Melatonin promotes cardiomyogenesis of embryonic stem cells via inhibition of HIF-1α stabilization

Melatonin, a molecule involved in the regulation of circadian rhythms, has protective effects against myocardial injuries. However, its capability to regulate the maturation of cardiac progenitor cells is unclear. Recently, several studies have shown that melatonin inhibits the stabilization of hypoxia‐inducible factors (HIFs), important signaling molecules with cardioprotective effects. In this study, by employing differentiating mouse embryonic stem cells, we report that melatonin significantly upregulated the expression of cardiac cell‐specific markers (myosin heavy chains six and seven) as well as the percentage of myosin heavy chain‐positive cells. Importantly, melatonin decreased HIF‐1α stabilization and transcriptional activity and, in contrast, induced HIF‐2α stabilization. Interestingly, the deletion of HIF‐1α completely inhibited the pro‐cardiomyogenic effect of melatonin as well as the melatonin‐mediated HIF‐2α stabilization. Moreover, melatonin increased Sirt‐1 levels in a HIF‐1α‐dependent manner. Taken together, we provide new evidence of a time‐specific inhibition of HIF‐1α stabilization as an essential feature of melatonin‐induced cardiomyogenesis and unexpected different roles of HIF‐1α stabilization during various stages of cardiac development. These results uncover new mechanisms underlying the maturation of cardiac progenitor cells and can help in the development of novel strategies for using melatonin in cardiac regeneration therapy.

cardiomyogenesis; hypoxia‐inducible factor‐alpha; melatonin; mouse embryonic stem cells

The mammalian adult heart has a low regenerative capacity, which is directly associated with the very limited proliferative capacity of cardiac cells. Rather than making new, functional muscle, it is prone to scarring and hypertrophy.[1] Damaged areas are replaced by alternative, fibrotic tissue that often leads to fatal arrhythmias and subsequent heart failure.[2] However, increasingly evidence suggests the presence of postnatal mammalian cardiomyogenesis, which can be presented as a low‐rate division of pre‐existing cardiomyocytes.[1] , [2] A contribution of cardiac stem cells or other cardiac progenitors to the new cardiomyocytes formation has also been described.[1] , [2] The new information about the genesis of new cardiomyocytes in adult hearts brings a novel dimension to the heart self‐regeneration capacity. Importantly, the identification of the factors that regulate and significantly promote cardiomyocytes differentiation and maturation should bring essential information about their promising application as new treatments for damaged hearts.

Melatonin is a hormone with numerous important physiological functions.[3] , [4] , [5] , [6] Recently, the significant regulatory role of melatonin (N‐acetyl‐5‐methoxytryptamine) in cardiovascular functions was shown.[3] , [4] , [7] , [8] Melatonin can contribute to the protection of the heart against an ischemia/reperfusion injury, atherosclerosis, hypertension, and heart failure (see review[3] , [4] , [5] , [7] , [9] , [10] ). These effects are mostly credited to the melatonin antioxidant capacity.[6] , [8] , [11] , [12] In addition to its antioxidant activity, melatonin can bind to its specific membrane receptors, and both receptor‐dependent and receptor‐independent effects of melatonin have been described.[3] , [4] , [5] , [6] However, the exact molecular mechanisms of the melatonin‐mediated regulation of a variety of cell functions are not clear.

