Exercise Training Enhances Myocardial Mitophagy and Improves Cardiac Function via Irisin/FNDC5-PINK1/Parkin Pathway in MI Mice

Myocardial infarction is the major cause of death in cardiovascular disease. In vitro and in vivo models are used to find the exercise mode which has the most significant effect on myocardial irisin/FNDC5 expression and illuminate the cardioprotective role and mechanisms of exercise-activated myocardial irisin/FNDC5-PINK1/Parkin-mediated mitophagy in myocardial infarction. The results indicated that expression of irisin/FNDC5 in myocardium could be up-regulated by different types of exercise and skeletal muscle electrical stimulation, which then promotes mitophagy and improves cardiac function and the effect of resistance exercise. Resistance exercise can improve cardiac function by activating the irisin/FNDC5-PINK1/Parkin-LC3/P62 pathway, regulating mitophagy and inhibiting oxidative stress. OPA1 may play an important role in the improvement of cardiac function and mitophagy pathway in myocardial infarction mice by irisin-mediated resistance exercise. Resistance exercise is expected to become an effective therapeutic way to promote myocardial infarction rehabilitation.


Introduction
Myocardial infarction (MI) is a major cause of death in cardiovascular diseases [1]. During MI, the level of local myocardial oxidative stress elevated abnormally due to ischemia and hypoxia, resulting in the death of a large number of cardiomyocytes, myocardial fibrosis and eventually heart failure [2]. Exercise can improve cardiac function, rescue necrotic myocardium [3,4] and reduce the incidence of cardiovascular events by regulating myocardial oxygenation and ventricular remodeling after MI [5]. Mitophagy can remove damaged and aging mitochondria and maintain cell homeostasis [6]. Oxidative stress leads to abnormal mitophagy and cardiac dysfunction [7], and its effective therapeutic target and mechanism need to be further studied.
Irisin is a secreted peptide proteolytically processed from transmembrane protein fibronectin type III domain protein 5 (FNDC5) [8]. It was originally found in mice and human serum and can be secreted in skeletal muscle, myocardium, adipose and other tissues [9]. Irisin can reduce heart injury caused by ischemia and hypoxia through regulating mitochondrial function [10]. During myocardial ischemia, hypoxia and abnormal energy metabolism as well as impaired cardiac function, mitophagy could improve cardiac function by removing damaged mitochondria [11]. PTEN-induced kinase 1/ E3 ubiquitin ligase parkin (PINK1/Parkin) is the main pathway of mitophagy, it can induce mitophagy through recruiting microtubule-associated protein light chain 3 (LC3II/I) and SQSTM1/p62 (P62) at the mitochondria [12]. Exercise-activated mitophagy was mediated by the irisin/FNDC5-PINK1/Parkin-LC3/P62 pathway, which could significantly increase the level of myocardial anti-oxidation and improve cardiac function [13]. As the myogenic

Echocardiographic Measurements
To measure cardiac physiological functions, an ultrasound cardiotachograph (VINNO 6 VET, VINNO, Suzhou China) was used for the detection and analysis of echocardiography. After the exercise, the mice are anesthetized with isoflurane and fixed on the operating table after skin preparation. B-mode ultrasonography was applied in seeking the long axis of left ventricular, then 2-dimensional M-mode was transferred for data

Echocardiographic Measurements
To measure cardiac physiological functions, an ultrasound cardiotachograph (VINNO 6 VET, VINNO, Suzhou China) was used for the detection and analysis of echocardiography. After the exercise, the mice are anesthetized with isoflurane and fixed on the operating table after skin preparation. B-mode ultrasonography was applied in seeking the long axis of left ventricular, then 2-dimensional M-mode was transferred for data acquisition. Left ventricle internal dimension diastole (LVIDd), left ventricle internal dimension systole (LVIDs) and ejection fraction (EF) were directly measured by using the M-mode analysis. Fractional shortening (FS) was calculated by LVIDd and LVIDs (calculation formula: FS = (LVIDd − LVIDs)/LVIDd × 100%).
After 4 weeks of exercise training, the mice were anesthetized with isoflurane and fixed. Their heart was quickly collected with their chest opened in a low temperature environment and placed in liquid nitrogen for subsequent experiments. Heart tissue cryopreserved in liquid nitrogen is used for molecular testing.

