Promotion of maturation in CDM3-induced embryonic stem cell-derived cardiomyocytes by palmitic acid

Abstract

Background

Myocardial infarction leads to myocardial necrosis, and cardiomyocytes are non-renewable. Fatty acid-containing cardiomyocyte maturation medium promotes maturation of stem cell-derived cardiomyocytes.

Objective

To study the effect palmitic acid on maturation of cardiomyocytes derived from human embryonic stem cells (hESCs) to optimize differentiation for potential treatment of myocardial infarction by hESCs.

Methods

hESCs were differentiated into cardiomyocytes using standard chemically defined medium 3 (CDM3). Up to day 20 of differentiation, 200 Mm palmitic acid were added, and then the culture was continued for another 8 days to mimic the environment in which human cardiomyocytes mainly use fatty acids as the main energy source. Light microscopy, transmission electron microscopy, immunofluorescence, reverse transcription-polymerase chain reaction, and cellular ATP assays, were carried out to analyze the expression of relevant cardiomyocyte-related genes, cell morphology, metabolism levels, and other indicators cardiomyocyte maturity.

Results

Cardiomyocytes derived from hESCs under exogenous palmitic acid had an elongated pike shape and a more regular arrangement. Sarcomere stripes were clear, and the cells color was clearly visible. The cell perimeter and elongation rate were also increased. Myogenic fibers were abundant, myofibrillar z-lines were regularly, the numbers of mitochondria and mitochondrial cristae were higher, more myofilaments were observed, and the structure of round-like discs was occasionally seen. Expression of mature cardiomyocyte-associated genes TNNT2, MYL2 and MYH6, and cardiomyocyte-associated genes KCNJ4, RYR2,and PPARα, was upregulated (p < 0.05). Expression of MYH7, MYL7, KCND2, KCND3, GJA1 and TNNI1 genes was unaffected (p > 0.05). Expression of mature cardiomyocyte-associated sarcomere protein MYL2 was significantly increased (p < 0.05), MYH7 protein expression was unaffected (p > 0.05). hESC-derived cardiomyocytes exposed to exogenous palmitic acid produced more ATP per unit time (p < 0.05).

Conclusion

Exogenous palmitic acid induced more mature hESC-CMs in terms of the cellular architecture, expression of cardiomyocyte maturation genes adnprotein, and metabolism.

Keywords

myocardial infarction palmitic acid embryonic stem cells cell-derived cardiomyocytes maturation

Introduction

Cardiovascular disease has become a major health problem worldwide.¹ Myocardial infarction leads to myocardial necrosis, and cardiomyocytes are non-renewable,² making it particularly important to develop methods to repair the heart and cardiomyocytes. Human embryonic stem cells (hESCs) which can provide an unlimited number of cardiomyocytes for myocardial regeneration, drug screening, and disease modeling, are an ideal solution.

hESCs self-renew and generate new adult tissues through a pluripotent differentiation potentials.³ Induced cardiac differentiation of hESCs generates cardiomyocytes. However, increasing evidence suggsets that such cardiomyocytes exhibit the phenotype of immature fetal cardiomyocytes and behave differently from adult cardiomyocytes in terms of cellular metabolism and cytoarchitecture, thereby limiting clinical applications of hESC-CMs.⁴ Fatty acid-containing cardiomyocyte maturation medium promotes maturation of stem cell-derived cardiomyocytes by altering the sarcomere organization and increasing expression of cardiomyocyte maturation genes and metabolism levels in cardiomyocytes.⁵ Therefore, we investigated increasing hESC-CM maturation using chemically defined medium 3 (CDM3) to induce differentiation of hECSs into cardiomyocytes⁶ exogenous palmitic acid to mimic the environment in which human cardiomyocytes primarily use fatty acids as the major energy source.⁷ Increasing hESC-CM maturity has important implications in cardiac repair, disease modeling, and drug screening.

Materials and methods

Materials

The embryonic stem cells were purchased from WiCell Research Institute in the United States. Palmitic acid (CAS: 57-10-3) and its reference solvent were purchased from Xi’an Kunchuang Biotechnology Technology Co., LTD. Stem cell medium PSCeasy, cardiomyocyte differentiation medium, cardiomyocyte purification medium and stem cell digestive fluid EDTA were purchased from Beijing Cellapy Co., LTD. Cell primer Hesc-matrigrl from Corning Corporation, USA; cTnT, α-actin antibody and secondary antibody were purchased from Abcam, USA. DAPI dyeing solution, enhanced ATP test kit and RIPA buffer purchased from Shanghai Beyotime Biological Co., LTD. RNA primer was purchased from Shanghai Jierui Biological Engineering Co., LTD. RT2 First Strand Kit, Green PCR Master Mix and RNase-free water purchased from QIAGEN, Germany. The following materials are summarized in Table 1.