Interestingly, several studies have suggested that melatonin affects the intracellular levels of HIF‐1α, an important signaling molecule.[13] , [14] , [15] , [16] , [17] , [18] HIFs are heterodimeric protein complexes consisting of a constitutively expressed β‐subunit (HIF‐1β) and an oxygen‐dependent α‐subunit (HIF‐1α, HIF‐2α) with tightly regulated protein stability and transcriptional activity.[12] HIFs accumulation and activity are regulated by both oxygen‐dependent and oxygen‐independent signaling pathways. Melatonin was shown to inhibit HIF‐1α‐mediated pro‐angiogenic effects related to the inhibition of VEGF expression in various types of tumor cells through different mechanisms, including inhibition of HIF‐1α transcription and protein synthesis or decreasing HIF‐1α stability in the cytoplasm related to inhibition of the ubiquitin‐proteasome system.[12] It should be noted that melatonin‐mediated inhibition of VEGF was also observed under normoxic conditions.[19] More recently, Sohn et al.[20] reported the melatonin inhibition of mRNA of both HIF‐1α and HIF‐2α in prostate cancer cells through a microRNA‐dependent mechanism. In vitro observations are supported by studies from the group of Kaur et al. showing that melatonin administration in vivo reduced hypoxia‐induced VEGF concentrations in various tissues, including cerebellum,[21] pineal gland,[22] and retina.[23]

Importantly, both HIF‐1α and HIF‐2α are expressed in the heart with a demonstrated crucial role in cardiomyogenesis.[1] , [2] , [24] Moreover, the positive role of HIF‐1α stabilization in the cardioprotection of the stressed adult heart is suggested.[1] , [2] , [25] However, the impact of melatonin‐mediated HIFs modulation in cardiomyogenesis is unknown. Thus, the aim of this study was to determine the effect of melatonin on the differentiation and the maturation of cardiac progenitor cells. With this aim, an in vitro model of spontaneously differentiated mouse embryonic stem cells (mESC) was employed. Understanding the mechanisms underlying the melatonin effects on the maturation of cardiac progenitor cells will contribute to the development of novel strategies for using melatonin in cardiac regeneration therapy.

The mESC cell line R1 (HIF‐1α+/+) and R1‐derived HIF‐1α−/− (kindly provided by Peter F. Carmeliet; Vesalius Research Center, VIB, University of Leuven, 3000 Leuven, Belgium) were cultivated on gelatin‐coated dishes in Dulbecco′s modified Eagle′s medium (DMEM; HyClone, Logan, UT, USA) supplemented with 15% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 IU/mL penicillin and 0.1 mg/mL streptomycin (Sigma, St. Louis, MO, USA), 1× nonessential amino acid (Gibco), 0.05 mmol/L β‐mercaptoethanol (Fluka, Buchs, Switzerland), and 1000 U/mL of leukemia inhibitory factor (Chemicon, Temecula, CA, USA). The cells were maintained at 37°C in humidified air supplemented with 5% CO2.

Suspension of mESC (2.5 × 106 cells/mL) was seeded on the top of silicone mold preformed microwells in 1.5% agarose (VWR, Radnor, PA, USA) to form embryoid bodies (EBs; for more details see (Dahlmann J, 2013)). After 24 hours of incubation (day 0), the EBs were gently transferred onto an agar‐coated dish and cultivated in medium without leukemia inhibitory factor supplementation. On day 5 (5 days), the EBs were seeded on gelatin‐coated dishes into DMEM/F‐12 (1:1) medium (HyClone) supplemented with insulin‐transferrin‐selenium (Gibco) and antibiotics (specification above) and cultivated for a further two (5 + 2) or 10 (5 + 10) days (for details, see Figure [NaN] ).

For the treatment of the cells within EBs, the medium was supplemented with 100 μmol/L or 100 nmol/L melatonin dissolved in ethanol (<0.1%). Cells treated with ethanol in equivalent concentration were used as a control. Melatonin treatment was performed in three different time windows from day 0 to 5, day 6 to 10, and day 6 to 15. Melatonin had no toxic effect on the cells in the concentrations used, as proven by cell viability assay (see Figure S1).

