GSK3β inhibition and LEF1 upregulation in skeletal muscle following a bout of downhill running

Abstract

Canonical Wnt signaling is important in skeletal muscle repair but has not been well characterized in response to physiological stimuli. The objective of this study was to assess the effect of downhill running (DHR) on components of Wnt signaling. Young, male C57BL/J6 mice were exposed to DHR. Muscle injury and repair (MCadherin) were measured in soleus. Gene and protein expression of Wnt3a, active β-catenin, GSK3β, and LEF1 were measured in gastrocnemius. Muscle injury increased 6 days post-DHR and MCadherin protein increased 5 days post-DHR. Total and active GSK3β protein decreased 3 days (9-fold and 3.6-fold, respectively) post-DHR. LEF1 protein increased 6 days (5-fold) post-DHR. DHR decreased GSK3β and increased LEF1 protein expression, but did not affect other components of Wnt signaling. Due to their applicability, using models of physiological stimuli such as DHR will provide significant insight into cellular mechanisms within muscle.

Fig. 1

a Quantification of H&E staining as a marker of muscle injury. Two sections counted for every muscle (6–7 muscles/time point). Percentage of injured fibers relative to total fibers of cross section. Values are mean ± SE. *P < 0.001 vs. control, ^† P < 0.001 vs. 1D, ^# P < 0.05 vs. 2D, ^¥ P < 0.05 vs. control, ^§ P < 0.05 vs. 1D. b H&E representation of control muscle and 6 days post-DHR soleus muscle. D days

Fig. 2

a Immunofluorescence quantification of % MCadherin positive cells from total cells (DAPI-positive) on muscle cross-sections. Two sections counted for every muscle (6–7 muscles/time point). Values are mean ± SE. *P < 0.01 vs. baseline and 6D, ^† P < 0.05 vs. baseline and 6D, ^# P < 0.01 vs. 1D and 2D. b Immunofluorescence representation of mouse IgG isotype control with Alexa 546, with representative time points probed for MCadherin (red) and DAPI (blue). D days

Fig. 3

a qRT-PCR analysis of Wnt3a expression. Fold change relative to control. b Quantification of immunoblot probed for Wnt3a protein on lysates of gastrocnemius muscle that were exposed to DHR versus control. c Western blot representation of gastrocnemius lysates probed for Wnt3a antibody and striped and reprobed with GAPDH antibody

Fig. 4

qRT-PCR analysis of total β-catenin expression. Fold change relative to control

Fig. 5

a qRT-PCR analysis of total GSK3β expression. Fold change relative to control. b Quantification of immunoblot probed for total GSK3β protein on lysates of gastrocnemius muscle that were exposed to DHR vs. control. Values are mean ± SE. *P < 0.01 vs. control, ^† P < 0.01 vs. 5D, ^# P < 0.05 vs. 5D, ^¥ P < 0.05 vs. 1D. D days

Fig. 6

a Quantification of immunoblot probed for GSK3β (pY216) protein on lysates of gastrocnemius muscle that were exposed to DHR vs. control. Values are mean ± SE. *P < 0.01 vs. control, ^† P < 0.05 vs. control. b Western blot representation of gastrocnemius lysates probed for GSK3β (pY216 and total) and striped and reprobed with GAPDH antibody

Fig. 7

a qRT-PCR analysis of LEF1 expression. Fold change relative to control. b Quantification of immunoblot probed for LEF1 protein on lysates of gastrocnemius muscle that were exposed to DHR versus control. Values are mean ± SE. *P < 0.01 relative to baseline. c Western blot representation of gastrocnemius lysates probed for LEF1 antibody and striped and reprobed with GAPDH antibody