Introduction

Remarkable progress has been made over the past decade in the differentiation of human induced pluripotent stem cells (iPSCs) into functional iPSC-derived cardiomyocytes (iPSC-CMs). However, the resulting human iPSC-CMs are not fully comparable to their adult primary counterparts in terms of metabolism, structure, function, and electrophysiology [1]. Rather, they bear a strong resemblance to immature cardiomyocytes seen in the late foetal stage. This limits their applications, as most (genetic) cardiac diseases do not occur until middle-age [2,3,4,5]. Recently, maturation of iPSC-CMs through different approaches has gained traction, including prolonged time in culture [6, 7], use of specialized medium [8,9,10,11], activating specific metabolic pathways, electrical and/or mechanical stimulation, and encapsulation in a 3D environment [12,13,14,15]. Future progress will potentially come from identifying and mimicking developmental drivers [16]. During foetal development, cardiomyocytes show an extensive increase in contractile cytoskeleton protein content [17] and undergo ion channel remodelling [18]. Between the foetal stage and early adolescence, human cardiomyocytes undergo hypertrophy with an increase in myofibril mass, with cell sizes increasing 10- to 20-fold, and loss of self-depolarization outside the nodal population [19]. Furthermore, cardiomyocytes undergo remodelling of intercellular junctions, T-tubule formation, and increase in DNA content leading to polyploidy in a single nucleus or binucleation in about 60% of cells [19,20,21]. Cardiomyocytes experience an extensive increase in energetic demands leading to increased mitochondrial mass and structural changes, and a switch from anaerobic glycolysis to oxidative phosphorylation, in particular fatty acid (FA) oxidation (FAO) [22, 23]. Long-chain FAO produces 3–4 times more ATP per molecule compared to glucose oxidation, thus increasing energy supply, but at the cost of increased oxygen consumption [24].

We hypothesized that, next to providing the cells with the proper metabolic substrate—FA—a short treatment activating the peroxisome proliferator-activated receptor β/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway via the addition of small molecules could trigger FAO metabolism and improve cell maturation. Hence, we selected two small molecules—asiatic acid (AA) and GW501516—to specifically activate the PPAR/PGC-1α pathway in iPSC-CMs. T3 is a hormone involved in cardiac development, well known for its effect on iPSC-CMs maturation and has been shown to stimulate mitochondrial respiratory capacity and biogenesis, by activating p43, an activator of mitochondrial genome replication [11, 25,26,27]. In this study, T3 was used as a positive control due to its known increased respiratory capacity and electrophysiological maturation in iPSC-CMs, though the mechanism has not been established yet [28] (Fig. 1).

Fig. 1
figure 1

Schematic overview of the main cellular metabolic pathways with the proposed action mechanisms of small molecules asiatic acid and GW501516, as well as T3 hormone that was used as a positive control

Full size image

Asiatic acid and GW501516

Asiatic acid (AA; Fig. 1) is a naturally occurring pentacyclic triterpenoid, which is attributed a wide spectrum of biological activities [29,30,31]. In the context of metabolism, AA increases the activity of enzymes involved in lipid synthesis like 3-hydroxy-3-methyl-glutaryl-CoA-reductase and metabolic regulators like adenosine monophosphate-activated protein kinase (AMPK) [31]. AMPK regulates the switch between glycolytic and oxidative metabolism by controlling the FA availability via increased FA plasma membrane uptake and utilization. Furthermore, AMPK promotes FA entry into the mitochondria and β-oxidation via carnitine palmitoyl transferase (CPT1) [32]. In addition, AMPK activates PGC-1α, which in combination with peroxisome proliferator-activated receptors is responsible for the long-term stimulation of FAO in skeletal muscle and the heart [24, 33,34,35,36,37,38]. AA is also able to reduce oxidative stress and apoptosis via alpha-synuclein (α-syn) entry inhibition and release of cytochrome c from the mitochondria. Pre-treatment with AA was shown to increase peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) levels, enhancing mitochondrial biogenesis [21]. FAs constitute the main energy source necessary to support the high-energy demand of adult cardiomyocytes. Based on this, several groups demonstrated that a combination of galactose and FA leads to a glycolytic-oxidative metabolic shift and ultimately improving the iPSC-CM maturity [10, 71]. However, native mature cardiomyocytes maintain their metabolic flexibility allowing them to switch between different substrates, such as glucose, lactate, and glutamate [72]. In the present study, we directly targeted the FAO metabolism via GW or AA supplemented into the culture medium in the presence of FA. Activating the PGC1/PPAR pathway, resulted in an overall enhanced iPSC-CM maturation after only 10 days, thus indicating that cardiomyocyte maturation and FAO activation signalling events might be directly linked [25, 73, 74]. During maturation, the mitochondrial network of cardiomyocytes undergoes extensive remodelling to support the increased energy demand [75]. Kleiner et al. treated primary mouse myoblasts with 100 nM GW and demonstrated an increase in key FAO genes via PGC-1α; however, without effects on mitochondrial function [66]. Interestingly in our hands, GW increased both the expression of key regulators of cardiac metabolism and ATP5A content, indicating maturation of the mitochondrial network (Fig. 3). Previous findings suggested a neuroprotective effect of AA via mitochondrial biogenesis. Xu et al. showed how AA promotes a 1.5-fold increase in PGC-1α expression in vitro and restored lipid peroxidation in vivo [