Table 1

Summary table of experimental reagent materials

Materials	Information description
The embryonic stem cells	WiCell Research Institute in the United States
Palmitic acid (CAS: 57-10-3) reference solvent	Xi’an Kunchuang Biotechnology Technology Co., Ltd.
Stem cell medium PSCeasy cardiomyocyte differentiation medium cardiomyocyte purification medium stem cell digestive fluid EDTA	Beijing Cellapy Co., Ltd.
Cell primer Hesc-matrigrl	Corning Corporation, USA
cTnT, α-actin antibody secondary antibody	Abcam, USA
DAPI dyeing solution enhanced ATP test kit RIPA buffer	Shanghai Beyotime Biological Co., Ltd.
RNA primer	Shanghai Jierui Biological Engineering Co., Ltd.
RT2 First Strand Kit, Green PCR Master Mix RNase-free water	QIAGEN, Germany

hESCs culture

H9 hESCs were cultured in PSCeasy medium, 6-well plates coated with a 1:500 dilution of hESC-matrigel. H9 cells were passaged every 3–4 days with 0.5 mM EDTA at 70%–80% confluence. Cells were cultured at 37 °C in a 5% CO₂ incubator (Thermo Fisher Scientific, USA) (Fig. 1).

Fig. 1.

a. Characterization of hESCs by ×4 optical microscopy; b. Characterization of hESCs by ×10 optical microscopy; c. Characterization of hESCs by ×20 optical microscopy; d. Characterization of hESCs by ×40 optical microscopy.

Generation of CMs from hESCs

For cardiomyocytes differentiation, cardiomyocyte differentiation medium I was applied at 4 ml per well for 48 h, followed by 4 ml cardiomyocyte differentiation medium II for 48 h. Then, cardiomyocyte differentiation medium III was added at 4 ml per well for 48 h. Cardiomyocyte differentiation medium III was refreshed every 48 h. Four days after application of cardiomyocyte differentiation medium III, the cells began to beat. After the cells started beating, the culture was continued in cardiomyocyte differentiation medium III for another 96 h with two medium changes. After day 12 of differentiation, most cells had differentiated into cardiomyocytes with regular beating.

Purification of hESC-CMs

The culture was heterogeneous, containing various cell types such as fibroblasts, undifferentiated hESCs, and endothelial cells. Non-cardiomyocytes consume glucose as their main energy source. However, cardiomyocytes can derive energy from lactic acid.⁸ Therefore, cells were purified in cardiomyocyte purification medium 2 containing lactic acid for 4 days. After the culture was homogenous under light microscopy, the medium was changed to cardiomyocyte myoblast differentiation medium III with a medium change every 48 h.

Maturation of hESC-CMs

Purified cardiomyocytes continued to be cultured in cardiomyocyte differentiation medium III for 4 days until day 20 of differentiation. Then, 200 μM palmitic acid were applied for 8 day with medium changes every 48 h. Palmitic acid were omitted in the control group. Immunofluorescence, cell morphology analysis, Western blotting, reverse transcription-quantitative polymerization chain reaction (RT-qPCR), transmission electron microscopy, and cellular ATP assays were then carried out as described below.

Immunofluorescence

hESC-CMs were cultured on matrigel-coated coverslips. After fixing in 4% paraformaldehyde for 20 min at room temperature, 0.5% Triton-X 100 was applied to permeabilize the cells for 10 min at room temperature. Cells were blocked with 5% bovine serum for 1 h at room temperature, and then incubated with rabbit anti-rat primary antibodies against cTnT (1:200) and α-actinin (1:250) at 4 °C overnight. A goat anti-rabbit secondary antibody was then applied at 37 °C for 2 h while protected from light. Nuclei were counterstained with 4^′, 6- diamidino-2-phenylindole (DAPI) for 10 min at room temperature. The cells were washed three times with PBS for 5–10 min between each step. Fluorescence images were captured under a LEICADMI 4 000 –B inverted fluorescence microscope.

Transmission electron microscopy

After harvesting and washing, cells were fixed with electron microscope fixative (Servicebio, Wuhan, China) at room temperature for 2 h, and then stored at 4 °C until analysis. The cell structure was then observed by electron microscopy.