Blood Collection and Biochemical Index Measurement
After the mice were anesthetized with isoflurane, blood sample were collected from the orbit and stored in a 37 • C water pot for 4 h, and then centrifuged at 1000 rpm for 1 min. After centrifugation, the upper serum was transferred to a new EP tube and stored at −20 • C for subsequent experiments. All steps were strictly performed in accordance with the kit instructions.
By using the assay kits (Beyotime, Shanghai, China), we detected the renal dysfunction biomarkers including serum Total Superoxide dismutase (T-SOD), Malondialdehyde (MDA). All steps were carried out strictly according to kit instructions.

Extraction and Detection of Mitochondrial Protein
The tissue mitochondrial protein extraction kit (BB-31711, BestBio, Shanghai, China) was used to extract mitochondrial protein from mice myocardium. The protein concentration was determined by the bicinchoninic acid protein quantitative method. The protein was denatured at 100 • C, then stored at −80 • C for follow-up Western Blotting experiment. All steps were carried out strictly according to kit instructions.

RT-qPCR
Total RNA was extracted from frozen heart tissues (100 mg) and H9C2 cells with Trizol reagent (Invitrogen, Sao Paulo, Brazil). RNA concentration was determined spectrophotometrically at 260/280 nm wavelength using a commercial kit (BioTek Epoch, Winooski, VT, USA). First-strand cDNA was generated from RNA using Revertaid First Strand cDNA synthesis kit (TaKaRa, Kusatsu, Japan). PCR reactions were performed with quantitative PCR instrument (CFX connectTM Real-Time system, Bio-Rad, Singapore) using SYBR Green/ROX qPCR Master Mix (TaKaRa, Kusatsu, Japan) and quantified using Bio-Rad CFX manager. GAPDH was used as the internal control gene. The primer sequences used for RT-qPCR analyses are shown as follows:

Western Blotting
Total protein was extracted from the heart tissues collected from the peri-infarcted area as well as H9C2 cells harvested at the end of the vitro experiments using radioimmunoprecipitation assay (RIPA) lysate buffer (Roche, CA, USA). The protein concentration was determined by the bicinchoninic acid protein quantitative method. The tissue/cell lysate samples were separated by 8-12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) under 100 constant voltage for 1.5 h with electro-transferred (300 mA for 1.5 h at 4 • C) to nitrocellulose membranes (Millipore, MA, USA). The membranes were incubated with 3% BSA for 60 min at room temperature followed by incubation with one of the following primary antibodies overnight at 4 • C using the following dilution concentrations and commercial suppliers: and β-tubulin (1:5000) were used as internal control for equal sample loading at 4 • C overnight. On the second day, the membranes were incubated with the HRP-conjugated secondary antibody at room temperature for 2 h. After the membranes were washed with TBST for three times, ECL liquid (Bio-Rad, CA, USA) was added to the membranes for label observation. Finally, protein bands were detected by Image Systems (Bio-Rad, CA, USA).

Statistical Analysis
Statistical analysis was conducted using GraphPad Prism 7.01 analysis software. Three independent experiments were made. Data were expressed as Mean ± Standard Error (SEM). One-way ANOVA was used for evaluating the significant differences of the mean values. ROUT test is used to detect outliers (outliers were not found). Histograms were post hoc test. Differences were considered statistically significant at * p < 0.05, ** p < 0.01.

Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention Enhanced Myocardial Irisin/FNDC5 Expression and Cardiac Function
In non-MI mice, different types of exercise and skeletal muscle electrical stimulation intervention significantly up-regulated myocardial Irisin/FNDC5 mRNA and its protein expression (p < 0.01), There is no striking difference between groups, while the effect of resistance exercise group is more significant (Figure 2D-F). Left ventricular internal diameter at the end systole (LVIDs), left ventricular internal diameter at end diastole (LVIDd), ejection fraction (EF), and left ventricular fractional shortening (FS) are commonly used to reflect cardiac systolic function [22]. Echocardiography results showed different types of exercise and skeletal muscle electrical stimulation markedly decreased myocardial LVIDd and LVIDs, increased EF and FS (p < 0.01, Figure 2A-C). These results showed that different types of exercise and skeletal muscle electrical stimulation significantly upregulated myocardial irisin/FNDC5 expression, increased cardiac function. There is no striking difference between all groups, the effect of the resistance exercise group is more significant.

