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Hyperglycaemia-Induced Contractile Dysfunction and Apoptosis in Cardiomyocyte-Like Pulsatile Cells Derived from Mouse Embryonic Stem Cells

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Abstract

Hyperglycaemia, a key metabolic abnormality in diabetes mellitus, is implicated in pathological cardiogenesis during embryological development. However, the underlying mechanisms and potential therapeutic targets remain unknown. We, therefore, studied the effect of hyperglycaemia on mouse embryonic stem cell (mESC) cardiac differentiation. The mESCs were differentiated via embryoid body (EB) formation and cultured under conditions with baseline (25 mM) or high (50 mM) glucose. Time-lapse microscopy images of pulsatile mESCs and Ca2+ transients were recorded. Biomarkers of cellular changes were detected using immunocytochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and Western blot analyses. Differentiated, spontaneously beating mESCs stained positive for cardiac troponin T, α-actinin 2, myosin heavy chain, and connexin 43. Hyperglycaemia decreased the EB diameter and number of beating EBs as well as the cellular amplitude of contraction, the Ca2+ transient, and the contractile response to caffeine (1 mM), but had no effect on the expression of the sarco-endoplasmic reticulum calcium transport ATPase 2 (SERCA 2). Furthermore, hyperglycaemia decreased the expression of B cell lymphoma 2 (Bcl-2) and increased the expression of cytoplasmic cytochrome c and the number of TUNEL-positive cells, but had no effect on the expression of one of the mitochondrial fusion regulatory proteins, optic atrophy protein 1 (OPA1). Overall, hyperglycaemia suppressed the mESC cardiomyocyte-like differentiation and induced contractile dysfunction. The results are consistent with mechanisms involving abnormal Ca2+ handling and mitochondrial-dependent apoptosis, factors which represent potential therapeutic targets in developmental diabetic cardiac disease.

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Fig. 1

taken from undifferentiated mESCs. Inset: Negative (-ve) control in which the primary antibody was omitted. Arrowheads indicate nuclei of inactivated mouse embryonic fibroblasts (iMEFs) in the feeder layer. Scale bar = 50 µm. n = 3 replicates. c‒e Representative confocal microscopy images of βIII-tubulin, α-smooth muscle actin (α-SMA), α1-fetoprotein, Hoechst, and merged images. Insets: Negative (-ve) controls in which the primary antibodies were omitted. Scale bar = 10 µm. n = 3 replicates for each germ layer

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Fig. 10

taken from actively beating areas of embryoid bodies. Scale bar = 5 µm. c Quantitative analysis of the cellular Mitotracker Green fluorescence intensity (n = 4‒6 replicates per group). Data are shown as box plot and the mean (filled square). Glu, glucose

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Data and material available upon request.

References

  1. Aberg, A., Westbom, L., & Kallen, B. (2001). Congenital malformations among infants whose mothers had gestational diabetes or preexisting diabetes. Early Human Development, 61, 85–95

    Article  CAS  Google Scholar 

  2. Tinker, S. C., Gilboa, S. M., Moore, C. A., Waller, D. K., Simeone, R. M., Kim, S. Y., et al. (2020). Specific birth defects in pregnancies of women with diabetes: national birth defects prevention study, 1997–2011. American Journal of Obstetrics and Gynecology, 222, 176.e1-176.e11

    Article  Google Scholar 

  3. Loffredo, C. A., Wilson, P. D., & Ferencz, C. (2001). Maternal diabetes: an independent risk factor for major cardiovascular malformations with increased mortality of affected infants. Teratology, 64, 98–106

    Article  CAS  Google Scholar 

  4. Floris, I., Descamps, B., Vardeu, A., Mitic, T., Posadino, A. M., Shantikumar, S., et al. (2015). Gestational diabetes mellitus impairs fetal endothelial cell functions through a mechanism involving microRNA-101 and histone methyltransferase enhancer of zester homolog-2. Arteriosclerosis, Thrombosis, and Vascular Biology, 35, 664–674

    Article  CAS  Google Scholar 

  5. Kumar, S. D., Dheen, S. T., & Tay, S. S. (2007). Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development. Cardiovascular Diabetology, 6, 34

    Article  Google Scholar 

  6. Reinking, B. E., Wedemeyer, E. W., Weiss, R. M., Segar, J. L., & Scholz, T. D. (2009). Cardiomyopathy in offspring of diabetic rats is associated with activation of the MAPK and apoptotic pathways. Cardiovascular Diabetology, 8, 43

    Article  Google Scholar 

  7. Basu, M., & Garg, V. (2018). Maternal hyperglycemia and fetal cardiac development: clinical impact and underlying mechanisms. Birth Defects Research, 110, 1504–1516

    Article  CAS  Google Scholar 

  8. Narchi, H., & Kulaylat, N. (2000). Heart disease in infants of diabetic mothers. Images in Paediatric Cardiology, 2, 17–23