RNA isolation and RT-qPCR

hESC-CMs were lysed in QIAzol lysis reagent. The amount and purity of RNA were verified using an enzyme marker. DNA was synthesized using an RT2 First Strand Kit. For PCR analysis, cDNA was mixed with QuantiTect SYBR, Green PCR Master Mix, primers, and RNase-free water and run on an iCycler instrument (Bio-Rad, Hercules, CA, USA). PCR conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s). PCR were performed three times, and three wells of each sample were analyzed to average the cyclic threshold (Ct) value. Expression data were normalized to endogenous control glyceraldehyde 3-phosphate dehydrogenase and evaluated using the Ct method. Primer sequences are listed in Table 2.

Table 2

Primer sequences for reverse transcription-quantitative polymerase chain reaction

Gene	Forward primer sequence (5^′ to 3^′)	Reverse primer sequence (5^′ to 3^′ )	PCR product length (bp)
GAPDH	GTCTCCTCTGACTTCAACAGCG	ACCACCCTGTTGCTGTAGCCAA	131
MYL2	ACATCATCACCCACGGAGAAGAGA	ATTGGAACATGGCCTCTGGATGGA	247
KCNJ4	CCTTGGGATGTAGCGCC	GTCCGTGCATGTCCTGAAG	109
PPARα	ATTACGGAGTCCACGCGTGTG	TTGTCATACACCAGCTTGAGT	76
RYR2	AAGCCCTCTCGTCTGAAACA	CCACCCAGACATTAGCAGGT	193
TNNT2	AGCATCTATAACTTGGAGGCAGAG	TGGAGACTTTCTGGTTATCGTTG	112
MYH6	GCTGGCCCTTCAACTACAGA	CTTCTCCACCTTAGCCCTGG	106
MYH7	GAGGACAAGGTCAACACCCT	CGCACCTTCTTCTCTTGCTC	95
MYL7	CCGTCTTCCTCACGCTCTT	TGAACTCATCCTTGTTCACCAC	120
KCND2	CTACCTGTTCCGGTGATTGTATCC	TCTTTTGTGCCCTTCGTTTGT	82
KCND3	CCAATTCTAACCTGCCAGCTAC	CTGCTTTCAAATTAAGGCTGGA	122
GJA1	CAATCACTTGGCGTGACTTC	AAAGGCAGACTGCTCATCTC	211
TNNI1	CAGCTCCACGAGGACTGAAC	CTCTTCAGCAAGAGTTTGCG	101

Note: GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MYL2: myosin light chain2; KCNJ4: potassium inwardly rectifying channel subunit J4; PPARα: peroxisome proliferator-activator receptor alpha; RYR2: cardiac ryanodine receptor; TNNT2: troponin T2; MYH6: myosin heavy chain6; MYH7: myosin heavy chain7; MYL7: myosin light chain7; KCND2: potassium voltage-gated channel subfamily D2; KCND3: potassium voltage-gated channel subfamily D3; GJA1: gap junction protein, alpha 1; TNNI1: troponin I1.

Cellular ATP assay

Cellular ATP levels were assessed using an Enhanced ATP Assay Kit. After harvesting hESC-CMs were lysed in the lysis solution provided in the kit, and then centrifuged at 4 °C for 5 min at 12,000 rpm. The supernatant was collected and mixed with the ATP monitoring working solution. ATP concentrations were determined by a standard curves in accordance with the manufacturer’s instructions.

Western blotting

Cells were lysed using RIPA buffer, and then centrifuged at 4 °C for 15 min at 12,000 rpm to collect the supernatant. After measuring the protein concentration, protein samples were denatured in SDS-PAGE buffer at 100 °C for 10 min. Fifteen microliters of total protein were used for western blotting. Rabbit anti-myosin light chain 2 (MYL2), rabbit anti-myosin heavy chain protein 7 (MYH7), or glyceraldehyde 3-phosphate dehydrogenase antibodies were applied at 4 °C overnight on a shaker. After three rinses TBST, goat anti-rabbit IgG2 was applied for 1 h at room temperature. Labeled proteins were quantified using an Odyssey instrument.

Statistical analysis

Data were obtained from four independent cardiomyocyte differentiations. Values are the average with the standard error of the mean. Statistical analysis was performed using GraphPad Prism. The t-test was used for statistical analysis of differences. P-values less than 0.05 were considered statistically significant.

Fig. 2.