Statistical Analysis
Statistical analysis was conducted using GraphPad Prism 7.01 analysis software. Three independent experiments were made. Data were expressed as Mean ± Standard Error (SEM). One-way ANOVA was used for evaluating the significant differences of the mean values. ROUT test is used to detect outliers (outliers were not found). Histograms were post hoc test. Differences were considered statistically significant at * p < 0.05, ** p < 0.01.

Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention Enhanced Myocardial Irisin/FNDC5 Expression and Cardiac Function
In non-MI mice, different types of exercise and skeletal muscle electrical stimulation intervention significantly up-regulated myocardial Irisin/FNDC5 mRNA and its protein expression (p < 0.01), There is no striking difference between groups, while the effect of resistance exercise group is more significant (Figure 2D-F). Left ventricular internal diameter at the end systole (LVIDs), left ventricular internal diameter at end diastole (LVIDd), ejection fraction (EF), and left ventricular fractional shortening (FS) are commonly used to reflect cardiac systolic function [22]. Echocardiography results showed different types of exercise and skeletal muscle electrical stimulation markedly decreased myocardial LVIDd and LVIDs, increased EF and FS (p < 0.01, Figure 2A-C). These results showed that different types of exercise and skeletal muscle electrical stimulation significantly up-regulated myocardial irisin/FNDC5 expression, increased cardiac function. There is no striking difference between all groups, the effect of the resistance exercise group is more significant.

Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention Activated the Myocardial PINK1/Parkin Pathway and Enhanced Antioxidant Function
PINK1, Parkin, LC3 and P62 are the key proteins in mitophagy [23]. When mitochondrial damage occurs, phosphorylated PINK1/Parkin recruits P62 and binds to LC3 to maintain its function [12]. Western blotting results indicated that different types of exercise and skeletal muscle electrical stimulation intervention increased the phosphorylation level of AMPK (p < 0.01, Figure 3A,B), SOD2 expression in mice myocardium (p < 0.01, Figure 3A,C). Mitophagy related factors PINK1 and Parkin protein expressions were also significantly increased, and the effect of resistance exercise was more significant (p < 0.01, Figure 3A,D). In addition, different types of exercise and skeletal muscle electrical stimulation enhanced mice myocardial LC3II/I ratio (p < 0.01) and reduced P62 protein expression (p < 0.01, Figure 3A,E).

Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention Activated the Myocardial PINK1/Parkin Pathway and Enhanced Antioxidant Function
PINK1, Parkin, LC3 and P62 are the key proteins in mitophagy [23]. When mitochondrial damage occurs, phosphorylated PINK1/Parkin recruits P62 and binds to LC3 to maintain its function [12]. Western blotting results indicated that different types of exercise and skeletal muscle electrical stimulation intervention increased the phosphorylation level of AMPK (p < 0.01, Figure 3A,B), SOD2 expression in mice myocardium (p < 0.01, Figure 3A,C). Mitophagy related factors PINK1 and Parkin protein expressions were also significantly increased, and the effect of resistance exercise was more significant (p < 0.01, Figure 3A,D). In addition, different types of exercise and skeletal muscle electrical stimulation enhanced mice myocardial LC3II/I ratio (p < 0.01) and reduced P62 protein expression (p < 0.01, Figure 3A,E).

Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention Activated the Mitochondrial PINK1/Parkin Pathway and Enhanced Antioxidant Function
In order to reduce the other effective factors (the difference of membrane integrity or/and the openness of mitochondrial permeability transition pore) on mitophagy, external stimulation or other abnormal states can impair mitochondrial membrane integrity and lead to the abnormal opening of mitochondrial permeability transition pores, as well as leakage of mitochondrial cytokines (such as PINK1/Parkin, etc.) into the cytoplasm [24]. In order to reduce the influence of these factors on the level of mitophagy, mice myocardial mitochondrial protein was further extracted to verify the above results. Western blotting results showed the same trend of mitophagy-related proteins in PINK1/Parkin pathway and SOD2 expression both in the mitochondrial and myocardium (p < 0.01, Figure 4A-D). Among them, resistance exercise has the best effect.

Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention Activated the Mitochondrial PINK1/Parkin Pathway and Enhanced Antioxidant Function
In order to reduce the other effective factors (the difference of membrane integrity or/and the openness of mitochondrial permeability transition pore) on mitophagy, external stimulation or other abnormal states can impair mitochondrial membrane integrity and lead to the abnormal opening of mitochondrial permeability transition pores, as well as leakage of mitochondrial cytokines (such as PINK1/Parkin, etc.) into the cytoplasm [24]. In order to reduce the influence of these factors on the level of mitophagy, mice myocardial mitochondrial protein was further extracted to verify the above results. Western blotting results showed the same trend of mitophagy-related proteins in PINK1/Parkin pathway and SOD2 expression both in the mitochondrial and myocardium (p < 0.01, Figure  4A-D). Among them, resistance exercise has the best effect.

Effect of Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention on Myocardium L-OPA1/S-OPA1 Ratio and Its Correlation with Irisin/FNDC5
OPA1 mediates mitochondrial inner membrane fusion. There exist two forms: L-OPA1 and S-OPA1. L-OPA1 mediates mitochondrial fusion, while S-OPA1 inhibits mitochondrial fusion. The ratio of L-OPA1/S-OPA1 was often used to reflect the level of mitochondrial fusion. Western blotting results showed that mice myocardial L-OPA1/S-OPA1 ratio in resistance exercise group was significantly increased compared with the control group (p < 0.01, Figure 5A), Pearson correlation analysis was used to detect the correlation between OPA1 ratio and FNDC5 protein expression ( Figure 2F). The results showed that myocardial L-OPA1/S-OPA1 ratio had clear positive correlation with irisin/FNDC5 protein expression in the resistance exercise (r = 0.9053, p < 0.01, Figure 5B), and the other three intervention methods had no significant effect (r = 0.0896, p > 0.05; r = 0.4081, p > 0.05; r = 0.0288, p > 0.05).

Effect of Different Types of Exercise and Skeletal Muscle Electrical Stimulation Intervention on Myocardium L-OPA1/S-OPA1 Ratio and Its Correlation with Irisin/FNDC5
OPA1 mediates mitochondrial inner membrane fusion. There exist two forms: L-OPA1 and S-OPA1. L-OPA1 mediates mitochondrial fusion, while S-OPA1 inhibits mitochondrial fusion. The ratio of L-OPA1/S-OPA1 was often used to reflect the level of mitochondrial fusion. Western blotting results showed that mice myocardial L-OPA1/S-OPA1 ratio in resistance exercise group was significantly increased compared with the control group (p < 0.01, Figure 5A), Pearson correlation analysis was used to detect the correlation between OPA1 ratio and FNDC5 protein expression ( Figure 2F). The results showed that myocardial L-OPA1/S-OPA1 ratio had clear positive correlation with irisin/FNDC5 protein expression in the resistance exercise (r = 0.9053, p < 0.01, Figure 5B), and the other three intervention methods had no significant effect (r = 0.0896, p > 0.05; r = 0.4081, p > 0.05; r = 0.0288, p > 0.05).

