Effect of exercise training intensity on mitochondrial dynamics and mitophagy in post myocardial infarction rats

: Myocardial infarction (MI) is the most common type of heart disease. According to recent studies, mitochondrial dysfunction has been suggested as a central player in cardiac disease and evidences point out the association of mitochondrial morphology with development of heart diseases. Exercise training plays a protective role against cardiovascular disease. However, the role of exercise training on proteins involved in mitochondrial dynamics and mitophagy system are not well understood. Therefore, the aim of the present study was to investigate these on cardiac mitochondrial dynamic and mitophagy proteins in rats with myocardial infarction. The present study was post-test design experiment with the control group. after MI with ligation of the left anterior descending coronary artery (LAD) and ensuring the creation of MI by echocardiography, male rats were subjected to high intensity interval training (HIIT), moderate (MIIT), low (LIIT), sedentary myocardial infarction (SED-MI) and healthy control groups. After six weeks exercise, the levels of MFN2, DRP1, Parkin, P62 and PGC-1α proteins were measured by ELISA method. Data analysis showed that proteins levels of MFN2, PGC-1α, Parkin and P62 decreased significantly in SED-MI group compared to healthy control while DRP1 protein levels increased significantly (P≤0.05). Also, MFN2 and PGC-1α proteins increased in MIIT group compared with SED-MI group and DRP1 protein levels were significantly decreased (P≤0.05). Moderate-intensity interval training (MIIT) resulted to improve mitochondrial fusion and fusion proteins in rats with myocardial infarction. While high and low intensity interval training (HIIT, LIIT), despite increasing MFN2 and PGC-1α and reducing DRP1, failed to improve fusion and mitochondrial fission .


INTRODUCTION
MMP, Ca2+ overloading, or generation of ROS1. Ca2+ overloading has been shown to induce mitochondrial fragmentation in both neonatal and adult rat cardiomyocytes [1]. The function of mitochondria as cellular power plants is essential to meet the eukaryotic cells energy demands.
Nevertheless, the role of mitochondria is not limited to energy consumption supply, but also includes, for example, calcium storage, intracellular signaling and cell death [2]. Because of its wide range of cellular functions, the mitochondria have a central role in cell homeostasis; consequently, disturbance on mitochondrial function is associated with various disorders. Major chronic diseases such as neurodegenerative disorders, cancer, aging, diabetes, and heart failure are well known to be associated with mitochondrial dysfunction [2]. For instance, in myocardium disease, mitochondrion dysfunction leads to various deleterious consequences, including increased oxidative stress, contractile dysfunction and apoptosis [2].
Mitochondria have a complex functional morphology with, at basis, a compartmented double-membrane system and a usual tubular shape. However, this organelle presents a great morphological variability, which could be associated with different cellular environments in physiological conditions or pathological stress. In this sense, mitochondria may assume different shapes in various cell types, and this morphological change associated with distribution is generally termed mitochondrial dynamics [3].
Mitochondria dynamic has been well studies in exercise training research, as the majority of the literature on mitochondrial dynamics focused in skeletal muscle [4][5][6][7][8][9]. Nevertheless, emerging attention has been directed toward the cardiac muscle and the role of mitochondrial dynamics on cardiovascular diseases. Indeed, cardiomyocytes mitochondria occupy more than one-third of the cell volume, and produces more than 30 kg of ATP daily [3], which is crucial for normal heart function. Interestingly, mitochondrial dysfunction has been suggested as a central player in cardiac disease and evidences point out the association of mitochondrial morphology with development of heart diseases [10]. For instance, Schaper et al. observed that myocardial tissue from patients with dilated cardiomyopathy presented mitochondria in large clusters in cytoplasm free of myofibrils and they fall within a wide range of size from very small to very large, as well as the variance in their shape [11]. Similarly, it was shown that mitochondria in the heart failure (HF) were small and fragmented, in human HF [12]. Moreover, this change in mitochondria morphology was associated with mitochondrial fusion protein, suggesting that mitochondria fusion is decreased in HF, impairing mitochondrial function and resulting in the cell loss and downward progression of the HF [12]. In this line, other studies reported the disturbance on mitochondrial size and number in cardiac hypertrophy [13] and cardiac hypoxia [1].

reactive oxygen species
In addition to the mitochondrial dynamic, a growing body of evidence suggests that mitochondrial autophagy plays an essential role in the regulation of optimal performance of mitochondria population, which is commonly referred as mitochondrial quality control. Autophagy is a mechanism of cell destruction by lysosomes and particularly important for cell survival during energy stress, degradation of misfolded proteins, dysfunctional cytoplasmic organelles, as well as dysfunctional mitochondria, which is known as mitophagy [14]. Therefore, mitophagy is also essential to the maintenance of cellular metabolism and homeostasis. Indeed, it has been shown that the mitophagy is involved in the pathogenesis of cardiovascular disease, diabetes, inflammatory disorders, and cancer [14]. Therefore, it seems that clarifying the mechanisms involved in the regulation of mitochondrial function and fusion process, fission and mitophagy with exercise training intensity can provide a critical understanding about the role of regular exercise on improving of myocardial infarction and could be of high importance in the context of clinical settings. Therefore the purpose of this study was to investigate of exercise training intensity effect on mitochondrial dynamics and mitophagy system in infarcted rats.