Total RNA was extracted using the UltraClean Tissue & Cells RNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA). One microgram of total RNA was reversely transcribed into first strand cDNA using the Transcriptor First Strand cDNA Synthesis Kit according to the manufacturer's protocol (Roche, Basel, Switzerland). Real‐time quantitative PCR (RT‐qPCR) reactions were performed in a LightCycler480 instrument using LightCycler480® Probes Master solutions (Roche) and the following program: an initial denaturation step at 95°C for 10 minutes, followed by 45 cycles (95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 1 seconds), and a final cooling step at 40°C for 1 minutes. The sequences of primers and numbers of Universal Probe Library (UPL) probes are listed in Table [NaN] . Data were normalized to ribosomal protein L13A (Rpl13a) and presented as 2−∆∆cq.[26] Three independent experiments for each condition were carried out.

Sequence of primers used in quantitative RT‐PCR

Total protein lysates were prepared from mESC differentiated for particular time intervals (5 + 2d or 5 + 10d) using a lysis buffer (50 mmol/L Tris‐HCl, 100 mmol/L NaCl, 10% glycerol, 1% SDS, 1 mmol/L EDTA, with proteases and phosphatases inhibitors [Roche], pH 7.4). Protein concentrations were determined using BCA Protein Assay (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instructions. Twenty‐five micrograms of total protein was separated using a 10% SDS‐PAGE gel, transferred onto a polyvinyl difluoride membrane (Merck Millipore, Darmstadt, Germany), and blocked in 5% low‐fat milk in TBS‐T buffer (Tris, 0.05% Tween‐20). The following primary and secondary antibodies were used to detect particular proteins: rabbit anti‐HIF‐1α (GTX127309; GeneTex, Irvine, CA, USA), rabbit anti‐HIF‐2α (GTX30114; GeneTex), rabbit anti‐Sirt‐1 (#2028; Cell Signaling Technology, Danvers, MA, USA), mouse anti‐MF20 (MHCα/β; antibody detects both alpha and beta MHC isoforms; kindly provided by Dr. Donald Fischman, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and mouse anti‐β‐actin (sc47778; Santa Cruz Biotechnology, Dallas, TX, USA) as a loading control. Corresponding secondary HRP‐conjugated anti‐rabbit or anti‐mouse antibodies were employed, and HRP was detected using an ECL kit (Pierce Biotechnology) and radiographic film (AGFA, Gent, Belgium). Optical densities were quantified by scanning densitometry and expressed in arbitrary units determined by ImageJ software (NIH, Bethesda, MA, USA).

For immunofluorescent staining, 5 + 10d cells were digested with accutase solution (Biowest, Nuaillé, France) for 5 minutes at 37°C and plated on Falcon Culture Slides (Corning Incorporated, New York, NY, USA) coated with 0.1% gelatin. After 24 hours of incubation, the cells were washed in 1× phosphate‐buffered saline (PBS), fixed in 0.5% formaldehyde for 20 minutes at room temperature (RT), permeabilized with 0.1% TritonX‐100 for 30 minutes at RT, and then washed in 1× PBS. After 1 hour of blocking with 5% bovine serum albumin, cells were incubated for 2 hours at RT with the mouse anti‐MF20 (MHCα/β; the antibody detects both alpha and beta MHC isoforms; kindly provided by Dr. Donald Fischman, Developmental Studies Hybridoma Bank). Several washing steps later, the cells were incubated for 1 hour at RT with DyLight 488 conjugate goat anti‐mouse IgG (TF35502; Thermo Fisher Scientific, Waltham, MA USA). Nuclei were counterstained with DAPI (1 μg/mL), and cells were mounted on microscopic slides in Mowiol (Calbiochem, La Jolla, CA, USA) solution (10% Mowiol 4–88 prepared in 25% glycerol, 100 mmol/L Tris‐HCl, and 0.6% 1.4‐diazabicyclo‐[2.2.2]‐octane, pH 8.5). Images were acquired using a confocal microscope (TCS SP5; Leica, Wetzlar, Germany) equipped with a 63 × 1.4 oil immersion objective. The percentage of MHCα/β‐positive cells were analyzed using a confocal microscope (Fluoview FV10i; Olympus, Shinjuku, Tokyo, Japan) and determined using ImageJ software (NIH).

