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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.

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.

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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 (2021). https://doi.org/10.1038/s41596-020-00477-y

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