Electrical stimulation influences chronic intermittent hypoxia-hypercapnia induction of muscle fibre transformation by regulating the microRNA/Sox6 pathway

Abstract

Chronic obstructive pulmonary disease can cause muscle fibre transformation due to chronic intermittent hypoxia-hypercapnia (CIHH). Studies have shown that high expression of Sox6 in muscle could suppress type-I fibres through downregulating the PPARβ (peroxisome proliferator-activated receptor β)/ERRγ (oestrogen-related receptor γ)/microRNA pathway. However, whether this pathway is involved in CIHH-induced muscle fibre transformation is unknown. Electrical stimulation (ES) is an effective approach to ameliorate muscle dysfunction. Here, we explored the effects of ES on CIHH-induced muscle fibre transformation and the microRNA/Sox6 pathway. After CIHH exposure, both the soleus (SOL) and gastrocnemius (GC) muscles showed decreased type-I fibres. The PPARβ/ERRγ/mir-499&208b (PEM, for GC) and PPARβ/mir-499&208b (PM, for SOL) signalling cascades were suppressed, followed by elevated Sox6 expression. Low frequency electrical stimulation (LFES) activated the PEM/PM pathway and enhanced type-I fibre numbers through suppressing Sox6 in SOL and GC. High frequency electrical stimulation (HFES) promoted type-I fibre expression through activating the PEM pathway in GC. Although PPARβ expression and type-I fibres were suppressed in SOL after HFES, no significant change was found in mir-499&208b/Sox6 expression. These results suggest that the microRNA/Sox6 pathway is disturbed after CIHH. Both low and high frequency electrical stimulations induce muscle fibre transformation partly through regulating the microRNA/Sox6 pathway.

Figure 1. H & E staining of SOL.

These pictures show the pathological events in the SOL in each group. The scale bar represents 50 μm. The arrows indicate inflammatory cell infiltration. The triangles indicate internal nuclei. NC: normal control group; HS: hypoxia-hypercapnia+ sham stimulation group; HH: hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia + low frequency electrical stimulation group; HFES: hypoxia-hypercapnia + high frequency electrical stimulation group.

Figure 2. Fibre type change of each group in SOL.

(A) Immunochemical staining for type-I fibres in SOL. Scale bar = 50 μm; (B) Western blotting for MHC-I. The optical density values are normalised to their respective tubulin loading control, and the means ± SD are graphed (relative expression) to semi-quantitatively compare the protein levels; (C) Quantification of MHC-I, IIa, and IIx gene expression in SOL; (D) Average cross-sectional area (CSA) of type-I and type-II muscle fibres in SOL; *P < 0.05 vs. the NC group; ^#P < 0.05 vs. the HS or HH group. NC: normal control group; HS: hypoxia-hypercapnia+ sham stimulation group; HH: hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia+ low frequency electrical stimulation group; HFES: hypoxia-hypercapnia+ high frequency electrical stimulation group.

Figure 3. Electrical stimulation activated the microRNA/Sox6 pathway in SOL.

(A) Western blotting analysis of Sox6 expression. Tubulin was used as the loading control. The triangle shows the target band. (B) qRT-PCR analysis of mir-499 and mir-208b. Values are expressed as the mean ± SEM, *P < 0.05 vs. the NC group, ^#P < 0.05 vs. the HS or HH group. NC: normal control group; HS: hypoxia-hypercapnia + sham stimulation group; HH: hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia+ low frequency electrical stimulation group; HFES: hypoxia-hypercapnia+ high frequency electrical stimulation group.

Figure 4. Low electrical stimulation promotes PPARβ expression in SOL.

Western blotting was used to establish the expression of PPARβ and ERRγ. The values are normalised to their respective tubulin loading control, and the means ± SD are calculated to compare the protein levels. *P < 0.05 vs. the NC group, ^#P < 0.05 vs. the HS or HH group. NC: normal control group; HS: hypoxia-hypercapnia+ sham stimulation group; HH: hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia +low frequency electrical stimulation group; HFES: hypoxia-hypercapnia+ high frequency electrical stimulation group.

Figure 5. H & E staining of GC.

These pictures show the pathological characteristics of GC in each group. Scale bar = 50 μm. The arrows indicate the inflammatory cells. The triangles indicate internal nuclei. NC: the normal control group; HS: the hypoxia-hypercapnia + sham stimulation group; HH: the hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia + low frequency electrical stimulation group; HFES: the hypoxia-hypercapnia+ high frequency electrical stimulation group.

Figure 6. Type-I fibres in each group.

(A) Immunochemistry staining for type-I fibre in GC. Scale bar = 50 μm. (B) Representative Western blotting for MHC-I; the optical density values are normalised to their respective tubulin loading control and the means ± SD are graphed (relative expression) to semi-quantitatively compare the protein levels. (C) Average fibre cross-sectional area of type-I and type-II fibres. *P < 0.05 vs. the NC group; ^#P < 0.05 vs. the HS or HH group. NC: normal control group; HS: hypoxia-hypercapnia + sham stimulation group; HH: hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia+ low frequency electrical stimulation group; HFES: hypoxia-hypercapnia+ high frequency electrical stimulation group.

Figure 7. Both low and high electrical stimulation reduce Sox6 expression and induce mir-499 and mir-208b expression in GC.

(A) Western blotting analysis of Sox6 expression in GC. Tubulin was used as a loading control. (B) qRT-PCR analysis of mir-499 and mir-208b. (C) Cross-section of gastrocnemius stained for Sox6 and MHC-I by immunofluorescence. Green = MHC-I; Red = Sox6; Blue = DAPI. Scale bar = 50 μm. WG: white GC; *P < 0.05 vs. the NC group, ^#P < 0.05 vs. HS or the HH group;. All values represent the mean ± SEM. NC: normal control group; HS: hypoxia-hypercapnia+ sham stimulation group; HH: hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia+ low frequency electrical stimulation group; HF: hypoxia-hypercapnia+ high frequency electrical stimulation.

Figure 8. Electrical stimulation promotes PPARβ and ERRγ expression in GC.

Western blots for PPARβ and ERRγ are presented. Tubulin was used as a loading control. The values are expressed as the mean ± SEM. *P < 0.05 vs the NC group, ^#P < 0.05 vs the HB or the HH group. NC: normal control group; HS: hypoxia-hypercapnia+ sham stimulation group; HH: hypoxia-hypercapnia group; LFES: hypoxia-hypercapnia + low frequency electrical stimulation group. HFES: hypoxia-hypercapnia + high frequency electrical stimulation group.