Skeletal muscle HIF-1alpha expression is dependent on muscle fiber type

Abstract

Oxygen homeostasis is an essential regulation system for cell energy production and survival. The oxygen-sensitive subunit alpha of the hypoxia inducible factor-1 (HIF-1) complex is a key protein of this system. In this work, we analyzed mouse and rat HIF-1alpha protein and mRNA expression in parallel to energetic metabolism variations within skeletal muscle. Two physiological situations were studied using HIF-1alpha-specific Western blotting and semiquantitative RT-PCR. First, we compared HIF-1alpha expression between the predominantly oxidative soleus muscle and three predominantly glycolytic muscles. Second, HIF-1alpha expression was assessed in an energy metabolism switch model that was based on muscle disuse. These two in vivo situations were compared with the in vitro HIF-1alpha induction by CoCl(2) treatment on C(2)C(12) mouse muscle cells. HIF-1alpha mRNA and protein levels were found to be constitutively higher in the more glycolytic muscles compared with the more oxidative muscles. Our results gave rise to the hypothesis that the oxygen homeostasis regulation system depends on the fiber type.

Figure 1.

Comparison of HIF-1α expression in slow (soleus) versus fast (gastrocnemius, tibialis anterior, quadriceps) mouse skeletal muscles. (A) Protein analysis by Western blotting on skeletal muscle nuclear protein extracts. HIF-1α was detected as a specific 120-kD band, and an 80-kD nonspecific band stained with amido black is shown as an even-lane loading control. Lanes were evenly loaded with 25 μg of proteins. (B) RNA analysis performed by semiquantitative RT-PCR on skeletal muscle RNAs. Partial HIF-1α mRNAs were amplified and quantified and 18S rRNAs were used for normalization. The band intensity was measured, in linear PCR amplification range, and the results are expressed in arbitrary units after normalization. Histograms represent mean ± SEM of three independent experiments from two independent RNA or protein preparations. *, significantly different from soleus muscle (A, P < 0.001; B, P < 0.03).

Figure 2.

HIF-1α expression in differentiated C₂C₁₂ myotubes treated with CoCl₂. HIF-1α expression was assessed before treatment (T0) and after 30 min (T30') and 3 h (T 3h) in the presence of 200 μM CoCl₂. (A) Total cell proteins were extracted and analyzed by Western blotting using anti–HIF-1α antibody. An HIF-1α–specific 120-kD band was detected, and an 80-kD nonspecific band stained with amido-black is shown as an even-lane loading control. Lanes were evenly loaded with 20 μg of proteins. (B) RNAs were prepared and analyzed by semiquantitative RT-PCR. Primers specific to HIF-1α mRNA (for analysis) and to 18S rRNA (for normalization) were used. Band intensities were measured in the linear amplification range, and the results are expressed as ratios between the different time points and the T0-band intensities after normalization by 18S rRNA variations. The results represent mean ± SEM of three independent experiments.

Figure 3.

Comparison of HIF-1α mRNA expression in soleus and gastrocnemius muscles atrophied by unloading versus control muscles. Total RNAs isolated from rat muscle pools were analyzed by semiquantitative RT-PCR. HIF-1α mRNAs were amplified for analysis and 18S rRNAs were amplified for normalization. Band intensity was measured in the linear amplification range, and the results are expressed in an arbitrary unit after normalization by 18S rRNA variations. Histograms represent mean ± SEM of three independent RT-PCR experiments from two independent RNA preparations. *, significantly different from control (soleus muscle, P < 0.007).