Availability of data and materials

The datasets generated during and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Abbreviations

2DG:

2-Deoxy-d-glucose

AA:

Asiatic acid

ACTC1:

Actin alpha cardiac muscle 1

ADP:

Adenosine diphosphate

AMPK:

5ʹ Adenosine monophosphate-activated protein kinase

ATP:

Adenosine triphosphate

ATP5A:

ATP synthase

CACNA1C:

Calcium voltage-gated channel subunit alpha1 C

CAT:

Carnitine acetyltransferases

CPT:

Carnitine palmitoyltransferase

DMSO:

Dimethyl sulfoxide

DNA:

Deoxyribonucleic acid

ECAR:

Extracellular acidification rate

ERRa:

Estrogen-related receptor alpha

ETO:

Etomoxir

FA:

Fatty acid

FADH:

Flavin adenine dinucleotide

FAO:

Fatty acid oxidation

FCCP:

Carbonyl cyanide P-(tri-fluromethoxy) phenyl-hydrazone

GJA1:

Gap junction protein connexin 43

GLUT4:

Glucose transporter 4

GW:

GW501516

IMMT:

Inner mitochondrial membrane protein

iPSC:

Induced pluripotent stem cell

KCNQ1:

Potassium voltage-gated channel subfamily Q member 1

LDHB:

Lactate dehydrogenase B

MAPK:

Mitogen-activated protein kinase

mTOR:

Mammalian target of rapamycin

NADH:

Nicotinamide adenine dinucleotide

NRF:

Nuclear respiratory factor

OCR:

Oxygen consumption rate

PDK:

Pyruvate dehydrogenase kinase

PFK:

Phosphofructokinase-1

PGC:

Peroxisome proliferator-activated receptor-gamma coactivator

PPAR:

Peroxisome proliferator-activated receptor β/δ

PPARGC1A/PGC-1α:

Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha

Rot + A:

Rotenone and antimycin A

RPL32:

Ribosomal protein L32

RXR:

Retinoic acid receptors

SCN5A:

Sodium voltage-gated channel alpha subunit 5

SERCA:

Sarco/endoplasmic reticulum Ca2 + -ATPase

T3:

3,3ʹ-Triiodo-l-thyronine

TCA:

Tricarboxylic acid cycle

TGFb:

Transforming growth factor beta

TNNI3:

Cardiac troponin I

α-syn:

Alpha-synuclein

References

  1. Giacomelli E, Mummery CL, Bellin M. Human heart disease: lessons from human pluripotent stem cell-derived cardiomyocytes. Cell Mol Life Sci. 2017;74:3711–39. https://doi.org/10.1007/s00018-017-2546-5.

    Article  CAS  Google Scholar 

  2. Karbassi E, Fenix A, Marchiano S, et al. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol. 2020;17:341–59. https://doi.org/10.1038/s41569-019-0331-x.

    Article  Google Scholar 

  3. Guo Y, Pu WT. Cardiomyocyte maturation: new phase in development. Circ Res. 2020;126:1086–106. https://doi.org/10.1161/CIRCRESAHA.119.315862.

    Article  CAS  Google Scholar 

  4. van Mil A, Balk GM, Neef K, et al. Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential. Cardiovasc Res. 2018;114:1828–42. https://doi.org/10.1093/cvr/cvy208.

    Article  CAS  Google Scholar 

  5. Veerman CC, Kosmidis G, Mummery CL, et al. Immaturity of human stem-cell-derived cardiomyocytes in culture: Fatal flaw or soluble problem? Stem Cells Dev. 2015;24:1035–52. https://doi.org/10.1089/scd.2014.0533.

    Article  CAS  Google Scholar 

  6. Kamakura T, Makiyama T, Sasaki K, et al. Ultrastructural maturation of human-induced pluripotent stem cell-derived cardiomyocytes in a long-term culture. Circ J. 2013;77:1307–14. https://doi.org/10.1253/circj.cj-12-0987.

    Article  CAS  Google Scholar 

  7. Piccini I, Rao J, Seebohm G, et al. Human pluripotent stem cell-derived cardiomyocytes: Genome-wide expression profiling of long-term in vitro maturation in comparison to human heart tissue. Genom Data. 2015;4:69–72. https://doi.org/10.1016/j.gdata.2015.03.008.

    Article  Google Scholar 

  8. Wang L, Wada Y, Ballan N, et al. Triiodothyronine and dexamethasone alter potassium channel expression and promote electrophysiological maturation of human-induced pluripotent stem cell-derived cardiomyocytes. J Mol Cell Cardiol. 2021;161:130–8. https://doi.org/10.1016/j.yjmcc.2021.08.005.

    Article  CAS  Google Scholar 

  9. Feyen DAM, McKeithan WL, Bruyneel AAN, et al. Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes. Cell Rep. 2020;32: 107925. https://doi.org/10.1016/j.celrep.2020.107925.

