Abstract Body: Introduction
Cardiomyocyte proliferation is the fundamental cellular mechanism of heart growth and regeneration. In adult mammals, most cardiomyocytes are in a polyploid, post-mitotic state and heart regeneration does not occur. Indirect evidence suggests active cardiomyocyte proliferation in young humans; however, it is unclear until what age this occurs and how heart disease alters the process.

Methods
We conducted a prospective clinical study enrolling infants with tetralogy of Fallot (ToF) or severe heart failure (HF) listed for transplantation. Infants received oral ¹⁵N-thymidine for five consecutive days during which the label is incorporated into the DNA of cells undergoing S-phase. Myocardial pieces obtained at the time of ToF repair from the right ventricle and at time of ventricular assist device (LVAD) implantation or heart transplantation from the left ventricle were analyzed by multi-isotope imaging mass spectrometry (MIMS). Ventricular cardiomyocytes generated during the labeling period were identified by increased ¹⁵N/¹⁴N intensity on MIMS. Tissue sections adjacent to the MIMS sections were stained with the DNA-dye, Hoechst, to identify and quantify formation of cardiomyocytes with polyploid nuclei, i.e., a post-mitotic phenotype.

Results
Infants labeled under 3.5 months after birth (n=9 ToF; n=1 HF) generated approximately 0.2 billion cardiomyocytes per month, whereas infants labeled after 3.5 months (n=1 ToF; n=3 HF) generated approximately 0.04 billion per month—a fivefold reduction. Thus, during the first 3.5 months after birth, approximately 0.7 billion new ventricular cardiomyocytes were generated. Completed cardiomyocyte division accounted for ~29% of this increase, with generation of polyploid or multinucleated cardiomyocytes accounting for the remainder. The proportion of polyploid nuclei among newly generated cardiomyocytes in infants with heart disease (n=8 ToF; n=2 HF; 35.2 ± 8.0%) was significantly higher than that in infants without heart disease (n=5; 14.1 ± 4.0%) indicating that post-mitotic cardiomyocytes arise earlier in infants with heart disease.

Conclusions
These findings provide the first direct quantification of postnatal cardiomyocyte generation in humans with heart disease, identifying an early window of proliferative activity and a clinically applicable method to measure cardiac regeneration.