*3.3. E*ff*ects of Aging on Cleaved Caspase-3-Positive Cells and TUNEL-Positive Myonuclei in Cardiac Muscles*

The number of cleaved caspase-3-positive cells increased by 250% and 185% in the samples from the OG group (20.64 ± 1.96), compared with that in samples from the VYG (5.90 ± 0.85) and YG groups (7.24 ± 1.00), respectively (*p* < 0.05 in both comparisons; Figure 3A,C). Additionally, the number of TUNEL-positive myonuclei in the cardiac muscles of OG rats was significantly higher than that in the cardiac muscles of the VYG (by 2196%), YG (by 810%), and MG (by 107%) rats (mean ± SEM; 21.35 ± 0.42 vs. 0.93 ± 0.05, 1.92 ± 0.08, and 8.46 ± 0.35, respectively) (*p* < 0.05 in all comparisons; Figure 3B,D). The number of TUNEL-positive myonuclei was higher in the samples from the MG group than in those from the VYG and YG groups (*p* < 0.05 in both comparisons, Figure 3B,D). However, the samples from the VYG and YG groups did not show significant differences in the numbers of cleaved caspase-3-positive cells and TUNEL-positive myonuclei (*p* > 0.05).

**Figure 3.** Cleaved caspase-3–positive cells and TUNEL-positive myonuclei in the left ventricle (LV) tissues of rats from the very young (VYG), young (YG), middle-aged (MG), and old (OG) groups (scale bar: 100 µm and magnification: 40×). (**A**) Representative photographs of LV tissue sections stained with anti-cleaved caspase-3 antibodies (**B**) TUNEL staining images in which brown-stained regions represent TUNEL-positive myonuclei. Apoptotic myonuclei are indicated by arrows which are typically enlarged in the lower left corner. (**C**) Quantification of cleaved caspase-3–positive cells via immunohistochemical staining. (**D**) Quantification of TUNEL-positive myonuclei. Data are presented as the means ± SEMs. \* *p* < 0.05 vs. VYG. † *p* < 0.05 vs. YG. # *p* < 0.05 vs. MG.

#### **4. Discussion**

The main findings of this study were as follows: (i) cardiac muscle remodeling, assessed based on morphological changes in tissues, increased with advancing age; (ii) mitochondria-dependent apoptotic signaling (including Bax/Bcl-2 ratio, mPTP opening sensitivity, and cleaved caspase-3 protein levels) remarkably increased with advancing age; and (iii) apoptosis (including numbers of cleaved caspase-3-positive cells and TUNEL-positive myonuclei) also increased with advancing age. These results reveal that aging induces significant alterations in the myocardial structure and mitochondria-mediated apoptotic signaling in the rat heart, and that these changes are more drastic during the old-age phase and not in the developing-age phase.

To our knowledge, this is the first report regarding the changes in the myocardial structure and mitochondria-mediated apoptotic signaling, which occurs via Bcl-2 family proteins, in rat cardiac muscles throughout their lifespan—including each phase of growth (very young vs. young), development (young vs. middle-aged), and aging (middle-aged vs. old). As age-related changes in mitochondria-mediated apoptotic signaling are debatable, determination of such signals, as well as the myocardial structure at different ages will likely provide valuable information on the cellular and molecular mechanisms underlying aging in the heart.

Several studies using models of young and old animals have shown that cardiac remodeling and function deteriorate with age [18–20]. Consistent with previous findings, we also observed significant cardiac morphological changes, such as increased extramyocyte space and myocyte CSA, in aged rats, compared with younger animals during the growth and development phases. However, these changes seemed gradual between the development and aging phases. Aging is generally perceived as the most drastic morphological change in the cardiac muscle, with more subtle and progressive changes in the growth and development phases. Progressive aging of the heart is caused by the excessive deposition of extracellular matrix (ECM) elements, such as collagen and fibronectin, triggered by the uncontrolled activation of the fibrosis pathway and suppression of anti-fibrosis signals, which can lead to cardiac fibrosis [21]. Hence, aging may cause an excessive accumulation of ECM components in cardiomyocytes, leading to heart failure caused by pathological mechanisms, including diastolic decline and cardiac hypertrophy [22,23]. Our results suggest that aging is the main cause of increased extramyocyte space and myocyte CSA, and the reduced number of cardiomyocytes.

