Both iron excess and iron depletion impair viability of rat H9C2 cardiomyocytes and L6G8C5 myocytes

Abstract

A b s t r a c tBackground: Iron is presumed to play an important role in the functioning of cardiomyocytes and skeletal myocytes. There is scarcity of direct data characterising the cells functioning when exposed to iron depletion or iron overload in a cellular environment. There is some clinical evidence demonstrating that iron deficiency has serious negative prognostic consequences in heart failure (HF) patients and its correction brought clinical benefit. Aim:The viability of the cells upon unfavourable iron concentration in the cell culture medium and the presence of the molecular system of proteins involved in intracellular iron metabolism in these cells have been studied.Methods: H9C2 rat adult cardiomyocytes and L6G8C5 rat adult skeletal myocytes were cultured for 24 h in optimal vs. reduced vs. increased iron concentrations. Intracellular iron content was measured by flame atomic absorption spectroscopy (FAAS). We analysed the mRNA expression of: ferritin heavy and light chains (FTH and FTL; iron storage proteins), myoglobin (MB, oxygen storage protein) ferroportin type 1 (FPN1; iron exporter), transferrin receptor type 1 (TfR1; iron importer), hepcidin (HAMP; iron metabolism regulator) using qPCR, the level of respective proteins using Western Blot (WB), and the viability of the cells using flow cytometry and cell viability tetrazolium reduction assay (MTS).Results: Cardiomyocytes exposed to gradually reduced iron concentrations in the medium demonstrated a decrease in the mRNA expression of FTH, FTL, FPN1, MB, and HAMP (all R = -0.75, p < 0.05), indicating depleted iron status in the cells. As a consequence, the expression of TfR1 (R = 0.7, p < 0.05) was increased, reflecting a facilitated entrance of iron to the cells. The inverse changes occurred in H9C2 cells exposed to increased iron concentrations in the medium in comparison to control cells. The same pattern of changes in the mRNA expressions was observed in myocytes, and there was a strong correlation between analogous genes in both cell lines (all R > 0.9, p < 0.0001). WB analysis revealed the analogous pattern of changes in protein expression as an mRNA profile. Both iron depletion and iron excess impaired viability of cardiomyocytes and skeletal myocytes. Conclusions:Both rat cardiomyocytes and myocyte cells contain the set of genes involved in the intracellular iron metabolism, and both types of investigated cells respond to changing iron concentrations in the cultured environment. Both iron deficiency (ID) and iron overload is detrimental for the cells. This data may explain the beneficial effects of iron supplementation in patients with ID in HF.

Iron limitation promotes the atrophy of skeletal myocytes, whereas iron supplementation prevents this process in the hypoxic conditions

“…There is also a scarcity of data on the influence of both iron excess and iron deficiency on atrophy markers in skeletal myocytes when exposed to hypoxia. The present authors previously studied the influence of increased or reduced iron availability in hypoxic conditions on skeletal myocytes and demonstrated that, during hypoxia, the reduced iron concentration had a more negative impact on the viability and apoptotic activity of the studied cells as compared with elevated iron availability ( 24 , 42 ). The present authors' preliminary results also demonstrated that, in skeletal myocytes, the mRNA expression of muscle-specific atrophy marker Atrogin-1 was increased upon reduced iron availability, and was downregulated in increased iron concentrations ( 24 ).…”

Structural and functional abnormalities in iron-depleted heart

“…Interestingly, supplementation of transferrin-bound iron reversed all functional and morphological abnormalities. Additionally, our recent research showed that intracellular iron depletion is detrimental for functioning of cardiomyocytes and skeletal myocytes and leads to increased apoptosis and reduces cell viability [57, 58].…”

Effects of Iron Overload and Oxidative Damage on the Musculoskeletal System in the Space Environment: Data from Spaceflights and Ground-Based Simulation Models

“…Furthermore, treatment by antioxidant could abrogate iron loading-induced muscle atrophy by activating the Akt-FOXO3a pathway [ 105 ]. In vitro, Kasztura et al [ 106 ] used ferric ammonium citrate (FAC) to treat rat skeletal myocyte L6G8C5 cells, which showed reduced myoglobin levels and decreased proliferative activity in an iron overload condition. In hemodialysis patients, increased serum ferritin significantly correlated with handgrip strength and muscle quality, and thus suggested that iron overload should be concerned, to avoid its possibly detrimental effect on muscle in such patients [ 107 ].…”