Effect of exogenous palmitic acid on hESC-CM maturation. A. Immunofluorescence images of control and experimental hESC-CMs. Magnification ×40. a. α-Actinin immunofluorescence of the experimental group (green); b. cTnT immunofluorescence of the experimental group (red); c. Nuclei of the experimental group were counterstained by DAPI (blue); d. Merged images; e. α-Actinin immunofluorescence of the control group (green); f. cTnT immunofluorescence of the control group (red); g. Nuclei of the cells in the control group were counterstained by DAPI (blue); h. Merged image. The results showed that the cells in the experimental group exhibited more aligned myofilaments and more cTnT expression. B. Transmission electron microscopy of control and experimental hESC-CMs. a. Experimental group cells at magnification of ×3,000; b. Experimental group cells at magnification ×8,000; c. Experimental group cells at magnification of ×20,000; d. Control group cells at magnification of ×3000 times; e. picture shows the control group cells at a magnification of 8000 times; f. Control group cells at magnification of ×20,000. The results of transmission electron microscopy revealed regular myofilaments, more mitochondria and more mitochondrial cristae were seen in the hESC-CMs from the experimental group compared with those of the control group.

Results

Cell morphology

Control group cells showed a round shape with a smaller ratio of cell length to short axis, whereas experimental group cells showed a more elongated pike shape with a more regular arrangement, and a clearer myotome stripe and cells coloration (Fig. 2A). The circumference and elongation of the cells increased.

Cell microstructure

Cells cultured in medium with palmitic acid were rich in myogenic fibers with myofibrillar z-lines arranged in a more regular fashion, more mitochondria, and more mitochondrial cristae, more myofilaments, and occasionally in a round-like structure. Cardiomyocytes cultured in control CDM3 medium had fewer cellular myofibrils that were more randomly arranged with a smaller mitochondrial population, and most of them had fewer cristae in their inner mitochondria (Fig. 2B).

Cardiomyocyte-related gene expression

We found significant increases in expression of most mature cardiomyocyte-associated, sarcomere-encoding genes (TNNT2, MYL2 and MYH6) and cardiomyocyte-associated genes (KCNJ4 ion channel, cardiac beam junction protein receptor RYR2, and metabolism-associated PPARα) (p < 0.05). These data demonstrated that palmitic acid promoted maturation of hESC-CMs in terms of cellular morphology, sarcomere structure, ultrastructure, and gene expression (Fig. 3A). However, expression of MYH7, MYL7, KCND2, KCND3, GJA1, and TNNI1 genes was unaffected (p > 0.05) (Fig. 3B).

Fig. 3.

Gene expression in experimental and control groups. Real-time PCR was performed to analyze the effect of maturation medium on the expression of cardiomyocyte maturation-related genes. A. Genes with significant differences in expression; B. Genes with no significant differences in expression. n = 3, *p < 0.05 compared with control medium.

Cellular metabolic effects

As hESC-CMs mature, the ability of the cells to produce ATP increases. Therefore, we analyzed the concentration of ATP produced over a certain period. The experiment group produced more ATP per unit of time than the control group (p < 0.05), demonstrating that cardiomyocytes derived under palmitic acid treatments exhibited a more mature phenotype in terms of metabolism (Fig. 4).

Fig. 4.

Effect of addition of exogenous palmitic acid on the expression of ATP production by hESC-CMs. A. ATP production per unit of time between the experimental group and the control group. B. ATP-absorbance standard curve for this experiment (n = 3 *p < 0.05 vs. control medium), indicating that the cellular metabolic capacity of the experimental group was greater than that of the control group.

Cardiomyocyte-related protein expression

We found a significant increase in the expression of the mature cardiomyocyte-associated sarcomere protein MYL2, but expression of MYH7 protein was unaffected (p > 0.05). Their proteins expression was consistent with the gene expression levels (Fig. 5).

Fig. 5.

Effect of exogenous palmitic acid on expression of cardiomyocyte-related protein in hESC-CMs. A. Western blot analysis of the effect of maturation medium on MYH7 protein expression (p > 0.05). B. Western blot analysis of the effect of maturation medium on MYL2 protein expression (p < 0.05).

Discussion

The incidence of myocardial infarction as a major cardiovascular disease has shown a continuously increasing trend, and the age of onset is decreasing yearly.⁹ hESC transplantation has received increasing attention in the clinic, but studies have reported that hESC-CMs exhibit the phenotype of immature fetal cardiomyocytes and behave differently from adult cardiomyocytes in terms of cellular metabolism and structure. This limits their clinical application because immature hESC-CMs are unable to meet the appropriate human cardiac functional requirements, and their persistence in an injured heart may lead to arrhythmia. Therefore, it is clinically important to seek a method to promote maturation of hESC-CMs.