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Steinhauser, M. L., & Lee, R. T. (2011). Regeneration of the heart. EMBO Molecular Medicine, 3, 701–712

    Article  CAS  Google Scholar 

  10. Nag, A. C., Lee, M. L., & Sarkar, F. H. (1996). Remodelling of adult cardiac muscle cells in culture: dynamic process of disorganization and reorganization of myofibrils. Journal of Muscle Research and Cell Motility, 17, 313–334

    Article  CAS  Google Scholar 

  11. Wobus, A. M., Guan, K., Yang, H. T., & Boheler, K. R. (2002). Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods in Molecular Biology, 185, 127–156

    CAS  PubMed  Google Scholar 

  12. Shi, M., Tien, N. T., de Haan, L., Louisse, J., Rietjens, I., & Bouwmeester, H. (2020). Evaluation of in vitro models of stem cell-derived cardiomyocytes to screen for potential cardiotoxicity of chemicals. Toxicology In Vitro, 67, 104891

    Article  CAS  Google Scholar 

  13. Wang, X., & Yang, P. (2008). In vitro differentiation of mouse embryonic stem (mES) cells using the hanging drop method. Journal of Visualized Experiments. https://doi.org/10.3791/825

    Article  PubMed  PubMed Central  Google Scholar 

  14. Lee, M. Y., Cagavi Bozkulak, E., Schliffke, S., Amos, P. J., Ren, Y., Ge, X., et al. (2011). High density cultures of embryoid bodies enhanced cardiac differentiation of murine embryonic stem cells. Biochemical and Biophysical Research Communications, 416, 51–57

    Article  CAS  Google Scholar 

  15. Ali, N. N., Xu, X., Brito-Martins, M., Poole-Wilson, P. A., Harding, S. E., & Fuller, S. J. (2004). Beta-adrenoceptor subtype dependence of chronotropy in mouse embryonic stem cell-derived cardiomyocytes. Basic Research in Cardiology, 99, 382–391

    Article  CAS  Google Scholar 

  16. Guan, K., Furst, D. O., & Wobus, A. M. (1999). Modulation of sarcomere organization during embryonic stem cell-derived cardiomyocyte differentiation. European Journal of Cell Biology, 78, 813–823

    Article  CAS  Google Scholar 

  17. Crespo, F. L., Sobrado, V. R., Gomez, L., Cervera, A. M., & McCreath, K. J. (2010). Mitochondrial reactive oxygen species mediate cardiomyocyte formation from embryonic stem cells in high glucose. Stem Cells, 28, 1132–1142

    CAS  PubMed  Google Scholar 

  18. Han, S. S., Wang, G., Jin, Y., Ma, Z. L., Jia, W. J., Wu, X., et al. (2015). Investigating the mechanism of hyperglycemia-induced fetal cardiac hypertrophy. PLoS ONE, 10, e0139141

    Article  Google Scholar 

  19. Xu, X., Ruan, L., Tian, X., Pan, F., Yang, C., & Liu, G. (2020). Calcium inhibitor inhibits high glucoseinduced hypertrophy of H9C2 cells. Molecular Medicine Reports, 22, 1783–1792

    Article  CAS  Google Scholar 

  20. Yang, P., Chen, X., Kaushal, S., Reece, E. A., & Yang, P. (2016). High glucose suppresses embryonic stem cell differentiation into cardiomyocytes : High glucose inhibits ES cell cardiogenesis. Stem Cell Research & Therapy, 7, 187

    Article  Google Scholar 

  21. Grune, T., Ott, C., Haseli, S., Hohn, A., & Jung, T. (2019). The “MYOCYTER” - Convert cellular and cardiac contractions into numbers with ImageJ. Scientific Reports, 9, 15112

    Article  Google Scholar 

  22. Francis, M., Waldrup, J., Qian, X., & Taylor, M. S. (2014). Automated analysis of dynamic Ca2+ signals in image sequences. Journal of Visualized Experiments. https://doi.org/10.3791/51560

    Article  PubMed  PubMed Central  Google Scholar 

  23. Aboalgasm, H., Petersen, M., & Gwanyanya, A. (2020). Improvement of cardiac ventricular function by magnesium treatment in chronic streptozotocin-induced diabetic rat heart. Cardiovascular Journal of Africa, 31, 1–8

    PubMed  Google Scholar 

  24. Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., et al. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95, 379–391

    Article  CAS  Google Scholar 

  25. Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., et al. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell, 113, 643–655

    Article  CAS  Google Scholar 

  26. Chen, L., Gong, Q., Stice, J. P., & Knowlton, A. A. (2009). Mitochondrial OPA1, apoptosis, and heart failure. Cardiovascular Research, 84, 91–99

    Article  CAS  Google Scholar 

  27. Sutton-McDowall, M. L., Gilchrist, R. B., & Thompson, J. G. (2010). The pivotal role of glucose metabolism in determining oocyte developmental competence. Reproduction, 139, 685–695

