa, which was detected in both differentiated and undifferentiated cells (Figure 5C). To scrutinize at which time point of the differentiation process these isoforms initially appear, time-course experiments were performed, utilizing a recent and efficient protocol for cardiomyogenic differentiation.36 The intensity of the ∼55 kDa band increased from day 7 of differentiation onward not only in H3M (Figure 5D, upper panel) but also in H3 and I2 (Figure 5E,F, upper panel). Of note, NKX2.5, coanalyzed by Western blot, showed a similar time course of expression (Figure 5D−F, lower panel). PDLIM5 bands at ∼65 and ∼55 kDa were additionally quantified densitometrically (Supporting Information Figure S10). The appearance of both proteins, the PDLIM5 band at ∼55 kDa and NKX2.5, was accompanied by the appearance of beating areas in the cell culture. To more precisely determine the composition of the detected PDLIM5 isoforms, we pursued a RT-PCR study. According to the NCBI database (http://www.ncbi.nlm.nih. gov/protein/?term=PDLIM5), PDLIM5 is a complex tran-

Enrichment Analyses Reveal Alterations in Energy Metabolism during Cardiomyogenesis

Proteins that were identified in at least two replicates of each time point comparison of H3M and that were at least in one comparison (d20/d0 and/or d35/d0) significantly regulated >1.5-fold were chosen for further analyses. KEGG pathway enrichment analyses applying STRING (setting “whole genome” of Homo sapiens as statistical background, http:// string-db.org/)42 were performed with 512 proteins >1.5-fold down- (Figure 3A) and 626 proteins >1.5-fold up-regulated (Figure 3B) between d20 and d0, respectively (regardless of their d35/d0 regulation ratio). Moreover, we defined further regulation categories describing proteins being significantly >1.5-fold up-regulated on d20 and significantly >4-fold upregulated on d35 (“up/further-up”, Figure 3C), significantly >1.5-fold up-regulated on d20 and >1-fold up-regulated on d35, but not higher up-regulated than on d20 (“up/stays up”, Figure 3D), significantly >1.5-fold down-regulated on d20 and 1.5-fold upregulated on d20 and 90% pure hPSC-CMs (over 99% myosin heavy chain 6 (MYH6/αMHC)- and α-actinin-1 (ACTN1)positive cells described in literature).49 Importantly, the quantitative proteomic analysis carried out with samples without (d20) and after neomycin selection (d35) delivered data sets of high congruence, suggesting that even in the unselected samples (d20) CM proteins are highly represented. Even in the presence of oxygen, hPSCs are known to depend mainly on anaerobic metabolism with high glycolytic rates (Warburg effect),12 and we expected a shift from glycolysis based fermentation toward oxidative phosphorylation during cardiomyogenic differentiation. Thus it seems on the first glimpse to be contradictory that in our study nearly all enzymes of glycolysis were concertedly up-regulated at d20 and d35 of differentiation. However, we applied an EB-based differentiation protocol in our study, and it is widely accepted that 1143


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Figure 6. RT-PCR for PDLIM5 transcript variants in H3M, H3, and I2. (A) RT-PCR for variants 1 and 8 using primers fw_exon2 and rev_exon6a. A band corresponding to transcript variant 1 (expected product size: 492 bp) was visible in all samples. Variant 8 (340 bp) could not be detected. (B) RT-PCR for PDLIM5 transcript variant 2 using primers fw_exon5/6b and rev_exon6b/9. A PCR product of variant 2 (117 bp) was detectable from d6-d7 onward. (C) RT-PCR for PDLIM5 variants 6 and 7 using primers fw_exon8 and rev_exon15. A PCR product corresponding to variant 7 (353 bp) was visible from d7 onward. A weak band corresponding to the PCR product of variant 6 (749 bp) was detectable in the d15 samples. (D) RT-PCR for the important cardiogenic transcription factor NKX2.5 showing expression from around d6 onward. (E) RT-PCR for the housekeeping gene β-actin (ACTB, 296 bp) as loading control. (A−E) Intensities of agarose gel bands were quantified densitometrically using the open-access 1144


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Journal of Proteome Research Figure 6. continued

software ImageJ version 1.46r (retrieved from https://imagej.nih.gov/ij/). The ratio of the respective band of interest against the housekeeping gene ACTB was determined for the individual time points, and this ratio was set to one at d0.

