Electrical Stimulation Improves Rat Muscle Dysfunction Caused by Chronic Intermittent Hypoxia-Hypercapnia via Regulation of miRNA-Related Signaling Pathways

Abstract

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) patients is common. Neuromuscular Electrical Stimulation (NMES) is a powerful exercise training that may relieve muscle dysfunction in COPD. This study investigated whether electrical stimulation may have atypical adaptations via activation of miRNA related pathways in counteracting COPD muscle dysfunction. Forty-eight male Sprague-Dawley rats were randomly assigned to 3 groups. With the exception of the rats in the control group, the experimental rats were exposed to chronic intermittent hypoxia-hypercapnia (CIHH) (9∼11%O2,5.5∼6.5%CO2) for 2 or 4 weeks. Electrical stimulation was performed immediately after each CIHH session. Following assessment of the running capacity, biopsy samples were obtained from the gastrocnemius of the rats. The miR-1, miR-133a and miR-133b levels were measured, as well as their related proteins: phosphorylation of Akt (p-AKT), PGC-1alpha (PGC-1α), histone deacetylase 4 (HDAC4) and serum response factor (SRF). Myosin heavy chainIIa (MHCIIa) and myosin heavy chainIIb (MHCIIb) were also measured to assess fiber type changes. After 2 weeks, compared with the controls, only miR-1 and miR-133a were significantly increased (p<0.05) in the exposure group. After 4 weeks, the exposure group exhibited a decreased running distance (p = 0.054) and MHCIIa-to-MHCIIb shift (p<0.05). PGC-1α (p = 0.051), nuclear HDAC4 (p = 0.058), HDAC4, p-AKT, PGC-1α and SRF was also significantly decreased (p<0.05). In contrast, miR-1 and miR-133a were significantly increased (p<0.05). Four weeks of electrical stimulation can partly reversed those changes, and miR-133b exhibited a transient increase after 2 weeks electrical stimulation. Our study indicate miRNAs may have roles in the response of CIHH-impaired muscle to changes during electrical stimulation.

Fig 1. Effects of electrical stimulation on the CIHH-induced expression of the relative myosin heavy chain (MHC) protein content expression.

Gastrocnemius muscles were isolated from the lower limb of three groups of rats at 2 weeks (A) and 4 weeks (B). Analyses of the expression of MHCⅡa and MHCⅡb were performed by Western blotting. Tubulin was used as a loading control. The optical density values were normalized to their respective Tubulin loading control, and the means±SEMs were graphed (relative expression) to semi-quantitatively compare the protein levels. *p<0.05 (at least) vs. NC group; #p<0.05 (at least) vs. HH group. NC = normal control group; HH = hypoxia-hypercapnia group; HE = hypoxia-hypercapnia + electrical stimulation.

Fig 2. Effects of electrical stimulation on the CIHH-induced expression of miR-1, miR-133a and miR-133b.

Gastrocnemius muscles were isolated from the lower limb of three groups of rats at 2 weeks (A) and 4 weeks (B). Analyses of the expression of miR-1, miR-133a and miR-133b were performed by qRT-PCR and normalized to the expression of 5S RNA in the same sample, as described in the Methods section. *p<0.05 (at least) vs. NC group; #p<0.05 (at least) vs. HH group. Data are expressed as the means ±SEMs. NC = normal control group; HH = hypoxia-hypercapnia group; HE = hypoxia-hypercapnia + electrical stimulation.

Fig 3. Effects of electrical stimulation on the CIHH-induced reduction of PGC-1α and p-Akt protein.

Gastrocnemius muscles were isolated from the lower limb of three groups of rats at 2 weeks (A) and 4 weeks (B). Analyses of the expression of PGC-1α and p-Akt proteins were performed by Western blotting. Tubulin and GAPDH were used as loading controls. The optical density values were normalized to their respective Tubulin and GAPDH loading controls, and the means±SEMs were graphed (relative expression) to semi-quantitatively compare the protein levels. *p<0.05 (at least) vs. NC group; #p<0.05 (at least) vs. HH group. NC = normal control group; HH = hypoxia-hypercapnia group; HE = hypoxia-hypercapnia + electrical stimulation.

Fig 4. Effects of electrical stimulation on the CIHH-induced reduction of HDAC4 and nuclear HDAC4 protein.

Gastrocnemius muscles were isolated from the lower limb of three groups of rats at 2 weeks (A) and 4 weeks (B). Analyses of the expression of HDAC4 and nuclear HDAC4 proteins were performed by Western blotting. Tubulin and GAPDH were used as loading controls. The optical density values were normalized to their respective Tubulin and GAPDH loading controls, and the means±SEMs were graphed (relative expression) to semi-quantitatively compare the protein levels. *p<0.05 (at least) vs. NC group; #p<0.05 (at least) vs. HH group. NC = normal control group; HH = hypoxia-hypercapnia group; HE = hypoxia-hypercapnia + electrical stimulation.

Fig 5. Effects of electrical stimulation on the CIHH-induced reduction of protein expression of SRF.

Gastrocnemius muscles were isolated from the lower limb of three groups of rats. Analyses of the SRF protein expression were performed via Western blotting at 2 weeks and 4 weeks, and GAPDH was used as a loading control. The optical density values were normalized to their respective Tubulin and GAPDH loading controls, and the means±SEMs were graphed (relative expression) to semi-quantitatively compare the protein levels. *p<0.05 (at least) vs. NC group; #p<0.05 (at least) vs. HH group. NC = normal control group; HH = hypoxia-hypercapnia group; HE = hypoxia-hypercapnia + electrical stimulation.

Fig 6. Effects of electrical stimulation on the CIHH-induced nuclear localization change of SRF (4 weeks).

Gastrocnemius muscles were isolated from the lower limb of three groups of rats. Nuclear localization of SRF was determined by immunofluorescence staining; observations via confocal microscope indicate the localization of SRF in green fluorescence and the nuclei with 4,6-diamidino-2-phenylindole (DAPI) in blue fluorescence as a comparison(see S1 Fig). Arrowheads show the localization of nuclei staining and the inset shows perinuclear staining for SRF. NC group showed that the staining pattern of the factor was identified in the nucleus as well as in the cytoplasm (g). After 4 weeks, some parts of HH group showed exclusively cytoplasmic in HH group (h). In HE group nuclear localization change of SRF was partly reversed (i). Detailed images of both 2 weeks and 4 weeks are shown in S1 Fig. Original magnification 40×. NC = normal control group; HH = hypoxia-hypercapnia group; HE = hypoxia-hypercapnia + electrical stimulation.