**1. Introduction**

Aging is commonly characterized by a gradual deterioration of tissue and organ function, and has been identified as the main cause of cardiac dysfunction. Several factors, including mitochondrial dysfunction, genomic instability, defective proteostasis, and epigenetic changes, have been considered important contributors to aging [1,2]. In particular, aging causes significant alterations in the structure and function of the heart, especially in the left ventricle, in addition to increasing cardiac oxidative stress and inflammation [1,3]. Impaired cardiac structure and function with aging result in enhanced susceptibility to cardiovascular diseases (such as heart failure), which can contribute to morbidity and mortality.

Mitochondria are the energy power plants of cells, controlling their fate. They are an important player in various processes and have the ability to maintain metabolic homeostasis and manage aging-related mechanisms [2]. Several studies suggested that mitochondria have evolved to regulate other cellular functions, including those contributing to biological aging, such as the generation of reactive oxygen species (ROS), inflammation, senescence, and resultant necrotic and apoptotic cell death [4]. Our studies have revealed that aging mainly results in damage to the electron transport chain during mitochondrial respiration, due to a decrease in electron transport by complex II-IV, which can promote electron leakage and ROS production [5].

Apoptosis, which is known as programmed cell death, is an essential process for cellular and tissue homeostasis, maintaining cell growth and differentiation, and controlled tissue repair [4,6]. However, excessive apoptosis in the heart induces cardiac dysfunction [7]. In particular, it was reported that reduced heart muscle fiber mass due to apoptosis directly results in the development of myocardial contraction abnormalities, heart failure, and other cardiac conditions [8,9]. The mitochondrion-driven apoptotic pathway, regulated by members of the Bcl-2 family, plays a pivotal regulatory role in aging [10]. An imbalance between the pro-apoptotic proteins Bax and Bid and the anti-apoptotic proteins Bcl-2 and Bcl-xl provokes the opening of the mitochondrial permeability transition pore (mPTP), which, in turn, triggers the translocation of cytochrome c from the mitochondrial intermembrane to the cytoplasm [11]. The released cytochrome c binds to adenosine triphosphate, apoptotic protease-activating factor 1, and pro-caspase 9, which subsequently activates caspase-3, thereby causing DNA fragmentation [12–14]. Data from our group and other studies have shown that aging alters the mitochondrial apoptotic pathway in the skeletal and cardiac muscles of rats, resulting in increased Bax/Bcl-2 ratio, cleaved caspase-3 levels, and DNA fragmentation [14–16]. However, Nitahara et al. [17] reported that aging has no effect on mitochondria-dependent apoptosis, in particular, the expression of Bax and Bcl-2, in the rat heart. Nevertheless, aging induced cardiomyocyte apoptosis. Despite these findings, the effects of aging on the regulation of mitochondria-mediated apoptosis in the rat heart remain unclear.

In the present study, we aimed to determine whether mitochondria-mediated apoptosis on cardiac muscle would induce greater changes by aging. To distinguish the alterations that occur during biological aging (i.e., growth, development, and aging phases), we investigated the changes in the myocardial structure and mitochondria-mediated apoptotic signaling in the cardiac muscles of male Fischer 344 rats during the four stages of life, based on the lifespan characteristics of this rat strain, i.e., very young (VYG; 1 month old), young (YG; 4 months old), middle-aged (MG; 10 months old), and old (OG; 20 months old). We hypothesized that aging would induce more drastic changes in myocardial structure and apoptosis mediated through the mitochondrial pathway—including alterations in the levels of Bcl-2 family proteins, mPTP opening, cleaved caspase-3, and DNA fragmentation—that correspond to the growth and development phases of the rat heart.