Resistance Exercise Enhanced MI Myocardial Irisin/FNDC5 Expression, Promoted Mitophagy, Enhanced Antioxidative Capability and Alleviated the Levels of Oxidative Stress
Western blotting results showed that Irisin/FNDC5 mRNA and its protein expression was significantly decreased, PINK1/Parkin expressions and LC3II/I ratio significantly decreased in MI group (p < 0.01), while P62 expression clearly increased (p < 0.01) compared with the sham group. Resistance exercise training reverse these results (p < 0.01, Figure  7A-E). The irisin/FNDC5 expression had obvious positive correlation with EF (r = 5929, p < 0.05). It is found that the lower OPA1 level caused by MI can be reversed by resistance exercise training (p < 0.01, Figure 7A,F). The L-OPA1/S-OPA1 ratio had obvious positive correlation with irisin/FNDC5 expression (r = 0.8638, p < 0.01, Figure 7G).
Total superoxide dismutase (T-SOD) and malondialdehyde (MDA) are used to assess oxidative stress level [25]. The results of this study showed that MDA content induced by MI significantly enhanced, T-SOD and the protein expression of SOD2 were significantly reduced (p < 0.01). Resistance exercise training reversed these results. (p < 0.01, Figure  7A,H-J).

Resistance Exercise Enhanced MI Myocardial Irisin/FNDC5 Expression, Promoted Mitophagy, Enhanced Antioxidative Capability and Alleviated the Levels of Oxidative Stress
Western blotting results showed that Irisin/FNDC5 mRNA and its protein expression was significantly decreased, PINK1/Parkin expressions and LC3II/I ratio significantly decreased in MI group (p < 0.01), while P62 expression clearly increased (p < 0.01) compared with the sham group. Resistance exercise training reverse these results (p < 0.01, Figure 7A-E). The irisin/FNDC5 expression had obvious positive correlation with EF (r = 5929, p < 0.05). It is found that the lower OPA1 level caused by MI can be reversed by resistance exercise training (p < 0.01, Figure 7A,F). The L-OPA1/S-OPA1 ratio had obvious positive correlation with irisin/FNDC5 expression (r = 0.8638, p < 0.01, Figure 7G).
Total superoxide dismutase (T-SOD) and malondialdehyde (MDA) are used to assess oxidative stress level [25]. The results of this study showed that MDA content induced by MI significantly enhanced, T-SOD and the protein expression of SOD2 were significantly reduced (p < 0.01). Resistance exercise training reversed these results. (p < 0.01, Figure 7A,H-J).

Resistance Exercise Improved MI Mice Cardiac Function
Echocardiography results showed myocardial LVIDd and LVIDs increased (p < 0.01), EF and FS decreased (p < 0.01) in MI group. After resistance exercise intervention, LVIDd and LVIDs decreased (p < 0.01, Figure 8A,B), EF and FS increased (p < 0.01, Figure 8A,C). These results indicated that resistance exercise training improved MI cardiac function.

Resistance Exercise Improved MI Mice Cardiac Function
Echocardiography results showed myocardial LVIDd and LVIDs increased (p < 0.01), EF and FS decreased (p < 0.01) in MI group. After resistance exercise intervention, LVIDd and LVIDs decreased (p < 0.01, Figure 8A,B), EF and FS increased (p < 0.01, Figure 8A,C). These results indicated that resistance exercise training improved MI cardiac function.

Discussion
Exercise involves protecting impaired cardiac function caused by MI, but the exact mechanism is not clear yet. The present study showed that different types of exercise and skeletal muscle electrical stimulation intervention enhanced irisin/FNDC5 expression, activated PINK1/Parkin pathway mitophagy, enhanced antioxidant function and improved cardiac function, and the effect of resistance exercise is more significant. The mice myocardial OPA1 expression in resistance exercise group significantly increased, which had clear positive correlation with irisin/FNDC5 expression, while the other three intervention methods had no significant effect. In addition, resistance exercise also significantly increased MI myocardial irisin/FNDC5 and OPA1 expression, enhanced PINK1/Parkin pathway mitophagy, alleviated oxidative stress and improved MI cardiac function. These results suggested that resistance exercise inhibited oxidative stress and improved cardiac function in MI, partially via activating FNDC5/Irisin-PINK1/Pakin-LC3II/I-P62 signaling pathway. OPA1 may play a major role in this process.
Irisin is a novel myokine and adipokine in various body tissues, including skeletal muscle, myocardium and adipose tissue, which can be effectively induced by several kinds of stimulation such as exercise training [9,14]. Some research showed that many different types of exercise can significantly increase the myocardial Irisin expression [26][27][28][29]. This study found that the expression of myocardium irisin/FNDC5 was significantly up-regulated in different exercise intervention and skeletal muscle electrical stimulation models, among which the resistance exercise had the significant effects. Previous studies demonstrated that irisin/FNDC5 significantly inhibited cardiomyopathic hypertrophy, reduced cell apoptosis and alleviated myocardial fibrosis, and ultimately improved cardiac function in MI model [30][31][32]. Our present study confirmed that MI significantly decreased the irisin/FNDC5 expression in the myocardium. Resistance exercise training significantly up-regulated irisin/FNDC5 expression, improved the cardiac function in mice. Previous studies indicated that resistance exercise training stimulated the secretion of skeletal muscle-associated proteins and significantly promoted the skeletal muscle growth [33]. Skeletal muscle-derived irisin can be transported to the lungs via circulation,

