*3.1. E*ff*ects of Aging on Cardiac Muscle Morphology*

LV cross-sections were stained with hematoxylin and eosin to assess their morphology. Cardiac muscle remodeling was determined by measuring extramyocyte space, extent of apparent fibrosis, myocyte cross-sectional area (CSA), and the number of myocytes per 100,000 µm<sup>2</sup> (Figure 1A). We found that the samples from the OG group showed more extensive cardiac muscle remodeling compared with those from the other groups. No difference in the percentage of extramyocyte space in the samples from the VYG and YG groups was observed, while the samples from the OG group showed significantly more extramyocyte space than those from the VYG, YG, and MG groups; furthermore, the samples from the MG group had more extramyocyte space than those from the VYG and YG groups (mean ± SEM; VYG: 4.48 ± 0.47%; YG: 7.13 ± 0.83%; MG: 13.51 ± 0.97%; OG: 20.85 ± 1.23%)) (*p* < 0.05 in all comparisons, Figure 1B). The number of myocytes per 100,000 µm<sup>2</sup> in the LV tissues was significantly lower in case of the samples from the MG and OG groups compared with those from the VYG and YG groups (mean ± SEM; VYG: 113.70 ± 6.79; YG: 115.38 ± 5.10; MG: 72.17 ± 5.83; OG: 50.86 ± 3.83)) (*p* < 0.05 in all comparisons, Figure 1C). In contrast, the mean myocyte CSA in the combined myocardial and endocardial regions was significantly higher in the OG group than in the VYG, YG, and MG groups (mean ± SEM; 377.66 ± 28.38 µm<sup>2</sup> vs. 120.94 ± 9.34, 134.04 ± 10.79, and 264.22 ± 18.90 µm<sup>2</sup> , respectively) (*p* < 0.05 in all comparisons, Figure 1D), and higher in the

MG rats than in the VYG and YG animals (mean ± SEM; 264.22 ± 18.90 µm<sup>2</sup> vs. 120.94 ± 9.34 and 134.04 ± 10.79 µm<sup>2</sup> , respectively) (*p* < 0.05 in all comparisons, Figure 1D).

**Figure 1.** (**A**) Representative histological cross-sections of the left ventricle (LV) tissues of rats from the very young (VYG), young (YG), middle-aged (MG), and old (OG) groups stained with hematoxylin and eosin (amplification: 40×). Unstained areas indicate extramyocyte space. (**B**) Quantification of the percentage of extramyocyte space. (**C**) Number of myocytes per 100,000 µm<sup>2</sup> in the LV tissues. (**D**) Myocyte cross-sectional area (CSA) of LV tissue histological sections, in square micrometers. The scale bar indicates 100 µm. Data presented as the means ± SEMs. \* *p* < 0.05 vs. VYG. † *p* < 0.05 vs. YG. # *p* < 0.05 vs. MG.
