Rat H9C2 cardiomyocytes and rat L6G9C2 (L6) skeletal myocytes (Sigma-Aldrich, St. Louis, MO, USA) were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mmol/l glutamine, 10⁴ diluted 10,000 U/ml penicillin and 10 mg/ml streptomycin (all from Sigma-Aldrich). For passaging, the cells were washed with phosphate-buffered saline (PBS; without Ca²⁺ and Mg²⁺) and were released by trypsinization (Sigma-Aldrich). The cells were maintained according to manufacturer's protocols.

Rat H9C2 cardiomyocytes and rat L6G9C2 skeletal myocytes were cultured with an addition of deferoxamine (DFO; Sigma-Aldrich) or ammonium ferric citrate (AFC; Sigma-Aldrich) in order to change iron accessibility in hypoxia for those cells in the cultured medium during hypoxia treatment for 48 h. DFO is a selective iron chelator commonly used in the cell culture studies. It has been reported that addition of DFO into the culture medium reduces iron concentration both in the cellular environment and inside the cell, since DFO can be taken up by fluid phase endocytosis (30). AFC was applied in cell culture studies in order to induce intracellular iron accumulation (31,32).

H9C2 and L6 cells were seeded at 0.3×10⁶ cells/well in a volume of 2 ml in 6-well plates. The cells were cultured under hypoxic conditions (1% O₂, 5% CO₂, 94% N₂), which were generated in a standard cell culture incubator by displacing O₂ with infusion of N₂, which was supplied by an external high-pressure liquid nitrogen tank, in differential iron availability in the growth media for 48 h. The cells were separated into three groups: i) Optimal iron concentration (standard iron concentration in DMEM with 10% FBS); ii) reduced iron concentration (iron chelation using 100 µM DFO); iii) increased iron concentration (supplementation with 200 µM AFC). The compounds were added to the cells from 1,000X concentrated stocks diluted in culture medium.

Multiple parameters were assessed during the present study. These parameters reflected the cellular condition and iron status assessed in the states of different iron availability in culture medium of H9C2 and L6 lines. These parameters were looking at the expression levels of various genes in the following processes: i) Apoptosis, including B-cell lymphoma (Bcl)-2-associated X protein (Bax; inductor) (33), Bcl-2 (inhibitor) (34) and the Bax/Bcl-2 gene expression ratio (35); ii) proteolysis, including Atrogin-1, which is a marker of protein degradation in myocytes and a marker of muscle atrophy (36); iii) glycolysis, including pyruvate kinase (PKM2; marker of non-oxidative metabolism) (37); iv) intracellular iron metabolism, including transferrin receptor type 1 (TfR1; cellular iron importer) (38), ferroportin (FPN1; cellular iron exporter) (39), FTH heavy chain (iron storage protein) (40) and hepcidin (HAMP; iron metabolism regulator protein) (41).

The MTS assay is a colorimetric method used to determine the number of viable and metabolically active cells in proliferation and cytotoxicity assays, as previously described (42). MTS assays were performed, according to manufacturer's protocol (CellTiter 96^® AQueous One Solution Cell Proliferation Assay; Promega Corporation, Madison, WI, USA). Briefly, 2×10⁵ H9C2 or L6 cells were seeded into each well of 96-well plates and were treated for 48 h with DFO, AFC or PBS (control) in hypoxia or in normoxia, as described above. A total of 20 µl CellTiter 96^® AQueous One Solution reagent was added to each well and the absorbance at 490 nm was measured after 2 h incubation in 37°C (KCjunior™; BioTek Instruments, Inc., Winooski, VT, USA). The viability of the control cells was treated as 100%.

The total RNA was prepared from H9C2 or L6 cells harvested from 6-well tissue culture plates using the RNeasy Fibrous Tissue Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The protocol included an on-column DNase digestion to remove the genomic DNA. First-strand cDNA was synthesized using a SuperScript III First-Strand Synthesis system with oligo(dT)20 primer (Invitrogen; Thermo Fisher Scientific, Inc.).

Based on the rat genomic and cDNA sequences the following primers were used: Bcl-2, forward: 5′-AGCATGCGACCTCTGTTTGA-3′ and reverse 5′-TCACTTGTGGCCCAGGTATG-3′; Bax, forward: 5′-TGGCGATGAACTGGACAACA-3′ and reverse: 5′-CACGGAAGAAGACCTCTCGG-3′; Atrogin-1 forward: 5′AGCTTGTGCGATGTTACCCA-3′ and reverse 5′-GAGCAGCTCTCTGGGTTGTT-3′; PKM2, forward: 5′-TTAGGCCAGCAACGCTTGTA-3′ and reverse: 5′-AGCTGGGCTCTATTGCATGT-3′; TfR1, forward: 5′-GAGACTACTTCCGTGCTACTTC-3′ and reverse: 5′-TGGAGATACATAGGGTGACAGG-3′; FPN1, forward: 5′-TCGGTCTTTGGTCCTTTGATTTG-3′ and reverse 5′-GGCTGACTTTCATCTGTAACTTCC-3′; FTH, forward: 5′-GCCAAATACTTTCTCCATCAATCTC-3′ and reverse: 5′-CCGCTCTCCCAGTCATCAC-3′; HAMP, forward: 5′-GCAACAGACGAGACAGACTAC-3′ and reverse 5′-GCAACAGAGACCACAGGAG-3′. The primers were designed with Molecular Beacon Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers spanned exon junctions to prevent the amplification of genomic DNA. The reference genes, Actb and 18S-RNA, were selected for normalization of experimental conditions by means of geNorm analysis (PrimerDesign Ltd., Southampton, UK).