Discussion
Exercise involves protecting impaired cardiac function caused by MI, but the exact mechanism is not clear yet. The present study showed that different types of exercise and skeletal muscle electrical stimulation intervention enhanced irisin/FNDC5 expression, activated PINK1/Parkin pathway mitophagy, enhanced antioxidant function and improved cardiac function, and the effect of resistance exercise is more significant. The mice myocardial OPA1 expression in resistance exercise group significantly increased, which had clear positive correlation with irisin/FNDC5 expression, while the other three intervention methods had no significant effect. In addition, resistance exercise also significantly increased MI myocardial irisin/FNDC5 and OPA1 expression, enhanced PINK1/Parkin pathway mitophagy, alleviated oxidative stress and improved MI cardiac function. These results suggested that resistance exercise inhibited oxidative stress and improved cardiac function in MI, partially via activating FNDC5/Irisin-PINK1/Pakin-LC3II/I-P62 signaling pathway. OPA1 may play a major role in this process.
Irisin is a novel myokine and adipokine in various body tissues, including skeletal muscle, myocardium and adipose tissue, which can be effectively induced by several kinds of stimulation such as exercise training [9,14]. Some research showed that many different types of exercise can significantly increase the myocardial Irisin expression [26][27][28][29]. This study found that the expression of myocardium irisin/FNDC5 was significantly up-regulated in different exercise intervention and skeletal muscle electrical stimulation models, among which the resistance exercise had the significant effects. Previous studies demonstrated that irisin/FNDC5 significantly inhibited cardiomyopathic hypertrophy, reduced cell apoptosis and alleviated myocardial fibrosis, and ultimately improved cardiac function in MI model [30][31][32]. Our present study confirmed that MI significantly decreased the irisin/FNDC5 expression in the myocardium. Resistance exercise training significantly up-regulated irisin/FNDC5 expression, improved the cardiac function in mice. Previous studies indicated that resistance exercise training stimulated the secretion of skeletal muscle-associated proteins and significantly promoted the skeletal muscle growth [33]. Skeletal muscle-derived irisin can be transported to the lungs via circulation, which plays an important role in the improvement of lung injury caused by ischemia/reperfusion [34]. Therefore, it can be assumed that resistance exercise training might improve cardiac function in MI mice due to higher secretion of Irisin.
Autophagy is an important mechanism to maintain cell homeostasis, which can be divided into selective and non-selective pathway [35]. Mitophagy is a kind of selective autophagy which plays an indispensable role in maintaining cellular homeostasis under several conditions, and the PINK1/Parkin pathway is its main route [36]. When mitochondria function is impaired, PINK1 increased in outer mitochondrial membrane and combined with the transferring enzyme to promote itself phosphorylation, then recruited the activated cytoplasmic Parkin, and further recruited P62 to combine with LC3, prompted the damaged mitochondria to be degraded, eventually completed mitophagy [37]. PINK1 and Parkin expression could significantly decrease in the Irisin/FNDC5 knockout mice and accompanied with mitochondrial dysfunction in myocardium [38,39], which suggested that irisin/FNDC5 was positively correlated with PINK1 and Parkin expression. Our present study provided direct evidence for exercise induced up-regulated expression of Irisin and enhanced PINK1/Parkin-mediated mitophagy in the myocardium, which was consistent with the results of previous studies [40][41][42]. Our study also presented that resistance exercise group had better effect in different types of exercise and skeletal muscle electrical stimulation intervention. In addition, we further tested myocardial mitochondrial protein to verify the above results, which can reduce the other effective factors on mitophagy. The same trend of mitophagy-related proteins in PINK1/Parkin pathway expression was found both in the mitochondrial and in the myocardium, while the resistance exercise had better effect.
