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Use of stable isotope-tagged thymidine and multi-isotope imaging mass spectrometry (MIMS) for quantification of human cardiomyocyte division

Abstract

Quantification of cellular proliferation in humans is important for understanding biology and responses to injury and disease. However, existing methods require administration of tracers that cannot be ethically administered in humans. We present a protocol for the direct quantification of cellular proliferation in human hearts. The protocol involves administration of non-radioactive, non-toxic stable isotope 15Nitrogen-enriched thymidine (15N-thymidine), which is incorporated into DNA during S-phase, in infants with tetralogy of Fallot, a common form of congenital heart disease. Infants with tetralogy of Fallot undergo surgical repair, which requires the removal of pieces of myocardium that would otherwise be discarded. This protocol allows for the quantification of cardiomyocyte proliferation in this discarded tissue. We quantitatively analyzed the incorporation of 15N-thymidine with multi-isotope imaging spectrometry (MIMS) at a sub-nuclear resolution, which we combined with correlative confocal microscopy to quantify formation of binucleated cardiomyocytes and cardiomyocytes with polyploid nuclei. The entire protocol spans 3–8 months, which is dependent on the timing of surgical repair, and 3–4.5 researcher days. This protocol could be adapted to study cellular proliferation in a variety of human tissues.

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Fig. 1: MIMS analysis demonstrates proliferation of white blood cells (WBCs).
Fig. 2: MIMS analysis using ex vivo 15N-thymidine labeling of human fetal myocardium.
Fig. 3: Flowchart of the presented protocol to determine cardiomyocyte proliferation and formation of bi- and multinucleated cardiomyocytes and polyploid nuclei.
Fig. 4: Images of myocardial specimens and sections to highlight sample processing.
Fig. 5: MIMS.
Fig. 6: Integration of 31P, 12C14N, and 12C15N isotope signals to identify DNA synthesis in cardiomyocyte nuclei.
Fig. 7: Distinguishing cardiomyocyte from non-cardiomyocyte nuclei with additional analysis of 32S images.
Fig. 8: Alignment of 15N/14N image with Hoechst-stained serial sections for quantification of nuclear ploidy.
Fig. 9: Urinary 15N/14N ratio measurement confirms uptake after oral administration of 15N-thymidine.

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Data availability

Where possible, the data for this protocol are present either within this paper and its supporting documents or in the supporting primary research papers.

Code availability

Details on how to access the OpenMIMS software for MIMS image analysis are available at https://nano.bwh.harvard.edu/openmims.

References

  1. Duque, A. & Rakic, P. Different effects of bromodeoxyuridine and [3h]thymidine incorporation into DNA on cell proliferation, position, and fate. J. Neurosci. 31, 15205–15217 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Romar, G. A., Kupper, T. S. & Divito, S. J. Research techniques made simple: techniques to assess cell proliferation. J. Invest. Dermatol. 136, e1–e7 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Sullivan, B. A., Hollister-Lock, J., Bonner-Weir, S. & Weir, G. C. Reduced Ki67 staining in the postmortem state calls into question past conclusions about the lack of turnover of adult human b-cells. Diabetes 64, 1698–1702 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Peck, M. et al. Applications of PET imaging with the proliferation marker [18 F]-FLT. Q. J. Nucl. Med. Mol. Imaging 59, 95–104 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Costantini, D. L. et al. A pilot study of 18F-FLT PET/CT in pediatric lymphoma. Int. J. Mol. Imaging 2016, 1–5 (2016).

    Article  Google Scholar 

  6. Been, L. B. et al. [18F]FLT-PET in oncology: Current status and opportunities. Eur. J. Nucl. Med. Mol. Imaging 31, 1659–1672 (2004).

    Article  PubMed  Google Scholar 

  7. Senyo, S. E. et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature 493, 433–436 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Laflamme, M. A. & Murry, C. E. Heart regeneration. Nature 473, 326–335 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Eschenhagen, T. et al. Cardiomyocyte regeneration. Circulation 136, 680–686 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Mollova, M. et al. Cardiomyocyte proliferation contributes to heart growth in young humans. Proc. Natl Acad. Sci. USA 110, 1446–1451 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Austin, A., Fagan, D. G. & Mayhew, T. M. A stereological method for estimating the total number of ventricular myocyte nuclei in fetal and postnatal hearts. J. Anat. 187, 641–647 (1995).