    Article  CAS  Google Scholar 

  10. Correia C, Koshkin A, Duarte P, et al. Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Sci Rep. 2017;7:8590. https://doi.org/10.1038/s41598-017-08713-4.

    Article  CAS  Google Scholar 

  11. Yang X, Rodriguez M, Pabon L, et al. Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells. J Mol Cell Cardiol. 2014;72:296–304. https://doi.org/10.1016/j.yjmcc.2014.04.005.

    Article  CAS  Google Scholar 

  12. Abilez OJ, Tzatzalos E, Yang H, et al. Passive stretch induces structural and functional maturation of engineered heart muscle as predicted by computational modeling. Stem Cells. 2018;36:265–77. https://doi.org/10.1002/stem.2732.

    Article  CAS  Google Scholar 

  13. Tiburcy M, Hudson JE, Balfanz P, et al. Defined engineered human myocardium with advanced maturation for applications in heart failure modeling and repair. Circulation. 2017;135:1832–47. https://doi.org/10.1161/CIRCULATIONAHA.116.024145.

    Article  CAS  Google Scholar 

  14. Fong AH, Romero-Lopez M, Heylman CM, et al. Three-dimensional adult cardiac extracellular matrix promotes maturation of human induced pluripotent stem cell-derived cardiomyocytes. Tissue Eng Part A. 2016;22:1016–25. https://doi.org/10.1089/ten.TEA.2016.0027.

    Article  CAS  Google Scholar 

  15. Herron TJ, Rocha AM, Campbell KF, et al. Extracellular matrix-mediated maturation of human pluripotent stem cell-derived cardiac monolayer structure and electrophysiological function. Circ Arrhythm Electrophysiol. 2016;9: e003638. https://doi.org/10.1161/CIRCEP.113.003638.

    Article  CAS  Google Scholar 

  16. Spater D, Hansson EM, Zangi L, et al. How to make a cardiomyocyte. Development. 2014;141:4418–31. https://doi.org/10.1242/dev.091538.

    Article  CAS  Google Scholar 

  17. Wilson A, Schoenauer R, Ehler E, et al. Cardiomyocyte growth and sarcomerogenesis at the intercalated disc. Cell Mol Life Sci. 2014;71:165–81. https://doi.org/10.1007/s00018-013-1374-5.

    Article  CAS  Google Scholar 

  18. Zhao Z, Lan H, El-Battrawy I, et al. Ion channel expression and characterization in human induced pluripotent stem cell-derived cardiomyocytes. Stem Cells Int. 2018;2018:6067096. https://doi.org/10.1155/2018/6067096.

    Article  CAS  Google Scholar 

  19. Mollova M, Bersell K, Walsh S, et al. Cardiomyocyte proliferation contributes to heart growth in young humans. Proc Natl Acad Sci U S A. 2013;110:1446–51. https://doi.org/10.1073/pnas.1214608110.

    Article  Google Scholar 

  20. Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for cardiomyocyte renewal in humans. Science. 2009;324:98–102. https://doi.org/10.1126/science.1164680.

    Article  CAS  Google Scholar 

  21. Smolich JJ. Ultrastructural and functional features of the develo** mammalian heart: a brief overview. Reprod Fertil Dev. 1995;7:451–61. https://doi.org/10.1071/rd9950451.

    Article  CAS  Google Scholar 

  22. Ramachandra CJA, Mehta A, Wong P, et al. Fatty acid metabolism driven mitochondrial bioenergetics promotes advanced developmental phenotypes in human induced pluripotent stem cell derived cardiomyocytes. Int J Cardiol. 2018;272:288–97. https://doi.org/10.1016/j.ijcard.2018.08.069.

    Article  Google Scholar 

  23. Porter GA Jr, Hom J, Hoffman D, et al. Bioenergetics, mitochondria, and cardiac myocyte differentiation. Prog Pediatr Cardiol. 2011;31:75–81. https://doi.org/10.1016/j.ppedcard.2011.02.002.

    Article  Google Scholar 

  24. Palomer X, Barroso E, Zarei M, et al. PPARbeta/delta and lipid metabolism in the heart. Biochim Biophys Acta. 2016;1861:1569–78. https://doi.org/10.1016/j.bbalip.2016.01.019.

    Article  CAS  Google Scholar 

  25. Lin B, Lin X, Stachel M, et al. Culture in glucose-depleted medium supplemented with fatty acid and 3,3’,5-triiodo-l-thyronine facilitates purification and maturation of human pluripotent stem cell-derived cardiomyocytes. Front Endocrinol (Lausanne). 2017;8:253. https://doi.org/10.3389/fendo.2017.00253.

    Article  Google Scholar 

  26. Shi ST, Wu XX, Hao W, et al. Triiodo-L-thyronine promotes the maturation of cardiomyocytes derived from rat bone marrow mesenchymal stem cells. J Cardiovasc Pharmacol. 2016;67:388–93. https://doi.org/10.1097/FJC.0000000000000363.