The previous studies demonstrated that MI led to SOD inactivity, increased MDA and reactive oxygen species (ROS) accumulation, and broke the balance of oxidation and antioxidant [43][44][45]. Exercise intervention can significantly up-regulate the expression of Irisin and SOD activity in the heart, liver, kidney and other organs of mice under the circumstances of ischemia and hypoxia, while reduce the level of MDA and ROS [46][47][48]. Irisin contributed to the maintaining oxidative balance in organs and tissues [49]. It has been reported that both swimming and treadmill protocol enhanced the Irisin expression in MI rat's serum [50,51]. Similarly, previous studies in our laboratory confirmed that aerobic exercise can significantly increase the Irisin expression in the kidney of MI mice and simultaneously inhibit the oxidative stress [48]. Our current results showed that for MI mice, the resistance exercise significantly enhanced the expression of Irisin, increased the activity of T-SOD and SOD2 expression, decreased the content of serum MDA, and improved the level of oxidative stress in myocardium.
Recent research showed that OPA1 played a key role in enhancing myocardial mitophagy and improving cardiac function [40,41]. Different forms of exercise intervention could activate the OPA1 expression in the myocardium [9,52]. Meanwhile, Irisin activated mitophagy in MI cardiomyocytes induced by OPA1 [38,53,54]. Taken together, we focused on the role of OPA1 and Irisin in resistance exercise-induced cardio protection in the MI myocardium. This study compared the expression of OPA1 in myocardium after different kinds of exercise and skeletal muscle electrical stimulation intervention, and it was shown that resistance exercise can significantly up-regulate the expression of OPA1 in myocardium, which was correlated with the expression of Irisin positively. AICAR intervention to simulate exercise also significantly increased the expression of OPA1 in H9C2 cells. Furthermore, the results suggested the OPA1 expression trend was consistent with irisin/FNDC5 both in vitro and in vivo experiments. According to the above results, there possibly exists a speculated regulatory relationship among Irisin, OPA1 and mitophagy, which is affected by the forms of exercise intervention. Several studies have shown that exercise significantly increases AMPK phosphorylation, but the level of improvement may depend on exercise load [55]. The expression of OPA1 significantly increased in long-term exercise with moderate to high intensity, whereas exercise with acute or low intensity had no significant effect [52,54,56]. Therefore, we speculate that OPA1 expression may be affected by the phosphorylation of AMPK, but the potential mechanism remains to be further explored.
AMPK is a key energy sensor that regulates cell metabolism to maintain energy balance, and is closely related to mitophagy [57,58]. Inhibition of AMPK expression can lead to abnormal mitophagy, aggravate oxidative stress and apoptosis levels in the myocardium [59,60]. oleuropein, tilianin, metformin and AICAR have been shown to exert cardioprotective effects as AMPK agonists [61][62][63][64][65]. For example, oleuropein has a strong anti-inflammatory and antioxidant capacity, it can reduce myocardial infarction area and protect cardiac function by upregulating the expression of myocardial AMPK [61,62]. AICAR proved to play a cardioprotective role as an AMPK agonist in a variety of disease models (e.g., hypertensive [65], acute kidney injury [66], cardiac hypoxia [67]). The previous research also used AICAR as AMPK agonist to explore the protective effect of exercise on ischemic myocardium [44,48]. This study showed that AICAR could significantly up-regulate the expression of AMPK in H9C2 cells, activate the PINK1/Parkin-LC3/P62 signaling pathway, and enhance mitophagy.
This present study has several limitations: (1) Due to the wide variety of exercise parameters such as exercise modes, exercise intensity, exercise duration, etc., this experiment has not been fully involved. The corresponding intervention forms are based on the preliminary research of this laboratory and literature reports. (2) It has been well proved that exercise can activate the PINK1/Parkin-LC3/P62 pathway and regulate mitophagy level. This study lacks of endogenous intervention (irisin knockin or knockout) to further explore the irisin-mediated PINK1/Parkin-LC3/P62 mitophagy signaling pathway activated by exercise. (3) mPTP transition is an end-target of cardioprotection [68]. However, due to the difficulty of modeling, long cycle and small number of experimental samples in this study, the detection of mitochondrial function and mPTP transition was missing in this study. (4) Currently, many kinds of experimental methods can be used to detect mitophagy [69], this study lacks in verifying the changes of mitophagy from other perspectives.