Data are presented as mean ± standard error of the mean (SEM). Statistical differences between mean values were tested by Student's t‐test. P≤.05 was considered statistically significant.

To investigate the effect of melatonin on the differentiation and maturation of cardiac progenitor cells, the model of mESC‐derived cardiomyogenesis was employed (Figure [NaN] ). A development of cardiac cell phenotype was determined based on the gene expression of cardiac cell‐specific markers, such as NK2 transcription factor‐related locus 5 (Nkx2.5), myosin heavy chain 6 (Myh6), and myosin heavy chain 7 (Myh7), and on the differences in myosin heavy chain (MHC) intracellular localization in mESC‐derived cardiomyocytes (mESC‐CMs; Figure [NaN] ).

Primarily, the time‐specific impact of melatonin (100 μmol/L) was investigated by treating the cells for various periods during the differentiation process: from day 0 to 5, from day 6 to 10, and from day 6 to 15. The melatonin treatment from day 6 to 10 significantly upregulated the gene expression of all evaluated cardiac‐specific markers (Figure [NaN] A‐C), as well as the percentage of MHCα/β‐positive cells (Figure [NaN] D‐E). Moreover, melatonin‐treated mESC‐CMs revealed a higher organization of contractile apparatus with clearly visible sarcomeres, compared to spot localization of MHCα/β proteins in control mESC‐CMs (Figure [NaN] F). Interestingly, in contrast, treatment with the same concentration of melatonin from day 0 to 5 or from day 6 to 15 (see Figure S2) did not have any significant impact on the progress of cardiomyogenic differentiation. Importantly, a lower melatonin concentration (100 nmol/L) was also tested, however, without any significant effects on the mESC‐derived cardiomyogenesis (data not shown). Thus, 100 μmol/L of melatonin and the time window for treatment from day 6 to 10 were chosen for all further experiments.

To evaluate which factors are modulated by melatonin during cardiomyogenesis, the protein levels of HIF‐1α and HIF‐2α and the expression of putative HIF‐1α (carbonic anhydrase IX (CAIX)) and HIF‐2α (octamer‐binding transcription factor 4 [Oct4]) target genes were examined at day 7 of differentiation (Figure [NaN] ). As shown in Figure [NaN] A, melatonin significantly decreased HIF‐1α stabilization (Figure [NaN] A). This corresponded to the lower gene expression of CAIX (Figure [NaN] B). On the other hand, melatonin increased stabilization of HIF‐2α (Figure [NaN] C), accompanied by increased Oct4 gene expression (Figure [NaN] D). To further analyze long‐term effect of melatonin, the levels of HIF‐1α, HIF‐2α, and MHCα/β were detected at day 15 (see Figure S3). In comparison with the control cells, mESC‐CMs treated by melatonin revealed similar protein level of HIF‐2α, but a higher level of MHCα/β proteins. No HIF‐1α stabilization was detected in both tested groups (see Figure S3).