    Article  CAS  Google Scholar 

  27. Cini G, Carpi A, Mechanick J, et al. Thyroid hormones and the cardiovascular system: pathophysiology and interventions. Biomed Pharmacother. 2009;63:742–53. https://doi.org/10.1016/j.biopha.2009.08.003.

    Article  CAS  Google Scholar 

  28. Parikh SS, Blackwell DJ, Gomez-Hurtado N, et al. Thyroid and glucocorticoid hormones promote functional T-tubule development in human-induced pluripotent stem cell-derived cardiomyocytes. Circ Res. 2017;121:1323–30. https://doi.org/10.1161/CIRCRESAHA.117.311920.

    Article  CAS  Google Scholar 

  29. Yi CL, Si LJ, Xu J, et al. Effect and mechanism of asiatic acid on autophagy in myocardial ischemia-reperfusion injury in vivo and in vitro. Exp Ther Med. 2020;20: 54. https://doi.org/10.3892/etm.2020.9182.

    Article  CAS  Google Scholar 

  30. Lv J, Sharma A, Zhang T, et al. Pharmacological review on asiatic acid and its derivatives: a potential compound. SLAS Technol. 2018;23:111–27. https://doi.org/10.1177/2472630317751840.

    Article  CAS  Google Scholar 

  31. Ma ZG, Dai J, Wei WY, et al. Asiatic acid protects against cardiac hypertrophy through activating ampka signalling pathway. Int J Biol Sci. 2016;12:861–71. https://doi.org/10.7150/ijbs.14213.

    Article  CAS  Google Scholar 

  32. Bairwa SC, Parajuli N, Dyck JR. The role of AMPK in cardiomyocyte health and survival. Biochim Biophys Acta. 2016;1862:2199–210. https://doi.org/10.1016/j.bbadis.2016.07.001.

    Article  CAS  Google Scholar 

  33. Duncan JG, Finck BN. The PPARalpha-PGC-1alpha axis controls cardiac energy metabolism in healthy and diseased myocardium. PPAR Res. 2008;2008: 253817. https://doi.org/10.1155/2008/253817.

    Article  CAS  Google Scholar 

  34. Duncan JG. Peroxisome proliferator activated receptor-alpha (PPARalpha) and PPAR gamma coactivator-1alpha (PGC-1alpha) regulation of cardiac metabolism in diabetes. Pediatr Cardiol. 2011;32:323–8. https://doi.org/10.1007/s00246-011-9889-8.

    Article  Google Scholar 

  35. Lee WJ, Kim M, Park HS, et al. AMPK activation increases fatty acid oxidation in skeletal muscle by activating PPARalpha and PGC-1. Biochem Biophys Res Commun. 2006;340:291–5. https://doi.org/10.1016/j.bbrc.2005.12.011.

    Article  CAS  Google Scholar 

  36. Cheng L, Ding G, Qin Q, et al. Peroxisome proliferator-activated receptor delta activates fatty acid oxidation in cultured neonatal and adult cardiomyocytes. Biochem Biophys Res Commun. 2004;313:277–86. https://doi.org/10.1016/j.bbrc.2003.11.127.

    Article  CAS  Google Scholar 

  37. Cheng L, Ding G, Qin Q, et al. Cardiomyocyte-restricted peroxisome proliferator-activated receptor-delta deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy. Nat Med. 2004;10:1245–50. https://doi.org/10.1038/nm1116.

    Article  CAS  Google Scholar 

  38. Gilde AJ, van der Lee KAJM, Willemsen PHM, et al. Peroxisome proliferator-activated receptor (PPAR) alpha and PPAR beta/delta, but not PPAR gamma, modulate the expression of genes involved in cardiac lipid metabolism. Circ Res. 2003;92:518–24. https://doi.org/10.1161/01.Res.0000060700.55247.7c.

    Article  CAS  Google Scholar 

  39. Ding H, **ong Y, Sun J, et al. Asiatic acid prevents oxidative stress and apoptosis by inhibiting the translocation of alpha-synuclein into mitochondria. Front Neurosci. 2018;12:431. https://doi.org/10.3389/fnins.2018.00431.

    Article  Google Scholar 

  40. Lee KY, Bae ON, Serfozo K, et al. Asiatic acid attenuates infarct volume, mitochondrial dysfunction, and matrix metalloproteinase-9 induction after focal cerebral ischemia. Stroke. 2012;43:1632–8. https://doi.org/10.1161/Strokeaha.111.639427.

    Article  CAS  Google Scholar 

  41. Krishnamurthy RG, Senut MC, Zemke D, et al. Asiatic acid, a pentacyclic triterpene from centella asiatica, is neuroprotective in a mouse model of focal cerebral ischemia. J Neurosci Res. 2009;87:2541–50. https://doi.org/10.1002/jnr.22071.

    Article  CAS  Google Scholar 

  42. Alvarez-Guardia D, Palomer X, Coll T, et al. PPARbeta/delta activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells. Biochim Biophys Acta. 2011;1811:59–67. https://doi.org/10.1016/j.bbalip.2010.11.002.

    Article  CAS  Google Scholar 

  43. Dressel U, Allen TL, Pippal JB, et al. The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells. Mol Endocrinol. 2003;17:2477–93. https://doi.org/10.1210/me.2003-0151.

