Fibre-type specificity of interleukin-6 gene transcription during muscle contraction in rat: association with calcineurin activity

Abstract

In this study, we quantified the transcription of the interleukin-6 (IL-6) gene in individual fibres and the associated changes in calcineurin activity assessed at the cellular level during prolonged muscle contraction. Individual myofibres were isolated from plantaris and soleus muscles of rats at the end of an exhaustive running exercise test (n = 10), categorized according to their myosin heavy chain isoform content, and compared to those of resting rats (n = 10). Using real-time PCR analysis in individual fibres, a marked rise in IL-6 transcript levels occurred in type I and IIa fibres at the end of exercise (P < 0.05). Transcription of the gene encoding for the modulatory calcineurin-interacting protein-1 (MCIP-1), a sensitive indicator of calcineurin activity, also mainly increased in type I and IIa fibres (P < 0.05). Moreover, a slight increase in MCIP-1 mRNA levels was observed in type IIx (P < 0.05). Fibre types determined by immunohistochemistry were qualitatively examined for glycogen content using periodic acid-Shiff staining, and no direct relationship was found, at the cellular level, between glycogen content, fibre-type and IL-6 transcription. Our data clearly suggest that IL-6 gene transcription was mainly observed in early recruited myofibres and that contraction-induced IL-6 transcription could be associated with enhanced calcineurin activity.

Figure 1. Quantitative analysis of IL-6 mRNA levels by real-time RT-PCR in whole plantaris and soleus muscles of control non-exercised, active and recovering rats (arbitrary units)

Data are means ± s.e.m.*Significantly different from control values, P < 0.05; †significantly different from active group, P < 0.05.

Figure 2. Quantitative analysis of IL-6 mRNA levels by real-time RT-PCR in soleus (A) and plantaris (B) single fibres of control non-exercised and active rats (arbitrary units)

Plantaris fibres were categorized into four types: type I, IIa, IIx and IIb, according to their MHC isoform content. Data are means ± s.e.m.*Significantly different from control values, P < 0.05; †significantly different from IIa in active group values, P < 0.05.

Figure 3. Glycogen content in specific fibre types

Glycogen content in specific fibre types in soleus (A, B, E and F) and plantaris (C, D, G and H), before (A and C) and after exhaustive running exercise (B, D and E–H). Glycogen content was assessed by PAS staining (A–D) and fibre type by immunohistochemistry using antibodies directed against type I (F and H) and type IIa MHC isoforms (E and G). In soleus muscles, pure type IIa fibres (▪) or hybrid fibres comprising both type I and type IIa MHC isoforms (•) (E and F) are shown in B with a lower glycogen depletion than pure type I fibres (▴). In plantaris muscles, type IIa fibres (G) are shown in D with slight or no glycogen depletion (□); type I fibres (H) were markedly depleted (▵), while fibres negative for antibodies (i.e. fibres comprising either type IIx and/or type IIb MHC isoforms) (G and H) show a heterogeneity of glycogen depletion (○) (D). Scale bar, 100 μm.

Figure 4. Quantitative analysis of MCIP-1 mRNA levels by real-time RT-PCR in whole plantaris and soleus muscles (A) and in plantaris single fibres (B) of control non-exercised and active rats (arbitrary units)

For n and categorization of fibres see Figs 1 (A) and 2B (B). Data are means ± s.e.m.*Significantly different from control values (P < 0.05); **significantly different from control values (P < 0.01); †significantly different from IIx in the same group (P < 0.05); ‡significantly different from IIb in the same group (P < 0.05).