    Article  CAS  Google Scholar 

  28. Mochizuki, H., Ohnuki, Y., & Kurosawa, H. (2011). Effect of glucose concentration during embryoid body (EB) formation from mouse embryonic stem cells on EB growth and cell differentiation. Journal of Bioscience and Bioengineering, 111, 92–97

    Article  CAS  Google Scholar 

  29. Nakano, H., Minami, I., Braas, D., Pappoe, H., Wu, X., Sagadevan, A., et al. (2017). Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis. eLife, 6, e29330

    Article  Google Scholar 

  30. McKee, T. J., & Komarova, S. V. (2017). Is it time to reinvent basic cell culture medium? American Journal of Physiology Cell Physiology, 312, C624–C626

    Article  Google Scholar 

  31. Kolwicz, S. C., Jr., & Tian, R. (2011). Glucose metabolism and cardiac hypertrophy. Cardiovascular Research, 90, 194–201

    Article  CAS  Google Scholar 

  32. Hou, Q., Lei, M., Hu, K., & Wang, M. (2015). The effects of high glucose levels on reactive oxygen species-induced apoptosis and involved signaling in human vascular endothelial cells. Cardiovascular Toxicology, 15, 140–146

    Article  CAS  Google Scholar 

  33. Frezza, C., Cipolat, S., Martins de Brito, O., Micaroni, M., Beznoussenko, G. V., Rudka, T., et al. (2006). OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell, 126, 177–189

    Article  CAS  Google Scholar 

  34. Lu, S., Liao, Z., Lu, X., Katschinski, D. M., Mercola, M., Chen, J., et al. (2020). Hyperglycemia acutely increases cytosolic reactive oxygen species via o-linked glcnacylation and camkii activation in mouse ventricular myocytes. Circulation Research, 126, e80–e96

    Article  CAS  Google Scholar 

  35. Volpe, C. M. O., Villar-Delfino, P. H., Dos Anjos, P. M. F., & Nogueira-Machado, J. A. (2018). Cellular death, reactive oxygen species (ROS) and diabetic complications. Cell Death & Disease, 9, 119

    Article  Google Scholar 

  36. Kumar, S., Kain, V., & Sitasawad, S. L. (2012). High glucose-induced Ca2+ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways. Biochimica et Biophysica Acta - General Subjects, 1820, 907–920

    Article  CAS  Google Scholar 

  37. Choi, K. M., Zhong, Y., Hoit, B. D., Grupp, I. L., Hahn, H., Dilly, K. W., et al. (2002). Defective intracellular Ca2+ signaling contributes to cardiomyopathy in Type 1 diabetic rats. American Journal of Physiology Heart and Circulatory Physiology, 283, H1398-1408

    Article  CAS  Google Scholar 

  38. Zhang, L., Cannell, M. B., Phillips, A. R., Cooper, G. J., & Ward, M. L. (2008). Altered calcium homeostasis does not explain the contractile deficit of diabetic cardiomyopathy. Diabetes, 57, 2158–2166

    Article  CAS  Google Scholar 

  39. Bode, E. F., Briston, S. J., Overend, C. L., O’Neill, S. C., Trafford, A. W., & Eisner, D. A. (2011). Changes of SERCA activity have only modest effects on sarcoplasmic reticulum Ca2+ content in rat ventricular myocytes. The Journal of Physiology, 589, 4723–4729

    Article  CAS  Google Scholar 

  40. Rosen, M. R., Myerburg, R. J., Francis, D. P., Cole, G. D., & Marban, E. (2014). Translating stem cell research to cardiac disease therapies: pitfalls and prospects for improvement. Journal of the American College of Cardiology, 64, 922–937

    Article  Google Scholar 

  41. Kowalski, M. P., Yoder, A., Liu, L., & Pajak, L. (2012). Controlling embryonic stem cell growth and differentiation by automation: enhanced and more reliable differentiation for drug discovery. Journal of Biomolecular Screening, 17, 1171–1179

    Article  Google Scholar 

  42. Zhou, P., & Pu, W. T. (2016). Recounting cardiac cellular composition. Circulation Research, 118, 368–370

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Desiree Bowers for tissue-culture technical assistance and Susan Cooper for technical assistance in the HUB Confocal and Light Microscope Imaging Facility.

Funding

The study was funded by the South African Medical Research Council (MRC Grant Number 29841).

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Authors and Affiliations

  1. Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, 7925, South Africa

    Hamida Aboalgasm, Robea Ballo, Thulisa Mkatazo & Asfree Gwanyanya

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  1. Hamida Aboalgasm
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Correspondence to Asfree Gwanyanya.

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Aboalgasm, H., Ballo, R., Mkatazo, T. et al. Hyperglycaemia-Induced Contractile Dysfunction and Apoptosis in Cardiomyocyte-Like Pulsatile Cells Derived from Mouse Embryonic Stem Cells. Cardiovasc Toxicol 21, 695–709 (2021). https://doi.org/10.1007/s12012-021-09660-3

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