    PubMed  PubMed Central  Google Scholar 

  12. Arai, S. & Machida, A. Myocardial cell in left ventricular hypertrophy. Tohoku J. Exp. Med. 108, 361–367 (1972).

    Article  CAS  PubMed  Google Scholar 

  13. Linzbach, A. J. Die Anzahl der Herzmuskelkerne in normalen, uberlastet en, atrophischen und mit Carhorman behandelten Herzkammern. Z. Kreislaufforsch. 41, 641–658 (1952).

    CAS  PubMed  Google Scholar 

  14. Porrello, E. R. et al. Transient regenerative potential of the neonatal mouse heart. Science 331, 1078–1080 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang, H. et al. Natural heart regeneration in a neonatal rat myocardial infarction model. Cells 9, 229 (2020).

    Article  CAS  PubMed Central  Google Scholar 

  16. Polizzotti, B. D. et al. Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window. Sci. Transl. Med. 7, 281ra45–281ra45 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hirose, K. et al. Evidence for hormonal control of heart regenerative capacity during endothermy acquisition. Science 364, 184–188 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Liu, H. et al. Control of cytokinesis by β-adrenergic receptors indicates an approach for regulating cardiomyocyte endowment. Sci. Transl. Med. 11, eaaw6419 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Han, L. et al. Lamin B2 levels regulate polyploidization of cardiomyocyte nuclei and myocardial regeneration. Dev. Cell 53, 42–59.e11 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. González-Rosa, J. M. et al. Myocardial polyploidization creates a barrier to heart regeneration in zebrafish. Dev. Cell 44, 433–446.e7 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Patterson, M. et al. Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration. Nat. Genet. 49, 1346–1353 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wohlschlaeger, J. et al. Hemodynamic support by left ventricular assist devices reduces cardiomyocyte DNA content in the failing human heart. Circulation 121, 989–996 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Bergmann, O. et al. Dynamics of cell generation and turnover in the human heart. Cell 161, 1566–1575 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Linzbach, A. J. Heart failure from the point of view of quantitative anatomy. Am. J. Cardiol. 5, 370–382 (1960).

    Article  CAS  PubMed  Google Scholar 

  25. Steinhauser, M. L. & Lechene, C. P. Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry. Semin. Cell Devel. Biol. 24, 661–667 (2013).

    Article  CAS  Google Scholar 

  26. Zhang, Y. et al. Imaging mass spectrometry reveals tumor metabolic heterogeneity. iScience 23, 101355 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Steinhauser, M. L. et al. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism. Nature 481, 516–519 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Guillermier, C. et al. Imaging mass spectrometry reveals heterogeneity of proliferation and metabolism in atherosclerosis. JCI Insight 4, e128528 (2019).

    Article  PubMed Central  Google Scholar 

  29. Narendra, D. P. et al. Coupling APEX labeling to imaging mass spectrometry of single organelles reveals heterogeneity in lysosomal protein turnover. J. Cell Biol. 219, e201901097 (2020).

    Article  PubMed  Google Scholar 

  30. Zhang, D. S. et al. Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. Nature 481, 520–524 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. He, C. et al. NanoSIMS Analysis of intravascular lipolysis and lipid movement across capillaries and into cardiomyocytes. Cell Metab 27, 1055–1066.e3 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Messenger, S., Keller, L. P., Stadermann, F. J., Walker, R. M. & Zinner, E. Samples of stars beyond the solar system: silicate grains in interplanetary dust. Science 300, 105–108 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Nguyen, A. N. & Zinner, E. Discovery of ancient silicate stardust in a meteorite. Science 303, 1496–1499 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Guillermier, C., Poczatek, J. C., Taylor, W. R. & Steinhauser, M. L. Quantitative imaging of deuterated metabolic tracers in biological tissues with nanoscale secondary ion mass spectrometry. Int. J. Mass Spectrom. 422, 42–50 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lechene, C. et al. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J. Biol. 5, 20 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Gyngard, F. & Steinhauser, M. L. Biological explorations with nanoscale secondary ion mass spectrometry. J. Anal. At. Spectrom. 34, 1534–1545 (2019).

    Article  CAS  Google Scholar 

  37. Peteranderl, R. & Lechene, C. Measure of carbon and nitrogen stable isotope ratios in cultured cells. J. Am. Soc. Mass Spectrom. 15, 478–485 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Kleinfeld, A. M., Kampf, J. P. & Lechene, C. Transport of 13C-oleate in adipocytes measured using multi imaging mass spectrometry. J. Am. Soc. Mass Spectrom. 15, 1572–1580 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Lechene, C. P., Luyten, Y., McMahon, G. & Distel, D. L. Quantitative imaging of nitrogen fixation by individual bacteria within animal cells. Science 317, 1563–1566 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Musat, N. et al. A single-cell view on the ecophysiology of anaerobic phototrophic bacteria. Proc. Natl Acad. Sci. USA 105, 17861–17866 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Lovrić, J. et al. Nano secondary ion mass spectrometry imaging of dopamine distribution across nanometer vesicles. ACS Nano 11, 3446–3455 (2017).