To clarify the role of HIF‐1α in melatonin‐mediated differentiation and maturation of cardiac progenitor cells, the model of HIF‐1α‐deficient mESC differentiation was employed. First, the mRNA level of cardiac‐specific markers (Nkx2.5, Myh6, and Myh7) was analyzed at day 15 (Figure [NaN] A‐C). In comparison with the wild type (Figure [NaN] A‐C), HIF‐1α‐deficient mESC‐CMs were characterized by a lower expression of all selected genes (Figure [NaN] A‐C). Interestingly, the mRNA levels of these genes were not changed in HIF‐1α‐deficient cells after melatonin treatment. Additionally, the presence and the percentage of MHCα/β+ cells were similar in both the control‐ and melatonin‐treated HIF‐1α−/− mESC‐CMs (Figure [NaN] D‐E). Similar to wild‐type cells, the melatonin treatment at other time windows and in different concentrations did not change the mRNA levels of all tested genes in mESC‐CMs (see Figure S4). To further verify the impact of HIF‐1α deletion on the melatonin‐mediated regulation of selected factors, the changes in the expression profile of both HIF‐α responsive genes and the stabilization of HIF‐1α and HIF‐2α were analyzed in the control‐ and melatonin‐treated HIF‐1α−/− cells at day 7 of differentiation (Figure [NaN] ). The efficiency of HIF‐1α gene deletion in HIF‐1α−/− mESC‐CMs was clearly demonstrated via the lack of HIF‐1α protein stabilization (Figure [NaN] A). This was further proved by the unchanged mRNA level of CAIX in the melatonin‐treated cells in comparison with control HIF‐1α−/− mESC‐CMs (Figure [NaN] B). Interestingly, the stabilization of HIF‐2α (Figure [NaN] C) and the expression of Oct4 (Figure [NaN] D) were similar in both the control‐ and melatonin‐treated cells. Moreover, this effect was reflected in the subsequent significant reduction in HIF‐2α stabilization at day 15 of differentiation, where MHCα/β proteins had a similar protein profile in wild type and HIF‐1α‐deficient mESC‐CMs. Similarly, to wild‐type cells, no HIF‐1α stabilization was detected in both tested groups (see Figure S5).

As the melatonin regulatory effects are accompanied by the modulation of Sirt‐1, the impact of HIF‐1α deletion on the melatonin‐mediated regulation of Sirt‐1 was analyzed in the control‐ and melatonin‐treated wild type and HIF‐1α−/− cells at day 7 and at day 15 of differentiation. The protein level of Sirt‐1 was two times higher after melatonin treatment in wild‐type cells at day 7 of differentiation (Figure [NaN] A). However, the Sirt‐1 protein level was similar in the control‐ and melatonin‐treated wild‐type cells at day 15 of differentiation (Figure [NaN] B). Importantly, in agreement with other data, the level of Sirt‐1 was similar in both the control‐ and melatonin‐treated HIF‐1α−/− cells at day 7 of differentiation (Figure [NaN] C), as well as at day 15 of differentiation (Figure [NaN] D).

In this study, we show that time‐specific melatonin treatment promotes cardiac differentiation and the maturation of mESC. Although it has been reported that melatonin is able to protect the heart against myocardial injuries via its antioxidant capacity,[27] , [28] its role in the maturation of cardiac progenitor cells has not yet been investigated. Here, we show for the first time that melatonin significantly upregulates the expression profiles of cardiac‐specific markers (Nkx2.5, Myh6, Myh7) in differentiating mESC as well as increases the percentage of MHCα/β‐positive mESC‐CMs. Furthermore, the higher maturation status of mESC‐CMs, characterized by the presence of clearly visible sarcomeres within the contractile apparatus, was observed only in melatonin‐treated cells. Interestingly, these regulatory melatonin effects are dependent on the maturation status of the differentiating mESC. Overall, this is the first report demonstrating that melatonin significantly promotes both the differentiation and the maturation of mESC‐CMs.

Our present data show that melatonin promotes cardiomyogenesis in vitro via the reduction of protein expression and the transcriptional activity of HIF‐1α. The observed melatonin effects on HIF‐1α are in accordance with previous findings of other authors showing the melatonin‐mediated downregulation of HIF‐1α in a variety of cancer cells by various mechanisms including melatonin antioxidant action.[13] , [14] , [15] , [16] , [17] , [18] However, we suggest that the role of HIF‐1α in the regulation of cardiomyogenesis is complex, based on the timing of the HIF‐1α activity modulation during mESC differentiation. The positive effect of HIF‐1α stabilization and activation on cardiac cell differentiation was reported by Ng et al.,[29] who showed that overexpressed HIF‐1α in mESC significantly increased the percentage of beating EBs as well as mESC‐CMs maturation status. Controversially, we and other authors have shown that the complete HIF‐1α deficiency attenuates[30] or even inhibits[31] cardiomyogenesis in differentiating mESC. Importantly, similar to this contrasting regulatory role of HIF‐1α, also other signaling molecules are identified with a described dual role in cardiomyogenesis. For example, Wnt/β‐catenin signals have opposing roles during various stages of cardiac development.[32] This is in accordance with our observation that melatonin‐mediated HIF‐1α inhibition is dependent on the stage of progression of mESC differentiation. Thus, we suggest that HIF‐1α plays different roles at different time points of cardiac cells maturation. However, further research is required to uncover this effect.

