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

• Published in: Nature protocols Vol. 16; no. 4; pp. 1995 - 2022
• Main Authors: Yester, Jessie W., Liu, Honghai, Gyngard, Frank, Ammanamanchi, Niyatie, Little, Kathryn C., Thomas, Dawn, Sullivan, Mara L. G., Lal, Sean, Steinhauser, Matthew L., Kühn, Bernhard
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2021; Nature Publishing Group
• Subjects: 631/1647/296; 631/80/641/151/1431; 692/4019/592/2725; Analytical Chemistry; Biological Techniques; Biomedical and Life Sciences; Cardiomyocytes; Cardiovascular diseases; Cardiovascular research; Cell cycle; Cell Division; Cell Nucleus - metabolism; Cell Proliferation; Computational Biology/Bioinformatics; Confocal microscopy; Coronary artery disease; Female; Fetus - cytology; Health aspects; Heart cells; Heart diseases; Human tissues; Humans; Imaging; Imaging, Three-Dimensional; Infant; Infants; Isotope dilution techniques; Isotope Labeling - methods; Leukocytes - cytology; Life Sciences; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Methods; Microarrays; Myocardium; Myocardium - cytology; Myocytes, Cardiac - cytology; Nitrogen isotopes; Nitrogen Isotopes - urine; Organic Chemistry; Physiological aspects; Ploidies; Polyploidy; Pregnancy; Protocol; Sarcomeres - metabolism; Scientific imaging; Spectroscopy; Stable isotopes; Tetralogy of Fallot; Tetralogy of Fallot - pathology; Thymidine; Thymidine - metabolism; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/s41596-020-00477-y

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 15 Nitrogen-enriched thymidine ( 15 N-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 15 N-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. The dynamics of cardiomyocyte cell cycle activity and proliferation in humans are poorly understood. This protocol describes how to measure cell division in infant hearts by multi-isotope imaging spectrometry after incorporation of 15 N-thymidine.