We obtained eight-week-old male C57BL/6J mice from the Laboratory Animal Center at Xiangya Medical School in Changsha, China. The mice were kept in standard conditions with a temperature of 22 ± 2°C, humidity levels of 45–55%, and a regular 12-hour light-dark cycle. The padding was renewed on alternate days. Following a one-week feeding adaptation period, the mice were divided into three groups receiving different diets (n=10 per group) based on the different interventions they were assigned to receive. These interventions included a normal diet, a high-fat diet (HFD), and an HFD plus AE preconditioning. The normal diet group was fed standard chow consisting of 15% fat, 20% protein, and 65% carbohydrates. Mice in the HFD groups were fed an HFD containing 45% fat, 20% protein, and 35% carbohydrates (FBSH BIO-PHARMACEUTICAL, Shanghai, China) for eight weeks. Mice had free access to food and water. Mice subjected to AE preconditioning followed by our AE training protocol described in the section Aerobic Exercise Training Protocol below. The experimental design is illustrated in Figure 1. All protocols were approved by the Experimental Animal Welfare Ethics Committee of Central South University (approval number: CSU-2022-0282) and were in line with the Medicine Institute of Health’s guidelines for the use of live animals. All animal experiments complied with the ARRIVE guidelines.

The mouse AE training protocol followed the current guidelines for mice exercise and training (25) and has been reported in our previous study (12). Briefly, AE was conducted for 60 min per day, five days per week for eight weeks. Acclimatization training was also applied: one week before formal AE, the mice started with 10 min of swimming training on the first day, followed by daily increases in 10 min increments until they reached 60 min per day. After acclimatization, mice at nine weeks of age were forced to swim in a Morris water maze for 60 min per day with a wooden rod tied to its tail. All of the exercise training sessions were scheduled between 9 a.m. and 2 p.m.

Exhaustive exercise (EE) has been shown to induce fatal stress in mice. We previously successfully used this method in both non-obese (12) and obese mice (20). Briefly, after the 8-week intervention, 5 mice were randomly selected from each group to perform exhaustive exercise and forced weight-loaded swimming until exhaustion. The criteria for exhaustion were defined as sinking to the bottom of the Morris water maze and failing to hedge to the water surface to breathe for seven seconds. To force the mice to swim, a lead sheath load (five percent of each mouse’s weight) was bound to their tails. A session of adaptive weight-loaded swimming with an added load on the tail was provided to the mice before the day of the exhaustive exercise.

The maximal swimming time was noted as an indicator of exercise capacity (n=15, five per group) (11, 12). The echocardiography of the mice was performed prior to their sacrifice. M-mode echocardiography (Mindray Inc., Nanjing, China) under anesthetization was used to obtain the left ventricular ejection fraction (LVEF) and left ventricular internal dimensions. An experienced investigator, who was not aware of the treatment allocation, conducted all of the analyses. The formula V = [7.0/(2.4+D)]×D³ was utilized for calculating the left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV). Subsequently, the LVEF was calculated using the formula: LVEF (%) = (LVEDV-LVESV)/LVEDV×100%.

Body weight was measured and recorded weekly. Visceral fat weight, visceral fat mass/body weight ratio, and fasting blood glucose (FBG) levels were assessed at the end of the intervention in all mice (n=30, ten per group). We isolated and weighted visceral adipose tissues, consisting of perirenal, mesenteric, and unilateral epididymal adipose (n=30, ten per group). The visceral adipose was then fixed and embedded in paraffin for hematoxylin and eosin staining (n=15, five per group). The visceral adipocyte area was calculated by ImageJ as described previously (26). For FBG, blood was collected from the tail vein after 5 h of fasting and assayed for glucose content by Accu-Chek glucose meter (Roche Diagnostics, USA) (n=30, ten per group). Mice had free access to water during the fasting period.

Serum insulin levels were evaluated at the end of the intervention in mice without a fatal stress stimulus (n=15, five per group). Serum was obtained by removing the eyeball after euthanasia via pentobarbital sodium injection (150 mg/kg body weight). We used an enzyme-linked immunosorbent assay (ELISA) to measure insulin concentration (CSB-E05071m; Wuhan, China). Insulin resistance was estimated via the homeostasis model assessment for insulin resistance (HOMA-IR): HOMA-IR = FBG (mmol/L) × fasting insulin (mU/L)/22.5.

To verify fatal stress-induced myocardial injury, serum creatine kinase (CK) levels and myocardial ultrastructure were evaluated and compared between mice with (n=5 per group) and without (n=5 per group) fatal stress stimulus. The difference in response to fatal stress between normal-diet and HFD mice was also assessed. We used a commercial kit to measure serum CK levels (A032; Nanjing Jiancheng, China). The ultrastructure of the left ventricular myocardium was assessed using transmission electron microscopy (12).

To assess the degree of myocardial damage, we examined the activation of the apoptosis pathway using western blotting and by measuring the apoptotic rate using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) in left ventricular myocardium (n=5 for each group). TUNEL assays were performed using a commercial kit (40306ES50, Yeasen, Shanghai, China) following the manufacturer’s instructions. Apoptosis in the myocardium was observed under a fluorescence microscope (BA410T, Motic) at 400x magnification, and myocardial cells with green fluorescence staining were considered TUNEL-positive. TUNEL-positive cells were counted to assess the proportion of apoptotic cells using image analysis software (ImageJ, version 1.49, USA).

The expression of biomarkers of mitophagy, mitochondrial biogenesis, and mitochondrial fission and fusion (the components of MQC), as well as MIF and AMPK proteins, were determined using western blotting. Left ventricular myocardium protein extracts were obtained upon homogenization of tissues in RIPA lysis buffer (89901, Thermo Scientific, USA). After centrifuging the homogenates, the supernatants were analyzed as previously described (20). Polyvinylidene fluoride membranes with transferred proteins were blocked with 5% bovine serum albumin in phosphate-buffered saline plus 0.1% Triton X-100 (PBST) buffer before incubating with primary antibodies overnight at 4°C. The primary antibodies used were MIF (20415-1-AP, Proteintech, USA), AMPK (66536-1-Ig, Proteintech, USA), phosphorylated AMPK (pAMPK, ab23875, Abcam, UK), PTEN-induced kinase 1 (PINK1, 23274-1-AP, Proteintech, USA), microtubule-associated protein 1 light chain 3 (LC3, 14600-1-AP, Proteintech, USA), p62 (18420-1-AP, Proteintech, USA), mitofusin 1 (MFN1, 13798-1-AP, Proteintech, USA), DRP1 (12957-1-AP, Proteintech, USA), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α, 66369-1-Ig, Proteintech, USA), Sirtuin 1 (SIRT1, 13161-1-AP, Proteintech, USA), citrate synthase (CS, 16131-1-AP, Proteintech, USA), GAPDH (10494-1-AP, Proteintech, USA), cytochrome c (10993-1-AP, Proteintech, USA), B-cell lymphoma 2 (BCL-2, ab182858, Abcam, UK), and BCL-2-associated X protein (BAX, ab32503, Abcam, UK). Goat anti-Rabbit IgG (H+L) (AWS0002, Abiowell, CHN) was used as a secondary antibody, and the immunoblots were visualized and documented using an enhanced chemiluminescence substrate. Myocardial proteins were observed and compared with a common protein standard loaded onto the gel using Quantity ONE.

We extracted left ventricular myocardium mRNA using TRIzol reagent (15596026, Thermo, America). The concentration of mRNA was measured spectrophotometrically and RNA purity was assessed using the 260/280 ratio. RNA reverse-transcription, cDNA amplification, and PCR quantification were done by the PikoReal™ Real-Time PCR System. MIF gene expression was quantified using the 2^-ΔΔCt method, with GAPDH and β-actin as housekeeping genes. The used PCR primer sequences are listed in the Table 1.

Three types of interventions were introduced into mice: control, HFD, and HFD plus aerobic exercise, each contained 2 groups of control and exhaustive exercise. Thus, for each of the six subgroups, five mice were needed. The total sample size of 30 mice provided 89% power to detect differences among the means compared to the alternative of equal means, with a significance level of α 0.05, using an F test. The calculation was based on a pilot study that determined the size of variation in the means as 4.11 and assumed a common standard deviation of 5 within each group. The sample size was determined using PASS 15.0.5.

The Shapiro-Wilk test was conducted to assess the normality for continuous variables. To examine the differences in body weight over time among the groups, we employed repeated measures analysis of variance (ANOVA). In the subsequent analyses, normally distributed variables were assessed using ANOVA, followed by a 2-sample t-test for each pair with Bonferroni correction. For non-normally distributed variables, we employed the Kruskal-Wallis test, followed by the Wilcoxon rank-sum test for each pair with Bonferroni correction. The data were analyzed using SPSS Statistics version 25, and the threshold for statistical significance was set at P < 0.05 (two-sided).

Mice fed an HFD displayed a significant increase in body weight (Figure 2A), visceral fat mass (Figure 2B), and visceral fat mass/body weight ratio (Figure 2C), as well as increased adipocyte hypertrophy (Figures 2D, E), a typical obesity phenotype, compared to those fed a normal diet. The HFD-induced obese phenotype was significantly attenuated by AE (Figures 2A-E). Additionally, HFD-fed mice presented significantly higher serum FBG, insulin, and HOMA-IR levels than the mice in the control group, and these obesity phenotypes were also significantly attenuated by AE (Figures 2F-H).