Similarly, to HIF‐1α, HIF‐2α was also shown as an important modulator of cardiomyogenesis in vitro.[24] We clearly show that in mESC‐derived cardiac progenitor cells, melatonin increases HIF‐2α stabilization and transcriptional activity. This seems to be a cell‐specific effect, as melatonin attenuated the expression of HIF‐2α in the prostate cancer cell line.[20] Importantly, melatonin treatment was associated with increased levels of the NAD+ sensing deacetylase Sirt‐1, which is in accordance with the results of other authors.[9] , [33] HIF‐1α transcriptional activity was shown to repress Sirt‐1 activity via promoting glycolysis and subsequently decreasing the production of sirtuins cofactor NAD+.[10] Our data suggest the importance of melatonin‐mediated suppression of HIF‐1α activity in melatonin‐induced Sirt‐1 protein expression. This is supported by the observation that the lack of HIF‐1α fully suppressed the melatonin‐mediated upregulation of Sirt‐1 protein expression as well as HIF‐2α stabilization. These results provide new evidence showing that the melatonin‐mediated inhibition of HIF‐1α regulates the Sirt‐1 protein level and HIF‐2α stabilization. However, the exact mechanism needs to be elucidated. We speculate that observed effects are related to receptor‐independent action of melatonin, as the expression of melatonin receptors did not change during the differentiation process (see Figure S6). Among these, receptor‐independent effects can be melatonin‐mediated modulation of intracellular redox status, as the melatonin‐mediated effects on myocardium were, previously, mostly credited to melatonin antioxidant capacity.[6] , [8] , [12] However, the exact molecular mechanisms of the melatonin‐mediated regulation of variety of cell functions are not clear.

In summary, this is the first evidence describing the role of melatonin in the differentiation and maturation of mESC‐CMs. Melatonin significantly promotes both the differentiation and the maturation of mESC‐CMs via the partial, time‐dependent inhibition of HIF‐1α. Importantly, our data also uncovered unexpected different roles of HIF‐1α stabilization during various stages of cardiac development. These results uncover new mechanisms underlying the maturation of cardiac progenitor cells and can help in the development of novel strategies for using melatonin in cardiac regeneration therapy.

We would like to thank Peter F. Carmeliet of the Vesalius Research Center, VIB, University of Leuven, Belgium, for providing us with the R1 HIF‐1α−/− mESC line. Further, we would like to thank Donald A. Fishman for the development of the MF20 cell line which was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.

The authors declare no conflict of interests with regard to this study.

Graph: Schematic illustration of the protocol used for the in vitro differentiation of mESC. Suspension of mESC was seeded on the top of silicone mold preformed microwells to form embryoid bodies. After 24 hours of incubation (day 0), the EB s were gently transferred onto agar‐coated dish. On day 5 (5 days), the EB s were seeded on gelatin‐coated dishes and cultivated for a further 2 (5 + 2) or 10 (5 + 10) days. mESC —mouse embryonic stem cells; DMEM —Dulbecco's modified Eagle's medium; LIF —leukemia inhibitory factor; FBS —fetal bovine serum; ITS —insulin‐transferrin‐selenium

