Abstract Body (Do not enter title and authors here): Introduction:
Cell cycle entry and division of differentiated heart muscle cells (cardiomyocytes, CMs) is the evolutionarily conserved mechanism of heart development and regeneration. Quantification of CM proliferation in humans is a critical step towards developing new regenerative therapies for heart failure. To provide definitive evidence for CM proliferation and to characterize the generated phenotypes in humans, we implemented a new research approach using ¹⁵N-thymidine administration to mark cells in S-phase.

Tetralogy of Fallot (ToF) is the most common cyanotic congenital heart disease and heart failure is the most common indication for heart transplantation. We selected these patient populations because they undergo heart surgery or transplantation, which provide myocardial samples for research.

Research Goal:
To define CM generation in human infants.

Methods:
We administered ¹⁵N-thymidine on five consecutive days in infants who subsequently underwent heart surgery or heart transplantation. The retention of ¹⁵N was visualized by analyzing heart muscle sections with Multi-isotope Imaging Mass Spectrometry (MIMS). Tissue sections adjacent to the MIMS sections were stained with the DNA-dye, Hoechst, to identify and quantify formation of post-mitotic phenotypes, i.e., CMs with polyploid nuclei and multinucleated CMs. Quantitative results presented as mean ± SEM.

Results:
Infants of 2 months of age and younger at label administration generated 3.9 ± 1.3% new cardiomyocytes (n = 5) and infants between the ages of >2 to 4 months at label administration generated 2.9 ± 1.0% new cardiomyocytes (n = 6). Infants 4 months of age and older generated 0.1 ± 0.1% new cardiomyocytes (n = 3). In ToF (n = 9 infants), 36.0 ± 10.5% of generated cardiomyocytes were mononucleated with polyploid nuclei. Available results from four infants showed formation of binucleated CMs, with the majority having polyploid nuclei.

Conclusions:
Infants with ToF or heart failure showed a decrease in CM generation with increasing postnatal age. Infants with heart disease generated a high proportion of multinucleated CMs and CMs with polyploid nuclei, indicating increased formation of postmitotic CMs. In conclusion, this study provides definitive evidence for the generation of CMs in infants with heart disease, identifies formation of postmitotic CMs as targetable cellular mechanism, and defines the age window in which therapies to direct CM generation could be effective.