The EE was employed to induce fatal stress in mice (12, 20), and resulted in a significant reduction in LVEF, indicating impaired cardiac function. In addition, obesity exacerbated the decrease in LVEF, whereas AE preconditioning significantly mitigated this exacerbation (Figures 3A, B). Furthermore, we observed that obesity impaired exercise capacity, as evaluated by the swimming duration until exhaustion (Figure 3C). These findings indicate that AE preconditioning effectively alleviated fatal stress-induced impairments in cardiac function and overall function.

CK is an enzyme located in the skeletal and heart muscles that leak into the bloodstream when these muscles are damaged, and elevated CK levels indicate a high probability of acute myocardial damage in patients with chest pain (27). We observed an increase in serum CK levels using an ELISA (Figure 3D) and detected severe myocardial mitochondria and sarcomere damage using transmission electron microscopy (Figure 3E), indicating that exhaustive exercise-induced fatal stress resulted in myocardial damage.

The life-or-death decision for a cell is mainly determined by the interaction between BCL-2 family members. Among these, BCL-2 and BAX are the main pro-survival and pro-apoptotic mediators, respectively (28). Cytochrome c presents in the mitochondrial intermembrane and intercristae space in healthy conditions, but leaks from damaged mitochondria into the cytosol to activate the apoptotic signaling pathway (29). Overexpression of BCL-2 blocks cytochrome c release from the mitochondria (30) whereas BAX promotes cytochrome c leakage through BAX oligomeric pores (31). We detected elevated cytochrome c and BAX protein levels, decreased BCL-2 levels, and a decline in the ratio of BCL-2 to BAX upon exhaustive exercise (Figures 3F-I). These changes were accompanied by an increase in apoptosis, as evidenced by an increase in the percentage of TUNEL-positive cells (Figures 3J, K).

Together, these results indicate that obesity impaired cardiac and overall function, and exacerbated fatal stress-induced myocardial injury and apoptosis. AE preconditioning significantly alleviated fatal stress-induced cardiac injury and apoptosis and reversed the exacerbation caused by the HFD.

The quality of mitochondria plays a crucial role in both apoptosis and survival (32). Elevation of DRP1 in mice after cardiac ischemia-reperfusion injury leads to an increase in mitochondrial fission (33), which may counteract damage by promoting the separation and removal of injured mitochondrial components (12). Additionally, obesity disorders can suppress MFN1-mediated mitochondrial fusion in the heart (34, 35) in rats, leading to increased cardiomyocyte apoptosis (35). Furthermore, knockout of the mitophagy regulator PINK1 has been reported to increase serum CK levels and susceptibility to cardiac injury in response to exhaustive exercise (19). Moreover, impaired activity of the mitochondrial biogenesis pathway, SIRT1/PGC-1α, and reduced mitochondrial volume in adipose tissue have been observed in obese children (36). Here, we observed a lower conversion from LC3-I to LC3-II, a standard marker for autophagosomes, accompanied by the suppression of PNIK1 and higher levels of p62 in the mice myocardium after exhaustive exercise, indicating that obesity significantly suppressed mitophagy in response to fatal stress (Figures 4A-D). AE preconditioning significantly reversed obesity-induced suppression of myocardial mitophagy in mice after exhaustive exercise (Figures 4A-D). In addition, obese mice that suffered exhaustive exercise presented a deficit in mitochondrial biogenesis, as evidenced by significantly lower protein expression of SIRT1 (Figure 4E), PGC-1α (Figure 4F), and CS (Figure 4G), a marker of mitochondrial volume, compared to mice fed a normal diet; these phenotypes were also significantly reversed by AE (Figures 4E-G). Moreover, in response to fatal stress, obese mice exhibited significantly lower protein levels of MFN1 (Figure 4H), a promoter of mitochondrial fusion, and DRP1 (Figure 4I), an activator of mitochondrial fission, compared to the mice in the normal diet group, indicating impaired myocardial mitochondrial fusion and fission. Similar to the other phenotypes, this impairment was diminished by AE preconditioning (Figures 4H, I).

A previous report by Meng et al. suggested that impaired myocardial MIF/AMPK activation exacerbates ischemia-reperfusion injury in HFD-induced obesity (37). AMPK is a key molecular transducer of exercise and regulator of MQC (38). In the present study, obesity significantly suppressed the MIF/AMPK pathway response to exhaustive exercise-induced fatal stress, as indicated by the lower levels of MIF mRNA and protein (Figures 5A-C), pAMPK, and the ratio of pAMPK/AMPK (Figures 5D-F) in the myocardium of mice after exhaustive exercise. These effects were significantly rescued by AE (Figures 5A-F).