    Article  CAS  Google Scholar 

  44. Zizola C, Kennel PJ, Akashi H, et al. Activation of PPARdelta signaling improves skeletal muscle oxidative metabolism and endurance function in an animal model of ischemic left ventricular dysfunction. Am J Physiol Heart Circ Physiol. 2015;308:H1078–85. https://doi.org/10.1152/ajpheart.00679.2014.

    Article  CAS  Google Scholar 

  45. Hamad S, Derichsweiler D, Papadopoulos S, et al. Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations. Theranostics. 2019;9:7222–38. https://doi.org/10.7150/thno.32058.

    Article  CAS  Google Scholar 

  46. Lin Y, Linask KL, Mallon B, et al. Heparin promotes cardiac differentiation of human pluripotent stem cells in chemically defined albumin-free medium, enabling consistent manufacture of cardiomyocytes. Stem Cells Transl Med. 2017;6:527–38. https://doi.org/10.5966/sctm.2015-0428.

    Article  CAS  Google Scholar 

  47. Ruijter JM, Ruiz Villalba A, Hellemans J, et al. Removal of between-run variation in a multi-plate qPCR experiment. Biomol Detect Quantif. 2015;5:10–4. https://doi.org/10.1016/j.bdq.2015.07.001.

    Article  CAS  Google Scholar 

  48. Chou SJ, Yu WC, Chang YL, et al. Energy utilization of induced pluripotent stem cell-derived cardiomyocyte in Fabry disease. Int J Cardiol. 2017;232:255–63. https://doi.org/10.1016/j.ijcard.2017.01.009.

    Article  Google Scholar 

  49. Kim C, Wong J, Wen J, et al. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature. 2013;494:105–10. https://doi.org/10.1038/nature11799.

    Article  CAS  Google Scholar 

  50. Cerignoli F, Charlot D, Whittaker R, et al. High throughput measurement of Ca(2)(+) dynamics for drug risk assessment in human stem cell-derived cardiomyocytes by kinetic image cytometry. J Pharmacol Toxicol Methods. 2012;66:246–56. https://doi.org/10.1016/j.vascn.2012.08.167.

    Article  CAS  Google Scholar 

  51. Ong SB, Hausenloy DJ. Mitochondrial morphology and cardiovascular disease. Cardiovasc Res. 2010;88:16–29. https://doi.org/10.1093/cvr/cvq237.

    Article  CAS  Google Scholar 

  52. **n T, Lv W, Liu D, et al. Opa1 reduces hypoxia-induced cardiomyocyte death by improving mitochondrial quality control. Front Cell Dev Biol. 2020;8:853. https://doi.org/10.3389/fcell.2020.00853.

    Article  Google Scholar 

  53. Lopaschuk GD, Wall SR, Olley PM, et al. Etomoxir, a carnitine palmitoyltransferase I inhibitor, protects hearts from fatty acid-induced ischemic injury independent of changes in long chain acylcarnitine. Circ Res. 1988;63:1036–43. https://doi.org/10.1161/01.res.63.6.1036.

    Article  CAS  Google Scholar 

  54. Divakaruni AS, Paradyse A, Ferrick DA, et al. Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol. 2014;547:309–54. https://doi.org/10.1016/B978-0-12-801415-8.00016-3.

    Article  CAS  Google Scholar 

  55. Skorska A, Johann L, Chabanovska O, et al. Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach. Cell Mol Life Sci. 2022;79:149. https://doi.org/10.1007/s00018-022-04196-3.

    Article  CAS  Google Scholar 

  56. Hirose K, Payumo AY, Cutie S, et al. Evidence for hormonal control of heart regenerative capacity during endothermy acquisition. Science. 2019;364:184–8. https://doi.org/10.1126/science.aar2038.

    Article  CAS  Google Scholar 

  57. Bergmann O, Zdunek S, Felker A, et al. Dynamics of cell generation and turnover in the human heart. Cell. 2015;161:1566–75. https://doi.org/10.1016/j.cell.2015.05.026.

    Article  CAS  Google Scholar 

  58. Adler CP. Relationship between deoxyribonucleic acid content and nucleoli in human heart muscle cells and estimation of cell number during cardiac growth and hyperfunction. Recent Adv Stud Cardiac Struct Metab. 1975;8:373–86.

    CAS  Google Scholar 

  59. Liu J, Laksman Z, Backx PH. The electrophysiological development of cardiomyocytes. Adv Drug Deliv Rev. 2016;96:253–73. https://doi.org/10.1016/j.addr.2015.12.023.

    Article  CAS  Google Scholar 

  60. Otsuji TG, Minami I, Kurose Y, et al. Progressive maturation in contracting cardiomyocytes derived from human embryonic stem cells: Qualitative effects on electrophysiological responses to drugs. Stem Cell Res. 2010;4:201–13. https://doi.org/10.1016/j.scr.2010.01.002.