Mitochondrial dysfunction leads to an imbalance of Bax and Bcl-2 levels, which will, in turn, activate caspase-3, a pivotal protein involved in mitochondria-mediated apoptosis [14,24]. Oxidative stress is also known to cause the release of Bax into the cytoplasm, which will promote the mPTP opening sensitivity and the activation of caspase-9 and caspase-3. These signals eventually result in DNA fragmentation and programmed cell death [12,16,25]. We recently found increased mitochondrial hydrogen peroxide production with aging in rat cardiac muscles [5]. Interestingly, the current study revealed that the Bax/Bcl-2 ratio was markedly higher in the cardiac muscles of older rats, indicating that the aging phase was related to early-stage mitochondria-mediated apoptosis. Moreover, progressively increased mPTP opening sensitivity and the cleavage of caspase-3 were seen during the aging process, and they occurred more prominently in the aging phase. We believe that apoptosis is primarily driven by mitochondrial ROS accumulation and mPTP opening. Therefore, it is possible that aging could enhance mitochondria-mediated apoptotic signaling [26,27], which will contribute further towards cardiac muscle apoptosis.

Additionally, we found that the number of TUNEL-positive myonuclei, which is an apoptosis marker, was affected by aging, especially during the development and aging phases. Fannin et al. [28] investigated the mitochondria-mediated apoptotic pathway in aging female F344xBN rats and suggested that aging was associated with increases in the Bax/Bcl-2 ratio, caspase-3 activation, and the number of TUNEL-positive myonuclei. Apoptosis has an important homeostatic role in normal, healthy hearts; however, excessive apoptosis leads to pathological, life-threatening heart dysfunction during the aging process [29]. We did not measure the cardiac function in addition to it being relative to aging; however, apoptosis might have affected cardiac function [30]. Further studies are necessary to better understand the role of mitochondria-mediated apoptosis in aging-induced cardiac dysfunction.

The present study has some limitations. First, we did not include a histochemistry analysis of mitochondrial respiratory chain complexes and mitochondrial ultrastructure examination in heart tissues that can provide important information to prove the relationship between mitochondrial dysfunction and aging. Second, since female hormones (e.g., estrogen) regulate mitochondrial function [31], we used only male rats, including very young (1 months), young (4 months), middle-aged (10 months), and old (20 months) rats as animal models in this study.

#### **5. Conclusions**

This study showed that aging induces cardiac muscle remodeling in rats, promoting increases in extramyocyte space and the CSA and reducing the number of myocytes, which are key features involved in cardiac fibrosis. Additionally, aging induces mitochondria-mediated apoptotic signals (Bax expression, increased Bax/Bcl-2 ratio and mPTP opening sensitivity, and the activation of caspase-3) and apoptosis (TUNEL-positive myonuclei) in cardiac muscles. These results provide strong evidence supporting our hypothesis that aging would cause drastic changes in the myocardial structure, mitochondria-mediated apoptotic signaling, and DNA fragmentation in rat hearts. Nevertheless, morphological and molecular changes in cardiac muscles are substantially less prominent during the growth and development phases of the lifespan of rats.

**Author Contributions:** M.-H.N. and H.-B.K. contributed to the conception and design of the study. M.-H.N., J.-W.H. and E.-J.C. collected the data. M.-H.N., Y.C., J.C., D.-H.P., J.-H.K., C.-J.K., D.Y.S., J.H. and H.-B.K. conducted the critical discussion. M.-H.N., Y.C. and H.-B.K. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF-2016S1A5A8018954).

**Conflicts of Interest:** The authors declare no conflict of interest.