level. According to the NCBI database the human PDLIM5 protein exists in nine different isoforms, of which four isoforms have been previously described.21 The three LIM domaincontaining isoform a (transcript variant 1), which is homologous to the murine/rat ENH1, has a mass of ∼64 kDa and was suggested to be ubiquitously expressed in all tissues.57 Accordingly, we observed that this isoform was expressed in diverse cell types, namely, hPSCs, fibroblasts, and hPSC-CMs. The expression of shorter, LIM-less PDLIM5 isoforms, that is, of 40 kDa (homologous to murine ENH2), 33 kDa (homologous to murine ENH3), and 29 kDa (homologous to murine ENH4), occurs predominantly in skeletal or cardiac muscle21,23 and we detected bands likely corresponding to the 40 kDa (ENH2) and 33 kDa (ENH3) product in d20 and d35 hPSC-CMs as well. Interestingly, we have also observed a so far undescribed form of PDLIM5 at ∼55 kDa in d20 and d35 hPSC-CMs. The hPSC-CMs generated by the embryoid body-based48 differentiation protocol are comparable to an early, immature fetal phenotype, as described previously.33,48 Accordingly, we found that they express the embryonic myosins MYH3, MYH6, and MYL4 as well as MESP1, which is a transient marker for cardiac progenitors.58 To study expression of PDLIM5 in detail during the course of cardiomyogenic differentiation, we used the more recent and efficient small-molecule-based protocol described by Lian et al.36,59 Applying this protocol, we generally observed that large areas of the monolayer started beating upon 12 to 15 days of differentiation60 and that these cultures usually contained 70−90% cTnT-positive CMs on d15 of differentiation (data not shown). Similarly to the embryoid-bodyderived CMs, Lian et al. also described their hPSC-CMs as immature.36 Therefore, our time-course experiments for PDLIM5 expression rather reflect the very early stages of human embryonic heart development than the already described changes in PDLIM5 isoforms during embryonic and postnatal heart maturation in mice23 and rats,23,24 which might explain why the ∼55 kDa band, corresponding to isoforms b and g of PDLIM5, has not been detected before. From Western blot and RT-PCR experiments we could conclude that the isoforms b and g encoded by transcript variants 2 and 7, respectively, are both expressed in a timedependent manner, which was coincident with NKX2.5 expression starting on d6−d7 of cardiomyogenic differentiation. CM development severely relies on expression of the homeobox transcription factor NKX2.5 whose expression contributes to myogenic differentiation and maintains in embryonic, fetal, and adults hearts.61 Interestingly, both PDLIM5 and NKX2.5 mutations have been associated with adult-onset cardiomyopathy.22,62 PDZ-LIM domain proteins are considered as sequestration factors for nuclear transcription factors and act thereby beyond their structural function as signal modulators and control gene expression.63 Migfilin, a LIM-domain containing protein has been proposed to travel between the cytoplasm and the nucleus, where it can promote cardiac differentiation by interaction with NKX2.5, and this process is controlled by RNA splicing of migfilin.64 In that context there is room left for speculation about a direct or indirect crosstalk between PDLIM5 different splice variants and