Graph: Melatonin promotes cardiomyogenesis in vitro. Relative gene expression of selected cardiac markers (A) NK 2 transcription factor‐related locus 5 ( Nkx2.5 ), (B) myosin heavy chain 6 ( Myh6 ), and (C) myosin heavy chain 7 ( Myh7 ) was detected by qRT ‐ PCR in the mESC ‐derived cardiomyocytes (day 15 of differentiation) treated with (100 μmol/L) or without (−) melatonin. Data are expressed as mean ±  SEM from three independent experiments; * P <.05. (D) The presence and (E) the percentage of MHC α/β‐positive cells as well as (F) the localization of the myosin heavy chain α and β ( MHC α/β‐using anti‐ MF 20 antibody) were determined in mESC ‐derived cardiomyocytes at day 15, using confocal microscopy from at least (B‐C) three independent experiments. (E) Data are expressed as mean ±  SEM ; *** P <.001

Graph: Melatonin‐mediated inhibition of HIF ‐1α and HIF ‐2α stabilization and activity. Protein levels of (A) hypoxia‐inducible factor ( HIF )‐1α and (C) HIF ‐2α were detected by Western blot in the mESC ‐derived cardiomyocytes at day 7 of differentiation. The protein levels were normalized to the β actin signal. The densitometric analysis was calculated from three independent experiments, and data are expressed as means ±  SEM , * P <.05, ** P <.01. Relative gene expression of the HIF ‐1α target gene (B) carbonic anhydrase ( CAIX ) and the HIF ‐2α target gene (D) octamer‐binding transcription factor 4 (Oct4) was measured by qRT ‐ PCR at day 7 of differentiation. Data are expressed as mean ±  SEM from at least three independent experiments; * P <.05

Graph: Depletion of HIF ‐1α completely inhibits the pro‐cardiomyogenic effect of melatonin. Expression of selected cardiac markers (A) NK 2 transcription factor‐related locus 5 ( Nkx2.5 ), (B) myosin heavy chain 6 ( Myh6 ), and (C) myosin heavy chain 7 ( Myh7 ) was detected by qRT ‐ PCR in the HIF ‐1α‐deficient mESC ‐derived cardiomyocytes (day 15 of differentiation) treated with (100 μmol/L) or without (−) melatonin. Data are expressed as mean ±  SEM from three independent experiments. (D) The presence of myosin heavy chain α and β ( MHC α/β‐using anti‐ MF 20 antibody) protein and (E) the percentage of MHC α/β positive cells were determined in mESC ‐derived cardiomyocytes at day 15, using confocal microscopy. Data are expressed as mean ±  SEM from three independent experiments

Graph: Depletion of HIF ‐1α completely inhibits the melatonin‐mediated regulation of HIF ‐2α. Protein levels of (A) HIF ‐1α and (C) HIF ‐2α were detected by Western blot in the HIF ‐1α −/−mESC ‐derived cardiomyocytes at day 7 of differentiation. The protein levels were normalized to the β‐actin signal. The densitometric analysis was calculated from three independent experiments, and data are presented as means ±  SEM. Expression of HIF ‐1α target gene (B) carbonic anhydrase ( CAIX ) and HIF ‐2α target gene (D) octamer‐binding transcription factor 4 (Oct4) was detected by qRT ‐ PCR at day 7 of differentiation. Data are expressed as mean ±  SEM from at least three independent experiments

Graph: Melatonin‐mediated increase of Sirt‐1 is directly regulated by HIF ‐1α. Protein levels of sirtuin‐1 (Sirt‐1) were detected by Western blot in the (A‐B) wild type and (C‐D) HIF ‐1α −/−mESC ‐derived cardiomyocytes (A, C) at day 7 and (B, D) at day 15 of differentiation, treated with (100 μmol/L) or without (−) melatonin. The protein levels were normalized to the β‐actin signal. The densitometric analysis was calculated from three independent experiments, and data are expressed as means ±  SEM , * P <.05

Graph: Scheme indicating the possible role of melatonin in the promotion of cardiomyogenesis of mESC via inhibition of HIF ‐1α stabilization. Melatonin affects number of differentiated cardiac‐like cells and their maturation status including the presence of contractile apparatus. For more details, see Discussion

Graph