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. 2024 Mar 12;10(6):e27430.
doi: 10.1016/j.heliyon.2024.e27430. eCollection 2024 Mar 30.

Treadmill training impacts the skeletal muscle molecular clock after ischemia stroke in rats

Mai Li  1 , Yong Yin  2 , Dongdong Qin  3
Affiliations

Treadmill training impacts the skeletal muscle molecular clock after ischemia stroke in rats

Mai Li et al. Heliyon. .

Abstract

Objective: Stroke is frequently associated with muscle mass loss. Treadmill training is considered the most effective treatment for sarcopenia. Circadian rhythms are closely related to exercise and have been extensively studied. The skeletal muscle has its molecular clock genes. Exercise may regulate skeletal muscle clock genes. This study evaluated the effects of early treadmill training on the skeletal muscle molecular clock machinery in rats with stroke and determined the relationship of these changes with exercise-induced improvements in skeletal muscle health.

Materials and methods: Overall, 168 Sprague-Dawley rats were included in this study. We established an ischemic stroke rat model of sarcopenia. Finally, 144 rats were randomly allocated to four groups (36 per group): normal, sham, middle cerebral artery occlusion, and training. Neurological scores, rotating rod test, body weight, muscle circumference, wet weight, and hematoxylin-eosin staining were assessed. Twenty-four rats were used for transcriptome sequencing. Gene and protein expressions of skeletal muscles, such as brain muscle arnt-like 1, period 1, and period 2, were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays.

Results: Neurological function scores and rotating rod test results improved after treadmill training. Nine differentially expressed genes were identified by comparing the sham group with the hemiplegic side of the model group. Seventeen differentially expressed genes were identified between the hemiplegic and non-hemiplegic sides. BMAL1, PER1, and PER2 mRNA levels increased on both sides after treadmill training. BMAL1 expression increased, and PER1 expression decreased on both sides, whereas PER2 expression decreased on the hemiplegic side but increased on the non-hemiplegic side.

Conclusion: Treadmill training can mitigate muscle loss and regulate skeletal muscle clock gene expression following ischemic stroke. Exercise affects the hemiplegic side and has a positive regulatory effect on the non-hemiplegic side.

Keywords: Circadian rhythm; Ischemia stroke; Sarcopenia; Skeletal muscle molecular clock; Treadmill training.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Experimental design. Rats were subjected to middle cerebral artery occlusion (MCAO) after 3 days of pre-running training. MCAO rats underwent treadmill training beginning on day 3 and continuing until day 21. During the intervention, we measured neurological scores, rotating rod test, body weight, muscle circumference, and wet weight. After sacrifice, muscle tissues were obtained for laboratory testing, including transcriptome sequencing, quantitative real-time polymerase chain reaction, hematoxylin–eosin staining, and enzyme-linked immunosorbent assay.
Fig. 2
Fig. 2
Neurological scores on 2 h, 24 h, 3 days, and 7 days after MCAO. Two-way ANOVA. Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 3
Fig. 3
Rotating rod test in the MCAO and training groups. A. Rotate speed of the MCAO and training group. B. Test time of the MCAO and training group. Two-way ANOVA. Data are expressed as mean ± SEM. *P < 0.05.
Fig. 4
Fig. 4
Body weight were reduced after MCAO. (B, *MCAO group; #Training group). Two-way ANOVA. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ## <0.01, ### <0.001.
Fig. 5
Fig. 5
Muscle tissues of the gastrocnemius were reduced. A, and C. Muscle tissues loss of the hemiplegic side of MCAO and training group. B, and D. Muscle tissues loss of the non-hemiplegic side of MCAO and training group. Two-way ANOVA. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6
Fig. 6
Wet weight of the gastrocnemius were reduced after MCAO. B. Wet weight of the hemiplegic side of MCAO and training group. C. Wet weight of the non-hemiplegic side of MCAO and training group. Two-way ANOVA. Data are expressed as mean ± SEM. ***P < 0.001.
Fig. 7
Fig. 7
H&E staining showed sections of gastrocnemius in the hemiplegic side on days 10 and 21 (×200, scale bar = 50 μm). On 10 days, the muscle fibers were atrophied and varied in size.
Fig. 8
Fig. 8
Statistical results of cross section area of gastrocnemius muscle fibers stained by H&E. The cross-sectional area of muscle fibers was assessed on 10 and 21 days. A-D, One-way ANOVA; E-H, t-test. Data are expressed as mean ± SEM. **P < 0.01, *P < 0.05.
Fig. 9
Fig. 9
Differentially expressed genes. A. Volcano plot of the hemiplegic side of model/sham group. B. Heatmap of top 9 genes.
Fig. 10
Fig. 10
Differentially expressed genes. A. Volcano plot of the non-hemiplegic side of model/sham group. B. Heatmap of top 17 genes.
Fig. 11
Fig. 11
The mRNA levels of BMAL1, PER1, and PER2 in the hemiplegic (A, C, and E) and non-hemiplegic (B, D, and F) gastrocnemius muscles on 10 days. One-way ANOVA. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01.
Fig. 12
Fig. 12
The BMAL1 expression on 10 and 21 days following MCAO in the hemiplegic (A, B, C, and D) and non-hemiplegic (E, F, G, and H) gastrocnemius muscles. One-way ANOVA. Data are expressed as mean ± SEM. *P < 0.05, ***P < 0.001.
Fig. 13
Fig. 13
The PER1 expression on 10 and 21 days following MCAO in the hemiplegic (A, B, C, and D) and non-hemiplegic (E, F, G, and H) gastrocnemius muscles. One-way ANOVA. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01.
Fig. 14
Fig. 14
The PER2 expression on 10 and 21 days following MCAO in the hemiplegic (A, B, C, and D) and non-hemiplegic (E, F, G, and H) gastrocnemius muscles. One-way ANOVA. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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