    Article  CAS  Google Scholar 

  61. Garg P, Garg V, Shrestha R, et al. Human induced pluripotent stem cell-derived cardiomyocytes as models for cardiac channelopathies: a primer for non-electrophysiologists. Circ Res. 2018;123:224–43. https://doi.org/10.1161/Circresaha.118.311209.

    Article  CAS  Google Scholar 

  62. Krause U, Alflen C, Steinmetz M, et al. Characterization of maturation of neuronal voltage-gated sodium channels SCN1A and SCN8A in rat myocardium. Mol Cell Pediatr. 2015;2:5. https://doi.org/10.1186/s40348-015-0015-5.

    Article  Google Scholar 

  63. Crestani T, Steichen C, Neri E, et al. Electrical stimulation applied during differentiation drives the hiPSC-CMs towards a mature cardiac conduction-like cells. Biochem Biophys Res Commun. 2020;533:376–82. https://doi.org/10.1016/j.bbrc.2020.09.021.

    Article  CAS  Google Scholar 

  64. Buikema JW, Lee S, Goodyer WR, et al. Wnt activation and reduced cell-cell contact synergistically induce massive expansion of functional human iPSC-derived cardiomyocytes. Cell Stem Cell. 2020;27:50–63. https://doi.org/10.1016/j.stem.2020.06.001.

    Article  CAS  Google Scholar 

  65. Cheng CF, Ku HC, Lin H. PGC-1alpha as a pivotal factor in lipid and metabolic regulation. Int J Mol Sci. 2018. https://doi.org/10.3390/ijms19113447.

    Article  Google Scholar 

  66. Kleiner S, Nguyen-Tran V, Bare O, et al. PPAR{delta} agonism activates fatty acid oxidation via PGC-1{alpha} but does not increase mitochondrial gene expression and function. J Biol Chem. 2009;284:18624–33. https://doi.org/10.1074/jbc.M109.008797.

    Article  CAS  Google Scholar 

  67. Zhou Q, Xu H, Yan L, et al. PGC-1alpha promotes mitochondrial respiration and biogenesis during the differentiation of hiPSCs into cardiomyocytes. Genes Dis. 2021;8:891–906. https://doi.org/10.1016/j.gendis.2020.12.006.

    Article  CAS  Google Scholar 

  68. Bird SD, Doevendans PA, van Rooijen MA, et al. The human adult cardiomyocyte phenotype. Cardiovasc Res. 2003;58:423–34. https://doi.org/10.1016/s0008-6363(03)00253-0.

    Article  CAS  Google Scholar 

  69. Sheehy SP, Pasqualini F, Grosberg A, et al. Quality metrics for stem cell-derived cardiac myocytes. Stem Cell Reports. 2014;2:282–94. https://doi.org/10.1016/j.stemcr.2014.01.015.

    Article  CAS  Google Scholar 

  70. Yang X, Pabon L, Murry CE. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ Res. 2014;114:511–23. https://doi.org/10.1161/CIRCRESAHA.114.300558.

    Article  CAS  Google Scholar 

  71. Rana P, Anson B, Engle S, et al. Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening. Toxicol Sci. 2012;130:117–31. https://doi.org/10.1093/toxsci/kfs233.

    Article  CAS  Google Scholar 

  72. Lopaschuk GD, Jaswal JS. Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation. J Cardiovasc Pharmacol. 2010;56:130–40. https://doi.org/10.1097/FJC.0b013e3181e74a14.

    Article  CAS  Google Scholar 

  73. Horikoshi Y, Yan YS, Terashvili M, et al. Fatty acid-treated induced pluripotent stem cell-derived human cardiomyocytes exhibit adult cardiomyocyte-like energy metabolism phenotypes. Cells. 2019;8:1095. https://doi.org/10.3390/cells8091095.

    Article  CAS  Google Scholar 

  74. Gentillon C, Li D, Duan M, et al. Targeting HIF-1alpha in combination with PPARalpha activation and postnatal factors promotes the metabolic maturation of human induced pluripotent stem cell-derived cardiomyocytes. J Mol Cell Cardiol. 2019;132:120–35. https://doi.org/10.1016/j.yjmcc.2019.05.003.

    Article  CAS  Google Scholar 

  75. Dorn GW 2nd, Vega RB, Kelly DP. Mitochondrial biogenesis and dynamics in the develo** and diseased heart. Genes Dev. 2015;29:1981–91. https://doi.org/10.1101/gad.269894.115.

    Article  CAS  Google Scholar 

  76. Xu MF, **ong YY, Liu JK, et al. Asiatic acid, a pentacyclic triterpene in Centella asiatica, attenuates glutamate-induced cognitive deficits in mice and apoptosis in SH-SY5Y cells. Acta Pharmacol Sin. 2012;33:578–87. https://doi.org/10.1038/aps.2012.3.

    Article  CAS  Google Scholar 

  77. Luo C, Widlund HR, Puigserver P. PGC-1 coactivators: shepherding the mitochondrial biogenesis of tumors. Trends Cancer. 2016;2:619–31. https://doi.org/10.1016/j.trecan.2016.09.006.

