Functional and biochemical responses of skeletal muscle following a moderate degree of systemic iron loading in mice

An elevated iron status in the skeletal muscle was reported to occur in animal models of various pathophysiological states as well as aging. Notably, skeletal muscle iron levels ranging from approximately 1.4- up to 7-fold greater were observed in models of diabetes and aging, respectively. Such increment in the muscle iron level could be detrimental, as associations with increased muscle fatigability, atrophy, and oxidative stress were reported. Previous studies demonstrated direct effects of iron on muscle strength and its relevance to oxidative stress utilized a high degree of muscle iron loading (i.e. 60-fold). However, the skeletal muscle functional and redox status implications of a lower degree of muscle iron loading remain unclear. Thus, the aim of this study was to characterize the impact of a moderate degree of iron loading on changes in muscle mass and function, along with the underlying mechanism. In this experiment twenty, twelve-week-old male mice received either iron dextran (4 mg iron/200 µL i.p.) or vehicle (dextrose i.p.) 5 days/week for a period of two weeks (n = 10/group). Twenty-four hours after the last injection, soleus, extensor digitorum longus (EDL), gastrocnemius, and liver were harvested. Soleus muscle contractile properties were assessed. Muscle and liver non-heme iron (NHI) level, oxidative damage, antioxidant status, protein degradation markers, and stabilizing protein of ryanodine receptor (RyR1) were examined as potential mediators of iron-induced muscle dysfunction. No difference in animal body mass or soleus muscle mass was observed (p\u3e0.05). Maximal specific tension of the soleus was lower among iron-loaded animals (22.4±0.1 N/cm2) compared to controls (26.2±0.9 N/cm2; p\u3c0.05). In addition, specific tension (30, 50, 80, 120, 250 Hz) of soleus muscle was lower among iron-loaded animals compared to controls. NHI was approximately 4-fold greater in muscles and liver of mice that received iron compared to vehicle. Soleus lipid peroxidation (4-hydroxynonenal adducts) and protein oxidation (protein carbonyls) levels were similar between groups. In gastrocnemius muscles, a greater protein expression level of endogenous thiol antioxidant thioredoxin (TRX) was observed among mice that received iron treatment while its endogenous inhibitor thioredoxin-interacting protein (TXNip) and a redox-sensitive signaling complex TRX/TXNip was similar between groups. Glutaredoxin2 (GRX2), a thiol-disulfide oxidoreductase activated by GSSG-induced destabilization of its iron-sulfur [2Fe-2S] cluster, was lower following iron loading. Reduced glutathione (GSH) and glutathione peroxidase (GPX) activity were not different between the groups but glutathione disulfide (GSSG) and the ratio of GSSG to GSH in iron-loaded muscle were greater than controls. Protein expression level of antioxidant enzymes catalase (CAT), copper-zinc superoxide dismutase (Cu-ZnSOD), and manganese superoxide dismutase (MnSOD) was similar, as well as the activity of total superoxide dismutase (SOD). In addition, cytoskeletal protein level of II-spectrin at 240 kDa and -actinin were lower in the iron group compared to controls. Ryanodine receptor (RyR1) stabilizing subunit calstabin1, responsible for maintaining RyR1 channel in a closed state, was also lower following iron loading. In conclusion, a moderate degree of systemic iron loading impaired muscle contractile function but did not result in muscle atrophy. This was accompanied with an altered redox status, increased proteolytic response, and abnormal RyR1channel modification in the absence of overt oxidative damage. Further research should be aimed at exploring the potential mechanism(s) responsible for the specific tension deficits, as well as examine the importance of iron in mediating skeletal muscle adaptation responses over a longer period of time