In the present study, we exploited the unique energy substrates of cardiomyocytes, because glycolysis is the main metabolic mode in immature cardiomyocytes,^10–12 contributing to the cell being in a value-added state.¹³ With maturation of cardiomyocytes, the oxidative capacity of mitochondria increases, and fatty acid β-oxidation becomes the main energy source to satisfy the high energy metabolism of cardiomyocytes.^14,15 We found that hESC-CMs induced in the presence of palmitic acid exhibited a more mature phenotype in terms of their microstructure, cellular morphology, gene expression, and metabolic capacity.

hESC-CMs differentiated by 2D monolayer-based protocols have more mature structures, phenotypes, greater expression of cardiomyocyte maturation-related genes, and a higher metabolic capacity.^16,17 In the present study, we used a CDM3 differentiation culture protocol to induce differentiation into more mature and stable cardiomyocytes and assessed the effect of exogenous palmitic acid on maturation of hESC-CMs. To avoid contaminating cells, we purified the culture and observed autonomous contraction of the cells and expression of troponin T.

Cardiomyocyte maturation is usually defined by cell morphology, structure, fiber structure, gene expression levels, metabolism, electrophysiology, and protein expression levels.^18,19 In this study, we assessed cells by their cell morphology, structure, organelles, metabolism, and expression of cardiomyocyte maturation genes and proteins. In terms of cell morphology, cardiomyocytes derived from hESCs exhibit a round-like shape, and as the cells mature, they gradually elongate into a pike-like shape.²⁰ We found that cardiomyocytes differentiated under exogenous palmitic acid had a more elongated morphology. Moreover, myocyte nodules, which are the contractile units of cardiomyocytes, remained highly regular and densely packed in mature cardiomyocytes, whereas naive hESC-CMs showed disorganized myofibrils and a low number of myogenic fibers. During maturation of cardiomyocytes, expression of functional structure genes increases, such as cardiac T2 (TNNT2).²¹ Troponin T-positive sarcomere streaks are also more regular. In this study, expression of sarcomere-related genes, such as MYL2 and MYH6, was upregulated in some mature cardiomyocytes. However, expression of MYH7, MYL7, KCND2, KCND3, GJA1, and TNNI1 genes was unaffected. We also found a significant increase in the expression of mature cardiomyocyte-associated sarcomere protein MYL2, but expression of MYH7 protein was unaffected. The expression of these two proteins was consistent with their gene expression levels.⁴

Cardiomyocyte potentials are the main mechanism to control cardiac function. Normal cardiomyocyte potentials are ensured mainly by ion channels. In our study, expression of cardiomyocyte-associated ion channel proteins, such as KCNJ4 related to the α-subunit of the potassium channel,²² was significantly increased, suggesting that the maturation medium promoted maturation of cardiomyocytes derived from hESCs in terms of molecular and electrophysiological aspects.

Cardiomyocytes prefer the β-oxidation mode of energy metabolism through consumption of fatty acids,²³ and mitochondria are the main metabolic organelle.⁷ We found that cardiomyocytes derived from hESCs under exogenous palmitic acid had a greater number of mitochondria with more mitochondrial cristae, i.e., more surface area where cellular metabolism occurs, demonstrating a stronger metabolic potential.^24–26 Expression of the PPARα gene related to fatty acid metabolism was higher in the experimental group, indicating a more mature phenotype for fatty acid metabolism.^10–12 Furthermore, hESCs-CMs derived under exogenous palmitic acid produced more ATP per unit of quantity per unit of time, suggesting a higher metabolism and ability to use substrates for metabolism.^30,31

Conclusion

In conclusion, our data suggest that culturing hESC-CMs by the addition of exogenous palmitic acid enables them to behave in a more mature manner. These cultures also provide a valuable model for subsequent culturing of more mature hESCs for the study of drugs or treatment of heart-related diseases. This project is expected to provide a better therapeutic tool for the clinical treatment of myocardial infarction and improve the remodeling of infarcted myocardium.

Footnotes

Author contributions

MJS and GZ carried out the molecular genetic studies, participated in the immunoassays and drafted the manuscript. BP carried out the flow cytometric. YB participated in the design of the study and performed the statistical analysis. MJS conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethics statement

This study has been approved by the ethics committee of Beijing Anzhen Hospital, Capital Medical University (batch number GZR-3-072). Beijing Anzhen Hospital and national animal care and use guidelines were followed. This study does not involve any living invertebrate, human participants or patient data.

Availability of supporting data

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Funding

The study was financially supported by the 2022 National Natural Science Foundation (82270255).

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