*3.2. E*ff*ects of Aging on Mitochondria-Mediated Apoptotic Signaling in Cardiac Muscles*

The expression of the pro-apoptotic Bax protein increased by 1960%, 78%, and 69% in the samples from the OG group (4.12 ± 0.35), compared with that in samples from the VYG (0.20 ± 0.04), YG (2.32 ± 0.34), and MG groups (2.44 ± 0.31), respectively (*p* < 0.05 in all comparisons, Figure 2A). The samples from the YG and MG groups showed similar Bax levels (*p* > 0.05). In contrast, the levels of the anti-apoptotic Bcl-2 protein were reduced by 80%, 82%, and 70% in the samples from the OG group (0.29 ± 0.04), compared with those in the samples from the VYG (1.46 ± 0.17), YG (1.61 ± 0.22), and MG (0.98 ± 0.08) groups, respectively (*p* < 0.05 in all comparisons, Figure 2B), while the samples from the VYG and YG groups showed similar Bcl-2 levels (*p* > 0.05). The Bax/Bcl-2 ratio, which is important during the early stage of mitochondria-mediated apoptosis, increased more significantly in the samples from the OG group than in those from the VYG, YG, and MG groups (*p* < 0.05, Figure 2C). Analysis of mPTP opening sensitivity in the VYS group was set to 100.00; the mPTP opening sensitivity was increased by 154%, 303%, and 587% in the samples from the OG group (687.19 ± 60.83), compared with that in the samples from the VYG (100.00), YG (254.16 ± 23.76), and MG groups (403.55 ± 68.45), respectively (*p* < 0.05 in all comparisons, Figure 2D). Furthermore, the mPTP opening sensitivity was higher in the samples from the MG group than in those from the VYG group (*p* < 0.05, Figure 2D), while no significant difference was seen between the mPTP opening sensitivity in the samples from the VYG and YG groups (100.00 vs. 254.16 ± 23.76; Figure 2D). Analysis of the expression of cleaved

caspase-3, which is present downstream to the Bcl-2 family in the mitochondria-mediated apoptotic pathway, revealed that its levels were significantly higher in OG rats (1.05 ± 0.08) than in the VYG (0.20 ± 0.03), YG (0.54 ± 0.08), and MG (0.76 ± 0.05) rats (*p* < 0.05 in all comparisons, Figure 2E). Furthermore, the levels of cleaved caspase-3 were higher in the samples from the YG and MG groups than in those from the VYG group (*p* < 0.05 in both comparisons, Figure 2E).

**Figure 2.** Mitochondria-mediated apoptotic signaling in the left ventricle (LV) tissues of rats from the very young (VYG), young (YG), middle-aged (MG), and old (OG) groups. (**A**) Bax levels detected by Western blotting with actin as the normalization control. (**B**) Representative Western blot results and relative quantification of Bcl-2 levels in LV tissues. (**C**) Representative Bax/Bcl-2 ratios in LV tissues. (**D**) Representative mPTP opening sensitivity with that of the VYG samples as the normalization control. (**E**) Cleaved caspase-3 levels detected by Western blotting, with actin as the normalization control. Data are presented as the means ± SEMs. \* *p* < 0.05 vs. VYG. † *p* < 0.05 vs. YG. # *p* < 0.05 vs. MG.
