This study aimed to determine the effects of a single bout exercise on mitochondria-mediated apoptotic signaling in cardiac and skeletal muscles. Fischer 344 rats (4 months old) were randomly divided into the control or a single bout of exercise group (n=10 each). The rats performed a single bout of treadmill exercise for 60 min. Mitochondria-mediated apoptotic signaling (e.g., Bax, Bcl-2, mitochondrial permeability transition pore [mPTP] opening, cytochrome c, and cleaved caspase-3) was measured in cardiac (e.g., left ventricle) and skeletal (e.g., soleus and white gastrocnemius) muscles. A single bout of exercise significantly decreased mPTP opening sensitivity in all tissues. However, a single bout of exercise did not show any statistical differences in Bax, Bcl-2, cytochrome c, and cleaved caspase-3 in all tissues measured. A single bout of exercise did not show definite results on characteristics of mitochondria-mediated apoptotic signaling. Therefore, further research is necessary to provide a more mechanistic understanding of the apoptosis pathway.
Mitochondria are the powerhouses responsible for the energy production that occurs in cells called adenosine triphosphate (ATP) synthesis (
Mitochondria can serve as mediators, coordinating a highly programmed cell death referred to as apoptosis (
It is well known that exercise is vital for health and fitness because it improves immune function, cardiovascular system function, and metabolic function (
Four-month-old Fischer 344 rats were randomly divided into 2 groups (n=10 per group): control group (CON) and a single bout of exercise group (EX). Cardiac (left ventricle) and skeletal muscles (e.g., soleus, type I fiber and white gastrocnemius, type IIb fiber) were collected from the respective groups and protein levels of mitochondria-mediated apoptotic signaling (e.g., Bax, Bcl-2, cytochrome c, and cleaved caspase-3) were measured via Western immunoblot analysis.
All procedures for animal experiments adhered to the stipulations of the National Institutes of Health and the guidelines of the Korean Academy of Medical Science. Rats were housed under controlled environmental conditions with constant illumination (12:12-hr light/dark) and room temperature (20°C±2°C). Additionally, food and water were made available to the rats
The treadmill exercise was performed following an adaptation period of about 10 min for a week. The single bout of EX received a single bout of treadmill exercise training at 20 m/min for 1 hr. The incline of this exercise was 0% and the intensity was approximately ~60% of the rats’ maximum oxygen uptake (VO2max), which corresponds with a moderate intensity (
The level of proteins involved in mitochondria-mediated apoptotic signaling was determined. Left ventricle, soleus, and white gastrocnemius tissues were collected and frozen immediately at −80°C. About 30 μg of tissues were homogenized with lysis buffer and centrifuged at 14,000 rpm for 20 min and 10 min, respectively. Protein levels were quantified by bicinchoninic acid assay method and concentration was confirmed by enzyme-linked immunosorbent assay Reader. Protein was denatured at 95°C for 5 min, separated on an sodium dodecyl sulfate-polyacrylamide gel with running buffer at 100 V for 2 hr, and then transferred onto a nitrocellulose membrane with transfer buffer at 170 mA for 1.5 hr. After Ponceau S staining, the membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 2 hr. The membranes were incubated overnight at 4°C with the following primary antibodies: GAPDH, Bax, Bcl-2, cytochrome c (Santa Cruz Biotechnology, Dallas, TX, USA) and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA). The membranes that reacted with the primary antibody were incubated at room temperature for 1 hr with horseradish peroxidase-conjugated anti-mouse (Vector Laboratories, Inc., Burlingame, CA, USA) for GAPDH, Bax, Bcl-2, and cytochrome c and anti-rabbit (Vector Laboratories, Inc.) for cleaved caspase-3. The bands were detected by using the enhanced chemiluminescence detection reagent kit (Thermo Fisher Scientific, Santa Clara, CA, USA) and expressed through Chemidoc (Bio-Rad, Hercules, CA, USA).
Data were presented as mean±standard error of the mean. An independent
In cardiac and skeletal muscles the levels of Bax, a pro-apoptotic protein, were not significantly increased in EX compared with CON (
The mPTP opening sensitivity was significantly decreased in both cardiac and skeletal muscles by a single bout of exercise (
The purpose of this study was to investigate mitochondria-mediated apoptotic signaling responses to a single bout of exercise. We observed that a single bout of exercise significantly decreased mPTP opening sensitivity in all tissues (left ventricle, soleus, and white gastrocnemius). In contrast, a single bout of exercise did not show any statistical differences in Bax, Bcl-2, Bax/Bcl-2 ratio, cytochrome c, and cleaved caspase-3 between groups in both cardiac and skeletal muscles.
As mentioned before, mitochondria play a vital role in governing cellular events (survival and death) because they coordinate a process known as the mitochondria-mediated apoptotic pathway (
Moreover, it was demonstrated that pro-apoptotic proteins increased following strenuous acute exercise in human (
In conclusion, a single bout of exercise did not result in observable changes in Bax, Bcl-2, Bax/Bcl-2 ratio, cytochrome c, and cleaved caspase-3 in cardiac and skeletal muscle. However, a significant decrease in mPTP opening sensitivity in both cardiac and skeletal muscle was observed. Further research is necessary to provide a more mechanistic understanding of mitochondria-mediated apoptosis in the future.
This work was supported by the Inha University Research Grant (2018).
No potential conflict of interest relevant to this article was reported.
Effects of a single bout of exercise on Bax protein level in left ventricle (A), soleus (B), and white gastrocnemius (C). The data are shown as mean±standard error of the mean. CON, control group; EX, exercise group; AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Effects of a single bout of exercise on Bcl-2 protein level in left ventricle (A), soleus (B), and white gastrocnemius (C). The data are shown as mean±standard error of the mean. CON, control group; EX, exercise group; AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Effects of a single bout of exercise on Bax/Bcl-2 ratio in left ventricle (A), soleus (B), and white gastrocnemius (C). The data are shown as mean±standard error of the mean. CON, control group; EX, exercise group.
Effects of a single bout of exercise on mitochondrial permeability transition pore (PTP) opening sensitivity in left ventricle (A), soleus (B), and white gastrocnemius (C). The data are shown as mean±standard error of the mean. CON, control group; EX, exercise group; AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *
Effects of a single bout of exercise on cytochrome c protein level in left ventricle (A), soleus (B), and white gastrocnemius (C). The data are shown as mean± standard error of the mean. CON, control group; EX, exercise group; AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Effects of a single bout of exercise on cleaved caspase-3 protein level in left ventricle (A), soleus (B), and white gastrocnemius (C). The data are shown as mean±standard error of the mean. CON, control group; EX, exercise group; AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.