Conclusions
Different types of exercise and skeletal muscle electrical stimulation can increase the expression of myocardial irisin/FNDC5, promote mitophagy and improve cardiac function in the normal mice, and resistance exercise has the most significant effects. Resistance exercise significantly inhibits oxidative stress, regulates mitophagy and improves cardiac function in MI mice via activating myocardial irisin/FNDC5-PINK1/Parkin-LC3/P62 pathway, OPA1 may plays an important role in this series of protective effects (Figure 9). Biomedicines 2021, 9, x FOR PEER REVIEW 13 of 17 be affected by the phosphorylation of AMPK, but the potential mechanism remains to be further explored. AMPK is a key energy sensor that regulates cell metabolism to maintain energy balance, and is closely related to mitophagy [57,58]. Inhibition of AMPK expression can lead to abnormal mitophagy, aggravate oxidative stress and apoptosis levels in the myocardium [59,60]. oleuropein, tilianin, metformin and AICAR have been shown to exert cardioprotective effects as AMPK agonists [61][62][63][64][65]. For example, oleuropein has a strong antiinflammatory and antioxidant capacity, it can reduce myocardial infarction area and protect cardiac function by upregulating the expression of myocardial AMPK [61,62]. AICAR proved to play a cardioprotective role as an AMPK agonist in a variety of disease models (e.g., hypertensive [65], acute kidney injury [66], cardiac hypoxia [67]). The previous research also used AICAR as AMPK agonist to explore the protective effect of exercise on ischemic myocardium [44,48]. This study showed that AICAR could significantly up-regulate the expression of AMPK in H9C2 cells, activate the PINK1/Parkin-LC3/P62 signaling pathway, and enhance mitophagy.
This present study has several limitations: (1) Due to the wide variety of exercise parameters such as exercise modes, exercise intensity, exercise duration, etc., this experiment has not been fully involved. The corresponding intervention forms are based on the preliminary research of this laboratory and literature reports. (2) It has been well proved that exercise can activate the PINK1/Parkin-LC3/P62 pathway and regulate mitophagy level. This study lacks of endogenous intervention (irisin knockin or knockout) to further explore the irisin-mediated PINK1/Parkin-LC3/P62 mitophagy signaling pathway activated by exercise. (3) mPTP transition is an end-target of cardioprotection [68]. However, due to the difficulty of modeling, long cycle and small number of experimental samples in this study, the detection of mitochondrial function and mPTP transition was missing in this study. (4) Currently, many kinds of experimental methods can be used to detect mitophagy [69], this study lacks in verifying the changes of mitophagy from other perspectives.

Conclusions
Different types of exercise and skeletal muscle electrical stimulation can increase the expression of myocardial irisin/FNDC5, promote mitophagy and improve cardiac function in the normal mice, and resistance exercise has the most significant effects. Resistance exercise significantly inhibits oxidative stress, regulates mitophagy and improves cardiac function in MI mice via activating myocardial irisin/FNDC5-PINK1/Parkin-LC3/P62 pathway, OPA1 may plays an important role in this series of protective effects ( Figure 9).   Data Availability Statement: Data sharing not applicable. The data are not publicly available due to privacy restriction.

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

Abbreviations
The following abbreviations are used in this manuscript: