OPEN Experimental & Molecular Medicine (2016) 48, e254; doi:10.1038/emm.2016.70 & 2016 KSBMB. All rights reserved 2092-6413/16 www.nature.com/emm ORIGINAL ARTICLE Mitochondrial pyruvate dehydrogenase phosphatase 1 regulates the early differentiation of cardiomyocytes from mouse embryonic stem cells Hye Jin Heo1, Hyoung Kyu Kim1, Jae Boum Youm1, Sung Woo Cho2, In-Sung Song1, Sun Young Lee1, Tae Hee Ko1, Nari Kim1, Kyung Soo Ko1, Byoung Doo Rhee1 and Jin Han1 Mitochondria are crucial for maintaining the properties of embryonic stem cells (ESCs) and for regulating their subsequent differentiation into diverse cell lineages, including cardiomyocytes. However, mitochondrial regulators that manage the rate of differentiation or cell fate have been rarely identified. This study aimed to determine the potential mitochondrial factor that controls the differentiation of ESCs into cardiac myocytes. We induced cardiomyocyte differentiation from mouse ESCs (mESCs) and performed microarray assays to assess messenger RNA (mRNA) expression changes at differentiation day 8 (D8) compared with undifferentiated mESCs (D0). Among the differentially expressed genes, Pdp1 expression was significantly decreased (27-fold) on D8 compared to D0, which was accompanied by suppressed mitochondrial indices, including ATP levels, membrane potential, ROS and mitochondrial Ca2+. Notably, Pdp1 overexpression significantly enhanced the mitochondrial indices and pyruvate dehydrogenase activity and reduced the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate compared to a mock control. In confirmation of this, a knockdown of the Pdp1 gene promoted the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate. In conclusion, our results suggest that mitochondrial PDP1 is a potential regulator that controls cardiac differentiation at an early differentiation stage in ESCs. Experimental & Molecular Medicine (2016) 48, e254; doi:10.1038/emm.2016.70; published online 19 August 2016 INTRODUCTION Energy metabolic turnover, from anaerobic glycolysis to Embryonic stem cells (ESCs) are self-renewing and pluripotent oxidative phosphorylation, is predominantly regulated by and differentiate into diverse cell lineages.1 ESCs are a valuable mitochondria and is an essential step that triggers ESC tool for studying early cardiomyogenesis.2–4 Several molecular differentiation into cardiomyocytes.17 Therefore, the robust and conditional factors have been suggested to be major regulation of mitochondrial energy metabolism shifts is essen- inducers of ESC-derived cardiomyogenesis, including cardiac tial to meet energy demands during cell differentiation. transcription factors,5 reactive oxygen species (ROS)6 and The molecular mechanisms underlying mitochondrial hypoxic environments.7 Recent studies have suggested that energy metabolism during cardiomyocyte differentiation several mitochondrial factors regulate cardiomyocyte differen- remain unclear. This study aimed to investigate possible tiation, including mitochondrial DNA transcription factors,8 mitochondrial regulators that are necessary for cardiomyocyte mitochondrial ROS levels,6 and mitochondrial permeability differentiation from mESCs. Among mitochondrial regulators, transition pore (mPTP) states.9,10 Recently, it was reported that the pyruvate dehydrogenase (PDH) complex regulates the the inhibition of mPTP promotes early cardiomyocyte differ- acetyl-CoA conversion rate from pyruvate. The acetyl-CoA entiation from pluripotent stem cells and maturation in conversion rate directly affects tricarboxylic acid (TCA) cycle 10,11 developing embryos. activity, providing NADH2 to electron transfer chain com- These observations suggest that mitochondria can determine plexes. Thus, the PDH complex has been hypothesized to be a cardiomyocyte differentiation capacity in both human and mouse mitochondrial molecular regulator of cardiomyocyte differen- ESCs (mESCs) via the modulation of energy metabolism.12–16 tiation. In this study, we screened differentially expressed genes 1National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan, Korea and 2Laboratory of Vascular Biology and Stem Cell, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea Correspondence: Professor J Han, National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea. E-mail: [email protected] Received 27 January 2016; revised 2 March 2016; accepted 22 March 2016 Mitochondrial PDP1 regulates cardiac differentiation HJ Heo et al 2 related to energy metabolism during embryoid body (EB) Table 1 Quantitative real-time PCR primer sequences fi differentiation and found signi cantly modulated expression of α-actinin Forward 5′-AGCCAGGAACAGATGAACGA-3′ PDH complex components. Reverse 5′-AAGTCGATGAAGGCCTGGAA-3′ Among these regulators, we report that the expression of the α-MHC Forward 5′-GCCCAGTACCTCCGAAAGTC-3′ pyruvate dehydrogenase phosphatase catalytic subunit 1 (Pdp1) Reverse 5′-GCCTTAACATACTCCTCCTTGTC-3′ gene was significantly reduced during the early stages of cTnI Forward 5′-CGTGGAAGCAAAAGTCACCA-3′ mESC-derived EB differentiation. To examine whether Pdp1 Reverse 5′-GTCCTCCTTCTTCACCTGCT-3′ affects cardiomyocyte differentiation, we established mESCs cTnT Forward 5′-CAGAGGAGGCCAACGTAGAAG-3′ that transiently overexpressed or consistently inhibited Pdp1 in Reverse 5′-CTCCATCGGGGATCTTGGGT-3′ mESCs. In addition, we investigated PDH complex activity and Cx43 Forward 5′-ACGGCAAGGTGAAGATGAGA-3′ mitochondrial function changes in these cells. Reverse 5′-GAGAGACACCAAGGACACCA-3′ Drp1 Forward 5′-GGTGGTCAGGAACCAACAAC-3′′ MATERIALS AND METHODS Reverse 5′-TCACAATCTCGCTGTTCTCG-3′ Maintenance of undifferentiated mESCs Mef2c Forward 5′-ACCAGGACAAGGAATGGGAG-3′ ThemESClineR1(ATCC)wasroutinelyculturedandgrownonmito- Reverse 5′-GGCGGCATGTTATGTAGGTG-3′ tically arrested mouse embryonic fibroblasts (MEFs, 50 000 cells per MLC2 Forward 5′-GGCACCAAAGAAAGCCAAGA-3′ cm2) in 60-mm culture plates.18 For MEF exclusion, EMG7 (mESCs), Reverse 5′-GGACCTGGAGCCTCTTTGAT-3′ which have an α-myosin heavy chain (MHC) promoter controlling Mfn1 Forward 5′-GGCGTGATTTGGAAAACAGT-3′ green fluorescent protein (GFP) expression, were maintained in 0.1% Reverse 5′-TACTTGGTGGCTGCAGTTTG-3′ gelatin-coated 60-mm culture dishes without a feeder layer.10 Mfn2 Forward 5′-TGAAGACACCCACAGGAACA-3′ Reverse 5′-GCAGAACTTTGTCCCAGAGC-3′ Induction of mESC-derived cardiomyocytes using EBs GAPDH Forward 5′-CACCATCTTCCAGGAGCGAG-3′ The hanging-drop method was used to generate EBs from 750 cells per Reverse 5′-CCTTCTCCATGGTGGTGAAGAC-3′ 20 μl differentiation media (differentiation day 0, D0).18 On day 4 of differentiation, EBs were seeded in 1% gelatin-coated culture dishes μ for each experiment. During differentiation, the culture media were polymerase (Invitrogen) in the presence of 1 M oligonucleotide changed daily. Beginning on day 6 of differentiation (2 days after primers. Quantitative real-time PCR was performed using the iQ5 plating), EB growth was observed daily for self-beating capacity. For real-time system and iQ SYBR Supermix (Bio-Rad, Hercules, CA, analyses, EBs were dissociated with 0.1% collagenase (Worthington, USA). Expression of target mRNAs relative to housekeeping Lakewood, NJ, USA) plus 0.25% trypsin-EDTA (Gibco, Waltham, gene expression (glyceraldehyde 3-phosphate dehydrogenase MA, USA) for 30 min at 37 °C. (GAPDH) mRNA) was calculated using the threshold cycle –Δ(ΔCT) (CT)asr = 2 ,whereΔCT = CT target − CT GAPDH and Transient overexpression of Pdp1 in R1 mESCs Δ(ΔCT) = ΔCTD8 – ΔCTD0. The primer sequences are shown in The Pdp1 gene was subcloned into the EcoRI/XbaI enzyme sites of the Table 1 and Supplementary Table S1. FUW expression vector. FUW vectors with the Pdp1 coding sequence or empty FUW vectors were transfected into HEK293FT cells to Western blot analyses produce Pdp1-containing viruses. Two days after transfection, virus- Cells were homogenized in protein lysis buffer and centrifuged at containing media were collected to treat undifferentiated R1 cells. 10 000 g for 10 min at 4 °C. After centrifugation, protein samples After treatment with virus-containing media, the hanging-drop (50 μg) were transferred onto nitrocellulose membranes, which were method was employed to induce differentiation in mESC-derived then incubated with primary antibodies against sarcomeric α-actinin cardiomyocytes. (Sigma-Aldrich), cTnT (Abcam, Cambridge, UK), PDP1 (Abcam), α β Pdp1 PDH-E1 (Abcam) and -tubulin (Abcam). Immunoreactive protein Stable suppression of in EMG7 mESCs bands were detected using the SuperSignal West Pico system and For lentivirus-mediated Pdp1 gene transfer, the EMG7 mESC line visualized using LAS-3000 PLUS (Fuji Photo Film Company, Tokyo, was incubated for 15 min with 6 μgml− 1 Polybrene. Cells were Japan). transfected with 10 μlof1×106 TU ml − 1 of viral particles (Sigma-Aldrich, St Louis, MO, USA). Twenty hours after the first transfection, virus-containing media were removed and three times the Immunocytochemistry number of viral particles was added for the second virus transfection.19 Undifferentiated ESCs (D0) and differentiated EBs (D8) were cultured fi To establish a stable cell line, we purified transfected cells using a final on gelatinized 12-mm