    Article  PubMed  Google Scholar 

  42. Zhang, D.-S. et al. Multi-isotope imaging mass spectrometry (MIMS) reveals slow protein turnover in hair-cell stereocilia. Nature 481, 520–524 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Toyama, B. H. et al. Visualization of long-lived proteins reveals age mosaicism within nuclei of postmitotic cells. J. Cell Biol. 218, 433–444 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Vujic, A. et al. Exercise induces new cardiomyocyte generation in the adult mammalian heart. Nat. Commun. 9, 1659 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Kim, S. M. et al. Loss of white adipose hyperplastic potential is associated with enhanced susceptibility to insulin resistance. Cell Metab. 20, 1049–1058 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Guillermier, C. et al. Imaging mass spectrometry demonstrates age-related decline in human adipose plasticity. JCI Insight 2, e90349 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Stürup, S., Hansen, H. R. & Gammelgaard, B. Application of enriched stable isotopes as tracers in biological systems: A critical review. Analytical and Bioanalytical Chemistry 390, 541–554 (2008).

    Article  PubMed  Google Scholar 

  48. Schoenheimer, R. & Rittenberg, D. The application of isotopes to the study of intermediary metabolism. Science 87, 221–226 (1938).

    Article  CAS  PubMed  Google Scholar 

  49. Young, V. R. & Ajami, A. Isotopes in nutrition research. Proc. Nutr. Soc. 58, 15–32 (1999).

    Article  CAS  PubMed  Google Scholar 

  50. Picou, D. & Taylor-Roberts, T. The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein. Clin. Sci. 36, 283–296 (1969).

    CAS  PubMed  Google Scholar 

  51. Holden, L., Hoffbrand, A. V. & Tattersall, M. H. N. Thymidine concentrations in human sera: variations in patients with leukaemia and megaloblastic anaemia. Eur. J. Cancer 16, 115–121 (1980).

    Article  CAS  PubMed  Google Scholar 

  52. Grem, J. L., King, S. A., Sorensen, J. M. & Christian, M. C. Clinical use of thymidine as a rescue agent from methotrexate toxicity. Invest. New Drugs 9, 281–290 (1991).

    Article  CAS  PubMed  Google Scholar 

  53. Schreiber, F. et al. Phenotypic heterogeneity driven by nutrient limitation promotes growth in fluctuating environments. Nat. Microbiol. 1, 1–7 (2016).

    Article  Google Scholar 

  54. Kilburn, M. R. et al. Application of nanoscale secondary ion mass spectrometry to plant cell research. Plant Signal. Behav. 5, 760–762 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Trembath-Reichert, E. et al. Methyl-compound use and slow growth characterize microbial life in 2-km-deep subseafloor coal and shale beds. Proc. Natl Acad. Sci. USA 114, E9206–E9215 (2017).

    Article  CAS  PubMed  Google Scholar 

  56. Woebken, D. et al. Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis. ISME J. 6, 1427–1439 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Yester, J. W. & Kühn, B. Mechanisms of cardiomyocyte proliferation and differentiation in development and regeneration. Curr. Cardiol. Rep. 19, 13 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Borisov, A. B. & Claycomb, W. C. Proliferative potential and differentiated characteristics of cultured cardiac muscle cells expressing the SV40 T oncogene. Ann. NY Acad. Sci. 752, 80–91 (1995).

    Article  CAS  PubMed  Google Scholar 

  59. Rumyantsev, P. P. Interrelations of the proliferation and differentiation processes during cardiact myogenesis and regeneration. Int. Rev. Cytol. 51, 186–273 (1977).

    CAS  PubMed  Google Scholar 

  60. Bergmann, O. et al. Evidence for cardiomyocyte renewal in humans. Science 324, 98–102 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Steinhauser, M. L., Guillermier, C., Wang, M. & Lechene, C. P. Approaches to increasing analytical throughput of human samples with multi-isotope imaging mass spectrometry. Surf. Interface Anal. 46, 165–168 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Wildburger, N. C. et al. Amyloid-β plaques in clinical Alzheimer’s disease brain incorporate stable isotope tracer in vivo and exhibit nanoscale heterogeneity. Front. Neurol. 9, 14 (2018).