the oxygen availability is decreased for cells within multicellular spheroids.50,51 Oxygen deprivation-associated alterations in gene expression, for instance, by activation of hypoxia-inducible transcription factor 1 (HIF-1), could explain the observed upregulation of enzymes belonging to glycolysis during EB-based differentiation.11,50 Supportive of our observations, Chung et al. showed that several key glycolytic enzymes (ENO3, PFK, PGM, and GAPDH) were more highly expressed in CMs compared with murine ESCs.14 In addition to Chung et al., in our study, even further glycolytic enzymes were up-regulated (PGAM1, TPI) on d20 and d35 of differentiation. Interestingly, hexokinases, which are considered to catalyze rate-limiting steps in glycolysis,52 were down-regulated on d20 and d35 of differentiation. However, it has to be noted that hexokinases are generally more highly expressed in cardiac muscle than, for instance, in other types of muscle and that their expression is inversely correlated to the expression of other glycolytic enzymes.53 hPSCs have less complex and fewer mitochondria than their differentiated progeny, especially CMs,12 but expression of many factors of the electron transport chain (ETC) as well as F-Type ATPases was higher in hPSCs compared with d20 of differentiation. Supportive of our observations, Birket et al. have shown that in vitro the major proportion of ATP is synthesized in hPSCs by oxidative phosphorylation.54 However, it has to be considered that on d20 of differentiation the overall proportion of CMs was rather low and that the observed effects might be adulterated by other cell types including residual feeder cells. Nevertheless, our proteomics screen revealed that the oxidative phosphorylation is reconstituted in purified hPSC-CMs harvested on d35 upon selection. This is in accordance with the widely accepted observation that CMs have a high content of mitochondria due to their increased demand of ATP for muscle contraction.55,56 Taken together we propose a model, in which, according to literature, hPSCs rely on aerobic fermentation (Warburg effect), whereas cells of d20 aggregates suffer from oxygen deprivation and therefore depend on anaerobic fermentation (increased expression of glycolysis enzymes, decreased expression of enzymes/proteins belonging to TCA cycle and respiratory chain). Highly enriched CMs growing as cardiac bodies also suffer from oxygen deprivation, leading to increased expression of glycolysis enzymes, but on the other hand express high levels of mitochondria that cause increased expression of proteins belonging to TCA cycle and respiratory chain. Moreover, a KEGG pathway analysis of proteins that were highly up-regulated in hPSC-CMs revealed enrichment of proteins involved in “cardiac muscle contraction”, “hypertrophic cardiomyopathy”, and “dilated cardiomyopathy”. Upon rational screening the list of proteins up-regulated in hPSCCMs, PDLIM5 attracted our interest because this protein has not only structural but also regulatory functions and is associated with pathological transformations of the heart. PDLIM5 has been reported to contribute to a protein complex at Z-discs in heart muscle, and its deletion in mouse caused destabilization of this complex accompanied by dilated cardiomyopathy.22 We found that expression of PDLIM5 was up-regulated in H3M-CMs on the protein and gene expression 1145


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Journal of Proteome Research NKX2.5. One might speculate that PDLIM5 isoforms b and g (derived from splice variants 2 and 7) function as NKX2.5 binding partners and thereby support the nuclear import of NKX2.5 either directly or by preventing binding of NKX2.5 to other splice variants of PDLIM5 that preferentially interact with the cytoskeleton, which then might impact cardiomyogenic differentiation. This hypothesis is supported by the finding that different splice variants of PDLIM5 can even exert antagonistic physiological functions.23,24 Another LIM domain protein, CAL (CSX-associated LIM protein), can promote cardiac differentiation by interaction with NKX2.5.65 Furthermore, PDLIM5 was shown to sequestrate ID2 (inhibitor of differentiation 2),66 which is required for cardiac conduction system development in association with NKX2.5 and TBX5.67 Moreover, for another PDZ-LIM protein, PDLIM7, cytoplasmic sequestration of Tbx5 and reduction of its transcriptional activity have been shown during cardiogenesis in zebrafish.68 Because the identified shortened isoforms b and g of PDLIM5 contain the three LIM domains, it might be speculated that an interaction with specific transcription factors important for heart development takes place. However, at this point it is not clear how the shortened PDLIM5 isoforms influence the cardiomyogenic differentiation or whether the two isoforms b and g have different roles in the differentiation process.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49-511-532-8245. Fax: +49-511-532-8801. E-mail: [email protected]. ORCID

Falk F. R. Buettner: 0000-0002-8468-1223 Author Contributions

F.F.R.B. conceived and designed the study. S.A.K. and F.F.R.B. designed experiments; S.A.K., S.W., A.O., R.O., H.K., and M.J.A. performed research; A.P. contributed analytical tools; S.A.K., H.K., R.Z., and F.F.B analyzed data; S.A.K. and F.F.B. wrote the manuscript.

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CONCLUSIONS Embryonic stem cell cardiomyogenesis is accompanied by vast changes in expression of metabolic and cytoskeletal proteins. The Z-disc-associated PDZ and LIM domain containing protein PDLIM5 was up-regulated during cardiomyogenesis. Contemporaneously to onset of expression of the important cardiomyogenic transcription factor NKX2.5, specific PDLIM5 splice variants were produced during early CM development, suggesting a so far unrecognized regulatory function of PDLIM5 during early heart development.

