Abstract Body (Do not enter title and authors here): Background:
Cardiomyocyte proliferation, the fundamental mechanism of heart development and regeneration, is active in infants. We investigated cardiomyocyte proliferation in infants with heart disease.

Objective/Hypothesis:
Infants with tetralogy of Fallot (ToF, the most common type of cyanotic congenital heart disease) or heart failure (HF) exhibit altered cardiomyocyte proliferation.

Methods:
The DNA replication marker, ¹⁵N-thymidine, was administered to infants with unrepaired ToF (n = 13) and to infants with heart failure waiting for transplantation (n = 5). One of the infants with ToF was also treated with the beta-blocker, propranolol. Myocardial samples were ascertained at the time of ToF surgery, placement or removal of left ventricular assist devices (LVAD), and heart transplantation. Retention of ¹⁵N-thymidine in cells that had undergone DNA replication was assessed using Multi-Isotope Imaging Mass Spectrometry (MIMS). Polyploidy was quantified to identify post-mitotic, non-dividing cardiomyocytes.

Results:
Within the first 4 months after birth, 0.74 billion new cardiomyocytes were generated—3.4-fold higher than after the age of 4 months (P = 0.0417). Average per nucleus DNA content in ¹⁵N-labeled cardiomyocytes in ToF infants (n = 11) and HF infants (n = 3) was significantly greater (1.38-fold) than that in individuals without heart disease (n = 11, P = 0.0358), indicating increased formation of post-mitotic cardiomyocytes in diseased hearts. An infant with HF received ¹⁵N-thymidine while on LVAD. Despite clinical improvement and LVAD removal, cardiomyocyte generation remained below age-appropriate levels. In contrast, a ToF infant treated with propranolol, showed a reduced incidence of polyploid nuclei, suggesting enhanced cardiomyocyte division in response to β-blockade.

Conclusion:
Infants with heart disease generate new cardiomyocytes, with the majority of this occurring in the first four months after birth, which identifies a window for regenerative therapies. Beta-blockade may promote cardiomyocyte division, highlighting a direction for regenerative therapy.