    Article  Google Scholar 

  78. Zhou Q, Xu H, Yan L, et al. PGC-1 alpha promotes mitochondrial respiration and biogenesis during the differentiation of hiPSCs into cardiomyocytes. Genes Dis. 2021;8:891–906. https://doi.org/10.1016/j.gendis.2020.12.006.

    Article  CAS  Google Scholar 

  79. Liu Q, Wu H, Luo Q-J, et al. Tyrosine kinase inhibitors induce mitochondrial dysfunction during cardiomyocyte differentiation through alteration of GATA4-mediated networks. bioRxiv:2020.05.04.077024. 2020. https://doi.org/10.1101/2020.05.04.077024

  80. Caudal A, Ren L, Tu CY, et al. Human induced pluripotent stem cells for studying mitochondrial diseases in the heart. FEBS Lett. 2022;596:1735–45. https://doi.org/10.1002/1873-3468.14444.

    Article  CAS  Google Scholar 

  81. Gaspar JA, Doss MX, Hengstler JG, et al. Unique metabolic features of stem cells, cardiomyocytes, and their progenitors. Circ Res. 2014;114:1346–60. https://doi.org/10.1161/Circresaha.113.302021.

    Article  CAS  Google Scholar 

  82. Ye L, Zhang X, Zhou Q, et al. Activation of AMPK promotes maturation of cardiomyocytes derived from human induced pluripotent stem cells. Front Cell Dev Biol. 2021;9: 644667. https://doi.org/10.3389/fcell.2021.644667.

    Article  Google Scholar 

  83. Pour PA, Kenney MC, Kheradvar A. Bioenergetics consequences of mitochondrial transplantation in cardiomyocytes. J Am Heart Assoc. 2020;9: e014501. https://doi.org/10.1161/JAHA.119.014501.

    Article  Google Scholar 

  84. Zhang HL, Alder NN, Wang W, et al. Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte. Elife. 2020;9: e60827. https://doi.org/10.7554/eLife.60827.

    Article  CAS  Google Scholar 

  85. Xu GY, Sun W, Guo X, et al. Asiatic acid promotes liver fatty acid metabolism in diabetic models. Int J Clin Exp Med. 2018;11:11837–45.

    CAS  Google Scholar 

  86. Wen JY, Wei CY, Shah K, et al. Maturation-based model of arrhythmogenic right ventricular dysplasia using patient-specific induced pluripotent stem cells. Circ J. 2015;79:1402–8. https://doi.org/10.1253/circj.CJ-15-0363.

    Article  CAS  Google Scholar 

  87. Lauzier B, Vaillant F, Merlen C, et al. Metabolic effects of glutamine on the heart: anaplerosis versus the hexosamine biosynthetic pathway. J Mol Cell Cardiol. 2013;55:92–100. https://doi.org/10.1016/j.yjmcc.2012.11.008.

    Article  CAS  Google Scholar 

  88. Cao TT, Liccardo D, LaCanna R, et al. Fatty acid oxidation promotes cardiomyocyte proliferation rate but does not change cardiomyocyte number in infant mice. Front Cell Dev Biol. 2019;7: 42. https://doi.org/10.3389/fcell.2019.00042.

    Article  Google Scholar 

  89. Jiang YH, Wang HL, Peng J, et al. Multinucleated polyploid cardiomyocytes undergo an enhanced adaptability to hypoxia via mitophagy. J Mol Cell Cardiol. 2020;138:115–35. https://doi.org/10.1016/j.yjmcc.2019.11.155.

    Article  CAS  Google Scholar 

  90. Matsuyama D, Kawahara K. Oxidative stress-induced formation of a positive-feedback loop for the sustained activation of p38 MAPK leading to the loss of cell division in cardiomyocytes soon after birth. Basic Res Cardiol. 2011;106:815–28. https://doi.org/10.1007/s00395-011-0178-8.

    Article  CAS  Google Scholar 

  91. Becatti M, Taddei N, Cecchi C, et al. SIRT1 modulates MAPK pathways in ischemic-reperfused cardiomyocytes. Cell Mol Life Sci. 2012;69:2245–60. https://doi.org/10.1007/s00018-012-0925-5.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Not applicable

Funding

NC is supported by the Gravitation Program “Materials Driven Regeneration” by the Netherlands Organization for Scientific Research (RegmedXB #024.003.013) and the Marie Skłodowska-Curie Actions (Grant agreement RESCUE #801540). RM is supported by a grant of the PLN Foundation and HARVEY (18747 NWO OTP). AvM is supported by the EU-funded project BRAV3 (H2020, ID:874827). This work was supported by European Research Council (ERC) under the EVICARE Grant (number 725229) to JS.