    Article  Google Scholar 

  63. Fidziańska, A. et al. Obliteration of cardiomyocyte nuclear architecture in a patient with LMNA gene mutation. J. Neurol. Sci. 271, 91–96 (2008).

    Article  PubMed  Google Scholar 

  64. Hosios, A. M. et al. Amino acids rather than glucose account for the majority of cell mass in proliferating mammalian cells. Dev. Cell 36, 540–549 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Zhang, Y. et al. Targeting nuclear receptor NR4A1-dependent adipocyte progenitor quiescence promotes metabolic adaptation to obesity. J. Clin. Invest. 128, 4898–4911 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Gyngard, F., Trakimas, L. & Steinhauser, M. L. Methods in molecular biology. in Cardiac Regeneration Methods and Protocols (eds. Poss, K. D. & Kuhn, B.) 257–268 (Springer, 2021). https://doi.org/10.1007/978-1-0716-0668-1

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Acknowledgements

We would like to recognize the patients who participated in the study, and their families. Without their voluntary participation we would not have been able to develop this protocol. Their participation does not benefit them directly, but has opened a new avenue for understanding cardiomyocyte biology. We would also like to recognize the IRB (University of Pittsburgh), the Federal Drug Administration, and R. Sada and M. Cuda (UPMC Children’s Hospital of Pittsburgh) for their efforts related to research patient safety. We acknowledge M. Reyes-Mugica (UPMC Children’s Hospital of Pittsburgh) and the cardiothoracic surgeons at the UPMC Children’s Hospital of Pittsburgh for assistance in ascertaining human tissue samples. We are also grateful for the support from the research pharmacy at the UPMC Children’s Hospital of Pittsburgh, including M. Barlas and S. Ziobert. We would also like to recognize the Division of Cardiology and Cardiothoracic Surgery for their referrals of research subjects (UPMC Children’s Hospital of Pittsburgh). This research was supported by the NIH (R01HL151386, R01HL151415, and R01HL106302), the Department of Pediatrics, the Richard King Mellon Institute for Pediatric Research at UPMC Children’s Hospital of Pittsburgh, and HeartFest (to B.K.) and DP2CA216362 (to M.L.S.). The Leica Ultracut 7 was supported by NIH grant 1S10RR025488 to Simon Watkins (University of Pittsburgh). J.W.Y. was supported, in part, by the NIH (T32HD071834). H.L. was supported, in part, by a grant disbursed by the Research Advisory Committee of UPMC Children’s Hospital of Pittsburgh. S.L. was supported by an Australian Commonwealth Government Endeavour Fellowship. This publication was supported by the National Institutes of Health (NIH), National Center for Advancing Translational Sciences (NCATS) through grant numbers UL1 TR001857, KL2 TR001856, and/or TL1 TR001858.

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J.W.Y. developed the outline and wrote the first draft of the manuscript. N.A., K.C.L., D.T., and J.W.Y. developed the approach for ascertainment of myocardial samples. N.A. developed the protocol for curation of samples and sections. K.C.L. developed the approach for recruitment of eligible families and guiding them through the clinical research protocol. M.L.G.S. developed the protocol for sample processing. N.A. and S.L. completed the in vitro experiments. F.G. and M.L.S developed and wrote the NanoSIMS protocol. H.L. developed and wrote the protocol for analysis of ploidy. M.L.S. and B.K. conceived the research approach and protocol and wrote the manuscript. All authors edited the manuscript.

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Correspondence to Matthew L. Steinhauser or Bernhard Kühn.

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Peer review information Nature Protocols thanks Richard T. Lee, Yuki Sugiura and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Liu, H. et al. Sci. Transl. Med. 11, eaaw6419 (2019): https://stm.sciencemag.org/content/11/513/eaaw6419

Senyo, S. et al. Nature 493, 433–436 (2013): https://www.nature.com/articles/nature11682

Steinhauser, M. et al. Nature 481, 516–519 (2012): https://www.nature.com/articles/nature10734

Key data used in this protocol

Liu H. et al. Sci. Transl. Med. 11, eaaw6419 (2019): https://stm.sciencemag.org/content/11/513/eaaw641

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Yester, J.W., Liu, H., Gyngard, F. et al. Use of stable isotope-tagged thymidine and multi-isotope imaging mass spectrometry (MIMS) for quantification of human cardiomyocyte division. Nat Protoc 16, 1995–2022 (2021). https://doi.org/10.1038/s41596-020-00477-y

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