Oxidative and glycolytic skeletal muscles deploy protective mechanisms to avoid atrophy under pathophysiological iron overload

Abstract

Background: Iron excess has been proposed as an essential factor in skeletal muscle wasting. Studies have reported correlations between muscle iron accumulation and atrophy, either through ageing or by using experimental models of secondary iron overload. However, iron treatments performed in most of these studies induced an extra-pathophysiological iron overload, more representative of intoxication or poisoning. The main objective of this study was to determine the impact of iron excess closer to pathophysiological conditions on structural and metabolic adaptations (i) in differentiated myotubes and (ii) in skeletal muscle exhibiting oxidative (i.e. the soleus) or glycolytic (i.e. the gastrocnemius) metabolic phenotypes.

Methods: The impact of iron excess was assessed in both in vitro and in vivo models. Murine differentiated myotubes were exposed to ferric ammonium citrate (FAC) (i.e. 10 and 50 μM) for the in vitro component. The in vivo model was achieved by a single iron dextran subcutaneous injection (1 g/kg) in mice. Four months after the injection, soleus and gastrocnemius muscles were harvested for analysis.

Results: In vitro, iron exposure caused dose-dependent increases of iron storage protein ferritin (P < 0.01) and dose-dependent decreases of mRNA TfR1 levels (P < 0.001), which support cellular adaptations to iron excess. Extra-physiological iron treatment (50 μM FAC) promoted myotube atrophy (P = 0.018), whereas myotube size remained unchanged under pathophysiological treatment (10 μM FAC). FAC treatments, whatever the doses tested, did not affect the expression of proteolytic markers (i.e. NF-κB, MurF1, and ubiquitinated proteins). In vivo, basal iron content and mRNA TfR1 levels were significantly higher in the soleus compared with the gastrocnemius (+130% and +127%; P < 0.001, respectively), supporting higher iron needs in oxidative skeletal muscle. Iron supplementation induced muscle iron accumulation in the soleus and gastrocnemius muscles (+79%, P < 0.001 and +34%, P = 0.002, respectively), but ferritin protein expression only increased in the gastrocnemius (+36%, P = 0.06). Despite iron accumulation, muscle weight, fibre diameter, and myosin heavy chain distribution remained unchanged in either skeletal muscle.

Conclusions: Together, these data support that under pathophysiological conditions, skeletal muscle can protect itself from the related deleterious effects of excess iron.

Keywords: Disuse; Mitochondria; Myosin heavy chain; Sarcopenia; Typology.

Figure 1

Responses of C2C12 differentiated myotubes to FAC treatment. Differentiated C2C12 myotubes were exposed to various concentrations of FAC (10 and 50 μM) in serum‐free medium for 24 h (qPCR analyses) or 72 h (western blot analyses). (A) Iron metabolism gene expression in response to FAC 10 and 50 μM. (B) Iron metabolism protein expression in response to FAC 10 and 50 μM. (C) Representative blotting images used for the quantification shown in (B). Values are the mean ± SD. The experiments were performed on three independent replicates of three dependent samples for each condition. Significance was checked using Kruskal–Wallis and Dunn's multiple comparisons. Significant differences between conditions (*P < 0.05, **P < 0.01, and ***P < 0.001, respectively). Aco1, aconitase 1; Atg5, autophagy‐related 5; Atg7, autophagy‐related 7; Cyc1, cytochrome c; Dmt1, divalent metal transporter 1; Fpn, ferroportin; FtH, ferritin heavy chain; HSC70, heat shock cognate 71 kDa protein; TfR1, transferrin receptor 1; Zip14, ZRT/IRT‐like protein 14.

Figure 2

Impact of FAC treatment on myotube size and signalling pathway involved in muscle mass regulation and mitochondrial function. Differentiated C2C12 myotubes were exposed to various concentrations of FAC (10 and 50 μM) in serum‐free medium for 72 h. (A) Representative morphology of myotubes at 72 h after vehicle or FAC treatment. (B) Quantitative analysis of myotube diameter. (C) Protein expression or activation of key proteins involved in proteolysis, protein synthesis, and mitochondrial function in response to FAC 10 and 50 μM. (D) Representative blotting images used for the quantification shown in (B). Values are the mean ± SD. The experiments were performed on three independent replicates of three dependent samples for each condition. Significance was checked using Kruskal–Wallis and Dunn's multiple comparisons. Significant differences between conditions (^** P < 0.01). 4EBP1, eukaryotic translation initiation factor 4E‐binding protein 1; Akt, protein kinase B; COXIV, cytochrome c oxidase IV; Cyt c, cytochrome c; HSC70, heat shock cognate 71 kDa protein; Murf1, muscle RING‐finger protein‐1; MHC, myosin heavy chain; Nf‐κB, nuclear factor‐κB; Ub Prot, polyubiquitinated proteins.

Figure 3

Iron supplementation causes systemic and muscle iron overload. (A) Transferrin saturation. (B) Serum iron concentrations. (C) Liver iron concentrations. (D) Soleus and gastrocnemius iron concentration. Values are the mean ± SD (n = 12 per group). Significance was checked using Mann–Whitney U test, Student's unpaired t‐test, or two‐way ANOVA. Significant differences between conditions (^*** P < 0.001).

Figure 4

Muscle adaptations of iron metabolism under iron supplementation. (A) Iron metabolism protein gene expression in gastrocnemius and soleus. (B) Iron metabolism protein expression in gastrocnemius and soleus. (C) Representative blotting images used for the quantification shown in (B). (D) Iron metabolism protein gene expression in gastrocnemius in response to iron dextran treatment. (E) Iron metabolism protein expression in gastrocnemius in response to iron dextran treatment. (F) Representative blotting images used for the quantification shown in (E). (G) Iron metabolism protein gene expression in soleus in response to iron dextran treatment. (H) Iron metabolism protein expression in soleus in response to iron dextran treatment. (I) Representative blotting images used for the quantification shown in (H). Values are the mean ± SD (n = 6–12 per group). Significance was checked using Mann–Whitney U test or Student's unpaired t‐test. DMT1, divalent metal transporter 1; Fpn, ferroportin; FtH, ferritin heavy chain; HSC70, heat shock cognate 71 kDa protein; TfR1, transferrin receptor 1.

Figure 5

Iron supplementation does not induce oxidative damage in skeletal muscle. (A) 4‐HNE protein adducts and carbonylated proteins in gastrocnemius. (B) Representative blotting images for 4‐HNE protein adducts in gastrocnemius. (C) Representative blotting images for carbonylated proteins in gastrocnemius. (D) 4‐HNE protein adducts and carbonylated proteins in soleus. (E) Representative blotting images for 4‐HNE protein adducts in soleus. (F) Representative blotting images for carbonylated proteins in soleus. Values are the mean ± SD (n = 7–9 per group). Significance was checked using Mann–Whitney U test or Student's unpaired t‐test.

Figure 6

Iron supplementation does not impact skeletal muscle structure in mice. (A) Gastrocnemius and soleus weight‐to‐body weight ratio. (B) Minimal Feret's diameter of muscle fibres from gastrocnemius muscle. (C) Frequency distribution of muscle fibre's diameter in the gastrocnemius muscle. (D) Laminin representative staining of skeletal muscle section. Black scale bar corresponds to 100 μm. (E) Distribution of MHC isoforms in soleus muscle. (F) Distribution of MHC isoforms in gastrocnemius muscles. (G) Representative electrophoresis of MHC isoforms in soleus and gastrocnemius muscles. Values are the mean ± SD (n = 6–12 per group). Significance was checked using Mann–Whitney U test or Student's unpaired t‐test.