Author information

Author notes

  1. Nino Chirico and Elise L. Kessler have contributed equally and share first authorship

Authors and Affiliations

  1. Circulatory Health Laboratory, Regenerative Medicine Center Utrecht, University Utrecht, University Medical Center Utrecht, Utrecht, The Netherlands

    Nino Chirico, Elise L. Kessler, Renée G. C. Maas, Juntao Fang, Jiabin Qin, Inge Dokter, Mark Daniels, Jan-Willem Buikema, Zhiyong Lei, Pieter A. Doevendans, Joost P. G. Sluijter & Alain van Mil

  2. Laboratory of Experimental Cardiology, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands

    Nino Chirico, Elise L. Kessler, Renée G. C. Maas, Juntao Fang, Jiabin Qin, Inge Dokter, Mark Daniels, Klaus Neef, Jan-Willem Buikema, Zhiyong Lei, Pieter A. Doevendans, Joost P. G. Sluijter & Alain van Mil

  3. Center for Physiology and Pathophysiology, Institute for Neurophysiology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany

    Tomo Šarić

  4. Department of Cardiology, Amsterdam Medical Centre, 1105 AZ, Amsterdam, The Netherlands

    Klaus Neef

  5. Netherlands Heart Institute, Utrecht, The Netherlands

    Pieter A. Doevendans

Authors
  1. Nino Chirico
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elise L. Kessler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Renée G. C. Maas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Juntao Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiabin Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Inge Dokter
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mark Daniels
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tomo Šarić
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Klaus Neef
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jan-Willem Buikema
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Zhiyong Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Pieter A. Doevendans
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Joost P. G. Sluijter
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Alain van Mil
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AvM and JPGS designed and directed the project. AvM, NC, and ELK planned experiments. NC, ELK, ID, RM, JF, JQ, and MD carried out the experiments. TŠ and KN generated and characterized the hiPSC lines. NC and ELK drafted the manuscript. All authors provided critical feedback and helped shape the research, analysis, and manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Alain van Mil.

Ethics declarations

Ethics approval and consent to participate

All cell lines used in this study have been deposited in the European Bank for induced pluripotent Stem Cells (EBiSC, https://ebisc.org/) and are registered in the online registry for human pluripotent stem cell lines hPSCreg (https://hpscreg.eu/). All experiments were conducted according to the criteria of the code of proper use of human tissue used in the Netherlands.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1

. Overview of culture Media used during this study. Table S2. Primer list used for qPCR experiments. Table S3. Antibodies list used for western blot, flow cytometry and fluorescent Immunohistochemistry experiments. Fig. S2. Cell line-dependent small molecules dose titration. iPSC line-dependent dose–response effect on CMs at day 27 of differentiation (Fig. 2). For further experiments, the cell clone-dependent optimal concentrations (UKKi036-C: AA 2 and 1 μM, GW: 250 and 100 nM; and T3: 200, and 100 nM; UKKi032-C: AA 10 and 5 μM, GW: 1000 and 500 nM; and T3: 400, and 200 nM; UKKi037-C AA 10 and 5 μM, GW: 1000 and 500 nM; and T3 800 and 400 nM – Fig. S2) were used. Fig. S3. AA and GW treatment enhances levels of mitochondrial key enzymes expression. a) OGDH CTRL versus AA, GW, and T3, respectively: 1.110 (0.6000 – 1.800) versus 1.919 (1.285–2.375; p = 0.0078) versus 2.608 (2.035–2.963. n.s.) versus 2.287 (1.520–2.770; p =0.0156); b) NDUFV3 CTRL versus AA, GW, and T3, respectively: 1.037 (0.7100–1.400;) versus 1.738 (1,553 – 1.933, p =0,0078), 3.750 (1.738 – 4.390; n.s.). and 2.251 (1.950 – 2.510; p=0.0156); c) COX3 CTRL versus AA. GW. and T3, respectively: 1.187 (0.4200–1.830) versus 1.166 (0.8725–1.433; p = 0.0234) versus 2.860 (1.918–3.753; p = n.s.) versus 2.203 (1.760–2.880; p = 0.0156); d) COX5 CTRL versus AA. GW. and T3, respectively: 1.053 (0.7800–1.540) versus 2.471 (1.950–2.943; p = 0.0078) versus 9.758 (6.838–13.52; p = n.s.) versus 2.873 (2.460–3.320; p = 0.0156). Fig. S4. Experiments were performed using day 27 iPSC-CMs line UKKi036-C. (a) Raw extracellular acidification rate (ECAR) of iPSC-CMs cultured in the Seahorse medium with L-glutamine. Addition of ETO and 2DG block the FAO and glucose-dependent glycolysis, respectively. (b) Raw oxygen consumption rate (OCR) in cells at day 27 of differentiation after sequential administration of ETO and 2DG. Table S4. Relative DNA content per single nucleus measured on three independent experiments with iPSC lines: UKKi036-C UKKi032-C, and UKKi037-C combined n = 6–12. Fig. S5. Cell nuclei count per well measured by Hoechst staining and 20X magnification imaging using the Evos microscope and ImageJ show no significant changes in number of nuclei present. Fig. S6. Full-length blots of connexin-43 (left) and CaV1.2 (right) with marker and molecular weight of protein of interest in red. Western blots were performed on pooled proteins from all three cell lines. Equal amount of protein was loaded in each lane. Ponceau staining is shown underneath the western blot panel as a loading control.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chirico, N., Kessler, E.L., Maas, R.G.C. et al. Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes. Stem Cell Res Ther 13, 531 (2022). https://doi.org/10.1186/s13287-022-03209-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13287-022-03209-z

Keywords