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pattern of different cell lines and biological replicates. Table S2. Oligonucleotides used as primers for qPCR. Table S3. Antibodies used for Western blot, immunofluorescence microscopy, and flow cytometry. (PDF) Table S4. Data of MS analyses of γ-irratiated, SILAClabeled human fibroblasts (feeder cells). (XLSX) Table S5. Data of MS analyses of SILAC-labeled d20 and d0 samples of H3M, H3, and I2 cells. (XLSX) Table S6. Data of MS analyses of SILAC-labeled d35 and d0 samples of H3M cells. (XLSX) Table S7. Calculation of d35 vs d20 ratios of H3M. (XLSX)

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy, EXC 62/2). R.O. was funded by the Deutsche Zentrum für Lungenforschung (DLZ 2011-2015:82DLZ00201; 2016-2020:82DLZ002A1). R.Z. was funded by the German Research Foundation (DFG; including grants: Cluster of Excellence REBIRTH DFG EXC62/3 and ZW64/4-1), the German Ministry for Education and Science (BMBF; including grants: 13N12606, and 13N14086), StemBANCC (support from the Innovative Medicines Initiative joint undertaking under grant 115439-2, whose resources are composed of financial contribution from the European Union [FP7/2007-2013] and EFPIA companies inkind contribution), and TECHNOBEAT (European Union H2020 grant 668724). We thank Prof. Dr. Gerardy-Schahn, head of the Institute of Clinical Biochemistry, Hannover Medical School (MHH) for providing general laboratory equipment and for carefully reading this manuscript. We also thank Prof. Dr. Scheper (Institute of Technical Chemistry, Leibniz University of Hannover) for providing bFGF and the ROCK inhibitor Y27632, Prof. Dr. Martin and Dr. Haase (LEBAO, MHH) for providing hiPSCs, and Dr. Johannes Zeiser, Karsten Heidrich, Anne Höfer, and Anne Oltmanns for practical assistance.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00534. Figure S1. Confirmation of pluripotency of SILAClabeled hPSCs. Figure S2. Determination of SILAC amino acid incorporation for H3M, H3, and I2. Figure S3. SILAC amino acid incorporation efficiencies of human fibroblasts in comparison to hPSCs. Figure S4. Determination of cardiomyocyte content. Figure S5. Confirmation of cardiomyogenic differentiation. Figure S6.Comparison of regulation ratios of d20/d0 and d35/ d0 analyses for H3M. Figure S7. Bioinformatics analysis of the d35/d20 comparison for H3M. Figure S8. Combined bioinformatics analysis of H3M, H3, and I2. Figure S9. Regulation of metabolic enzymes in H3M, H3, and I2. Figure S10. Densitometric quantification of PDLIM5 Western blot bands. Figure S11. Schematic presentation of PDLIM5 exon structure. Figure S12. Identification of PDLIM5 transcript variant 1 in H3M by DNA sequencing. Figure S13. RT-PCR analyses of PDLIM5 variants. Figure S14. Identification of PDLIM5 transcript variant 2 in H3M by DNA sequencing. Figure S15. Identification of PDLIM5 transcript variant 7 in H3M by DNA sequencing. Table S1. SILAC labeling

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ABBREVIATIONS ACN, acetonitrile; AmBic, ammonium bicarbonate buffer; bFGF, basic fibroblast growth factor; d, day; DTT, dithiothreitol; EB, embryoid body; ETC, electron transport chain; h, hour; hCBiPSC2, human cord blood derived induced 1146


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pluripotent stem cell clone 2; hESCs, human embryonic stem cells; HIF-1, hypoxia-inducible transcription factor 1; hiPSCs, human induced pluripotent stem cells; hPSCs, human pluripotent stem cells; hPSC-CMs, human pluripotent stem cell-derived cardiomyocytes; IF, immunofluorescence; MCS, multicellular spheroid; MEF-CM, murine embryonic fibroblastconditioned medium; min, minute; MYH6, myosin heavy chain 6 (α-MHC); MYL4, myosin light chain 4 or embryonic myosin light chain 1; PPP, pentose phosphate pathway; qPCR, quantitative real-time polymerase chain reaction; RI, Rhoassociated coiled-coil kinase inhibitor Y27632; SILAC, stable isotope labeling by amino acids in cell culture; TCA cycle, tricarboxylic acid cycle

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Proteomic Analysis of Human Pluripotent Stem Cell

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