Abstract 20: Resident Cardiac Stem Cell Growth Determines the Number of Ventricular Cardiomyocytes in the Mouse Heart

Abstract

The objective of this study was to determine the role of c-kit-positive cardiac stem cells (CSCs) in the formation of the heart during prenatal life, and immediately after birth. Mice in which EGFP is under the control of the c-kit-promoter were employed to measure the number of CSCs (Ns), the fraction of cycling MCM5-positive CSCs (f) and the length of the cell cycle (Ts) in CSCs. The number of CSCs committed to the myocyte lineage (LCC: lineage committed cells) included myocyte progenitors (c-kit-positive, Nkx2.5-positive cells), myocyte precursors (c-kit-positive, Nkx2.5-positive, and α-sarcomeric actin-positive cells) and replicating amplifying myocytes (c-kit-negative, Nkx2.5-positive, α-sarcomeric actin-positive, and MCM5-positive cells). These variables derived from CSC growth and lineage specification were evaluated to define the rate of formation of terminally differentiated myocytes (r). Based on a hierarchically structured cell organization, the rate of entry (Rs) of CSCs into the cell cycle was computed from Rs = f x (Ns/Ts), and the rate of generation of mature myocytes, r, was obtained from r = Rs x 2 Gt = ((f x Ns)/Ts) x 2 Gt . The exponent Gt defines the number of transit generations, i.e., the number of divisions that one CSC has to go through before it acquires the terminally differentiated myocyte phenotype. To validate this scenario and establish the number of post-mitotic myocytes formed, the primary data listed above were collected at E9, E14, E19 and P1. The number of mature cardiomyocytes generated by 1 CSC in 1 day was 1.1 x 10 3 , 20 x 10 3 , 501 x 10 3 , and 440 x 10 3 at E9, E14, E19 and P1, respectively. The total number of myocytes (Nm) formed from E9 to E14, E19 and P1 was derived from an exponential equation with the best fit to the experimental data: Nm = exp (0.69 x t) where Nm is the number of myocytes and t is time in days. Accordingly, CSCs generated 1 x 10 5 , 1 x 10 6 and 1.8 x 10 6 myocytes at from E9 to E14, E19 and P1, respectively. These values accounted for all parenchymal cells present at mid and late gestation and in the neonatal heart measured morphometrically. Thus, the expansion of the myocyte mass during embryonic, fetal and immediate postnatal development is controlled by activation, growth and differentiation of resident c-kit-positive CSCs.