Document Type : Original Article
Authors
1 Master's student at Department of Exercise Physiology, A.C., Islamic Azad University, Arak, Iran.
2 Assistant Professor at Department of Exercise Physiology, A.C., Islamic Azad University, Arak, Iran.
Abstract
Background and Aim: Obesity is a major contributor to cardiovascular dysfunction and impaired angiogenesis (1). Vascular endothelial growth factor (VEGF) is a key promoter of blood vessel formation, while endostatin serves as an endogenous inhibitor of angiogenesis (8,9,11). High-intensity interval training (HIIT) has been shown to improve vascular health and cardiac function (8,13,15), and when combined with electrical stimulation (ES), may further enhance these effects (28,45). Caloric restriction (CR) is another well-established intervention that improves metabolic and cardiovascular outcomes(16,5). However, the combined impact of HIIT, ES, and CR on the expression of genes related to angiogenesis in cardiac tissue remains unclear. This study aimed to investigate the effects of four weeks of HIIT combined with ES under CR conditions on the expression of VEGF and endostatin genes in the heart tissue of obese male rats. The findings may provide insight into non-pharmacological strategies for improving cardiac health in obesity-related conditions.
Materials and Methods: This experimental study was conducted on 35 male Wistar rats (8 weeks old, 200 ± 19 g), purchased from Baqiyatallah University, Iran. The animals were housed in transparent polycarbonate cages under controlled conditions: a temperature of 22 ± 2 °C, a humidity of 50 ± 5%, and a 12-hour light/dark cycle. All rats had access to water and a specialized rodent diet. After a one-week acclimatization period, obesity was induced using a high-fat diet for 12 weeks, consisting of peanuts, milk chocolate, and sweet biscuits in a 3:1:1 ratio. The diet provided 20% protein, 60% fat, and 20% carbohydrates per 100 g (31,32). Following obesity induction, rats were randomly divided into five groups (n = 7 per group): 1- Obese control (CO), 2-Caloric restriction (CR), 3-CR + Electrical Stimulation (ES.CR), 4-CR + High-Intensity Interval Training (HIIT.CR), 5-CR + HIIT + Electrical Stimulation (HIIT.ES.CR). Caloric restriction was implemented using a time-restricted feeding model: 16 hours of food access followed by 8 hours of fasting daily. The standard diet used during this phase followed AIN-93 guidelines.HIIT was performed on a treadmill for four weeks, four sessions per week. Each session included alternating high-intensity intervals (25–30 m/min, 70–95% VO₂max) and low-intensity recovery intervals (21–23 m/min, 50–60% VO₂max), lasting 20–40 minutes. Warm-up and cool-down were performed at 10–12 m/min (30–50% VO₂max) (34,35). Electrical stimulation was applied using an R12 stimulator (Parto Danesh Co., Iran) at 0.5 mA for 20 minutes, three times per week (36,37). Electrodes were placed on the hind limbs, and stimulation was synchronized with post-exercise recovery. Two days after the final intervention, rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg), and cardiac tissue samples were collected. Samples were washed with phosphate-buffered saline (PBS), frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using standard protocols with LR buffer, β-mercaptoethanol, chloroform, and ethanol. cDNA synthesis was performed using reverse transcriptase (Fermentas, USA). Gene expression of VEGF and endostatin was quantified using real-time PCR (Kiagene, Iran), with GAPDH as the housekeeping gene. Primer sequences were designed based on NCBI data (36,37). Statistical analysis was conducted using GraphPad Prism (version 8). Normality was assessed with the Shapiro–Wilk test. One-way ANOVA followed by Tukey’s post hoc test was used to compare group means. A p-value < 0.05 was considered statistically significant.
Results: The results showed that the expression levels of the VEGF and Endostatin genes in the heart tissue of obese groups under calorie restriction combined with high-intensity interval training significantly decreased compared to the control group. The expression of the VEGF gene (X̅=0.05880) in this group showed a significantly greater decrease compared to the expression of the Endostatin gene (X̅=0.07296) (p<0.0001). Additionally, the expression of these two genes in obese groups under calorie restriction combined with electrical stimulation significantly decreased compared to the control group, and the expression of the VEGF gene (X̅=0.2718) showed a significantly greater decrease compared to the expression of the Endostatin gene (X̅=0.3256) in this group (p<0.0001). The combination of high-intensity interval training and electrical stimulation in obese individuals under calorie restriction resulted in a significant reduction in the expression of both VEGF and Endostatin genes compared to the control group. The expression of the VEGF gene (X̅=0.1260) showed a significantly greater decrease compared to the expression of the Endostatin gene (X̅=0.3006) in this group (p<0.0001). Conclusion: It appears that high-intensity interval training (HIIT) and electrical stimulation, along with calorie restriction, can cause a significant decrease in the expression of endostatin and VEGF genes in the cardiac tissue of obese individuals. The reduction of endostatin may be associated with the regulation of angiogenesis and increased myocardial oxygenation, while the decrease of VEGF can prevent abnormal vessel growth. However, determining the precise consequences of these changes and finding the optimal balance between pro-angiogenic and anti-angiogenic factors requires further research. Keywords: High-Intensity Interval Training, Electrical Stimulation, Obesity, Caloric Restriction, Angiogenesis
Ethical Considerations: This study was conducted according to ethical standards for biomedical research involving laboratory animals. All procedures were approved by the Ethics Committee of Islamic Azad University, Arak Branch (Ethics Code: IR.IAU.ARAK.REC.1403.293). Throughout the study, all efforts were made to minimize animal suffering and ensure humane treatment during housing, interventions, and sample collection.
Compliance with Ethical Guidelines: All experimental protocols were reviewed and approved by the institutional ethics board, and the study adhered to national and international guidelines for the care and use of laboratory animals.
Funding: This research did not receive any financial support from funding agencies or institutions.
Conflicts of Interest: The authors declare that there are no conflicts of interest associated with this study.
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