Study design and animals
A total of 60 adult male Wistar rats (320g ±20g ~16 weeks of age) were used with accessibility of food and water in ad libitum. To disinfect the surgical area betadine/alcohol wiping was utilized and the left anterior descending coronary artery (LAD) was ligated utilizing a 6-0 prolene (Ethicon Inc., USA) suture. [15]. At the end of operation, to recover the rats they were placed on the heating pad until anesthesia, warm sterile saline (0.5-1 mL, SC), Flunixin (1-2 mg/kg IM) and Cefazolin (25 mg/kg IM) were injected. By utilizing analgesia and hemodynamic monitoring for 24 h postoperative care was resumed [16,17]. to calculate the LV ejection fraction (EF) and fractional shortening (FS) the following formulas were applied [18,19] [%EF = (LVEDD2 -LVESD2)/ (LVEDD2) ×100] In this study the high degree myocardial damage rats which developed MI (FS ≤ 35%) based on echocardiographic result were included [20].

Training protocol
Thirty -five rats which met the inclusion criteria which survived a MI out of sixty MI-operated rats were randomly set to the low intensity training (low intensity training (LIT, n= 6), moderate intensity training (MIT, n = 6), and high intensity training (HIT, n=6), MI-Sedentary (MI-Sed, n = 6)) and Control (CNTRL, n = 6). By walking at a slow pace (5m/min) for 5 min/day, five days per week the MI-Trained groups familiarized with a motorized. Following this step, the rats rested for two days before maximal oxygen uptake (VO2 max). To measure VO2 max each maximal rat's running exercise test was utilized [21]. Control and MI-Sed animals did not received ET while LIT, MIT, and HIT animals were exercised according to the following table [22,23].
Running intensities increased to 0.02 m/s per week by considering previous studies [21,22]. During the exercise training, no treadmill slope were applied [23].

Myocardial mitochondria preparations
Mitochondria were isolated using the tissue around of the infarcted areas. In order to prepare the Myocardial mitochondria for extraction of proteins, the infarcted hearts were cut into pieces. The residual blood was The samples were then re-homogenized at 10.000×g for 10 min to attain the Mitochondria, contained in the final precipitation.
1 Enzyme-linked immunosorbent assay It should be noted that the whole aforementioned process were performed on ice and a BCA™ Protein Assay Kit (sc-202389, Santa Cruz Biotechnology, USA), with bovine serum albumin were employed at room temperature to determine the mitochondrial protein concentration [24].

ELISA1 analysis
Microtiter plates were coated with target protein diluted in 50 mM carbonate buffer at pH 9.0. Optimal concentrations determined by

STATISTICAL RESULTS
Weight changes, Ejection fraction (EF), and Fractional shortening (FS) among research groups at different stages of the study are presented in Table 1.   performance in response to the intensity and duration of exercise and more importantly is that these changes were not limited to a particular form of exercise [27][28][29]. It has been shown that exercise training improves mitochondrial dynamics and quality through enhanced mitophagy system. Evidence showed that mitophagy is regulated by AMPK, therefore, when mitochondria are disrupted, AMPK by increasing receptor p62/sequestosome triggers mitophagy system in two ways: First, by inhibiting mTOR3 through localizing to the lysosome, because mTOR through the phosphorylation inactivates the. If ULK1 is disabled, it will inhibits autophagy and mitophagy happens. Secondly, AMPK causing a progress in mitophagy through direct effect on ULK1.
Exercise may cause mitophagy through AMPK -ULK1 signaling pathways in skeletal muscle. But this hypothesis still is not agreed how   [30] . It has been suggested that the protective effects of exercise training due to improvement of glucose and fat metabolism is associated with increased mitochondrial biogenesis with activation of PGC-1α. Increased apoptosis and autophagy may be a sign of cardiomyocytes reduction that is caused by infarction. Accordingly, another way of protective activity of exercise training against myocardial infarction could be reduced apoptotic activity. Reduction in mitochondrial function and biogenesis after myocardial infarction is the main and dominant cause of heart failure development. It is postulated that PGC-1α could be used as a potential marker for heart recovery capacity after infarction. Finally, exercise with Beta-3 adrenergic receptor stimulation and activation of the signaling pathway of nitric oxide could also help protect against ischemia-reperfusion injury [30].
Generally, despite the lack of ROS measurements, it seems that interval is much more to be learned about the mechanisms responsible for exercise-induced cardioprotection against CAD.

CONFLICT OF INTERESTS STATEMENTS
The authors declare no conflict of interest.

ACKNOWLEDGEMENT
The writers would like to appreciate the faculty of sports sciences, University of Guilan.
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