Fig. 2

Number of mononucleate diploid ¹⁵N-labeled cardiomyocytes increases with exercise. a Serial sections (0.5–1 μm thickness) were processed to determine the ploidy status and number of nuclei of each ¹⁵N-labeled cardiomyocyte (large white arrow). Periodic acid Schiff staining (PAS) was performed on serial adjacent sections in both directions from the MIMS chip to define the number of nuclei contained in the cell and fluorescent in situ hybridization (Y-chromosome) was performed to identify ploidy status. A representative image series is shown from a mononucleate (PAS staining far left/right, scale bar = 10 μm) diploid (2 N, second from left, scale bar = 5 μm) ¹⁵N-labeled (MIMS second from right, scale bar = 10 μm) cardiomyocyte. ¹⁴N and ³¹P images are shown for subcellular resolution (center, scale bar = 10 μm). b Representative image of a ¹⁵N-labeled cardiomyocyte nuclei undergoing binucleation. Scale bar = 10 μm for mass image ¹⁴N and ¹⁵N/¹⁴N (left, center). Scale bar = 4 μm for mass image ¹⁵N/¹⁴N (right). c Bar graph showing the frequency of mononucleate/diploid vs. polyploid and/or multinucleate ¹⁵N-thymidine-labeled cardiomyocytes from each group (graph) and contingency table showing absolute numbers and percentage calculations of ¹⁵N-positive and all identified cardiomyocytes (sedentary: exercised = 0.25%:1.15%, n = 4 mice per group, *p = 0.01, Fisher’s exact test, OR = 4.695, CI 1.44–15.53). d Mononucleate ¹⁵N-thymidine labeled cardiomyocytes were significantly smaller than their binucleate counterparts in the same hearts (n = 3 mice per group, one-way ANOVA with Tukey’s correction for multiple comparisons (significance level p < 0.05). Error bars represent ± s.e.m