All samples were performed in triplicates. The qPCR assay was performed on the CFX Connect Real-Time PCR Detection system. The specificity of PCR was determined by melting curve analysis for each reaction. The amplification efficiency was estimated by running serial dilutions of a template. Successive dilutions were plotted against the appropriate Cq values to generate a standard curve. The slope calculated from the standard curve was used to determine the amplification efficiency (E) according to the formula: E=10-1/slope. The amplification efficiencies for the target amplicons, actb and 18srna, were not comparable and the Pfaffl method was used to determine the relative expression (43).

The data are presented as the mean ± standard deviation, unless otherwise indicated. Mann-Whitney U Test was used to compare the groups. All molecular assessments were performed in triplicates. R-value is a correlation coefficient between the expression of iron metabolism genes in three states of iron concentrations and the Bax/Bcl-2 gene expression ratio in those conditions. P<0.05 was considered to indicate a statistically significant difference.

Low oxygen treatment reduced the viability of cardiomyocytes and myocytes by 11 and 12%, respectively, as compared with the untreated cells (P<0.01 each). In each cell type, the decrement in viability was associated with an increased Bax/Bcl-2 gene expression ratio (P<0.05), reflecting an enhanced susceptibility of cardiomyocytes and myocytes to cellular programmed death (Fig. 1).

In both cardiomyocytes and myocytes, the exposition to low iron availability during hypoxia caused, as compared with the cells cultured during hypoxia and optimal iron concentration, a 15% loss in viability in each of the cell lines (P<0.01; Fig. 1A), and this change was associated with an increase in Bax/Bcl-2 gene expression ratio (P<0.001; Fig. 1B). Likewise, both H9C2 and L6 cells demonstrated an increase in Bax/Bcl-2 gene expression ratio (P<0.05 and P<0.01; Fig. 1B) during hypoxia, when exposed to the elevated iron availability, which was associated with 10 and 11% loss in viability in cardiomyocytes and myocytes, respectively (P<0.01; Fig. 1A). Notably, in each cell type during hypoxia, the increase in the Bax/Bcl-2 ratio (Fig. 1B) and associated viability loss (Fig. 1A) were greater upon exposure to low iron availability compared with that in the case of AFC treatment (P<0.05 each).

Hypoxic conditions of L6 cell culture caused an increase in the expression of PKM2, which responsible for the rate-limiting step of glycolysis, when compared with cells cultured in normoxia (P<0.01). Myocytes exposed to DFO during hypoxia demonstrated increased mRNA expression levels of PKM2 and Atrogin-1 (P<0.001 and P<0.05, respectively) as compared with the cells cultured in normal iron concentration, suggesting the further enhancement of non-oxidative glycolytic pathway and protein degradation in the cells. The different changes were observed in myocytes treated with AFC, since the increase in the expression of PKM2 (P<0.01) was accompanied by a decrease in the mRNA expression of Atrogin-1 (P<0.05) as compared with the cells cultured in optimal iron concentration. The augmented expression of PKM2 in myocytes during hypoxia was greater upon low iron availability compared with during ferric salt treatment (P<0.01). Notably, the expression of Atrogin-1 during hypoxia and concomitant elevated iron availability was decreased as compared with cells cultured in hypoxia and normal or decreased iron concentration (P<0.05 and P<0.01, respectively; Fig. 2).

Both L6 and H9C2 cell lines exposed to hypoxia demonstrated, as compared with the cells cultured in normoxia, a decrease in the expression TfR1 (P<0.05 each) and FPN1 (P<0.05 and P<0.01, respectively).

The addition of DFO to the culture medium of cardiomyocytes and myocytes during hypoxia caused an increased expression of TfR1 (P<0.01 and P<0.001, respectively), reflecting iron demand, thus facilitated entrance of iron to the cells. However, this did not affect the expression of FPN1 compared with the cells cultured in normal iron concentration.

The AFC treatment of cardiomyocytes and myocytes during hypoxia resulted in a decreased and increased, respectively, expression of TfR1 (P<0.05 each). Additionally, this resulted in an increased expression of FPN1 (P<0.05 each) in each cell line. Under hypoxic conditions with concomitant changing iron availability, the myocytes demonstrated low expression of FPN1 when compared with the cells cultured in normoxia (P<0.01) (Fig. 3A and B).

Low oxygen treatment of L6 cells caused, as compared with cells cultured in normoxia, an increased expression of the iron storage gene, FTH (P<0.05). Exposure to DFO of cardiomyocytes during hypoxia resulted, as compared with hypoxia-treated controls, in unchanged mRNA expression of FTH, while hypoxic culture of L6 cells exposed to low iron demonstrated an increase in the expression of the iron storage gene (P<0.01).

The different pattern of changes was observed upon ferric salt treatment during hypoxia. An increased expression of FTH in both cardiomyocytes and myocytes was observed compared with the cells cultured in normal iron concentration (P<0.05 and P<0.01, respectively) (Fig. 3C).

Low oxygen treatment of H9C2 and L6 cells caused an increment in the expression of iron regulator, HAMP (P<0.05 and P<0.01, respectively). An addition of DFO to the culture medium of both studied cell lines during hypoxia caused a further increase in the expression of HAMP (P<0.05 and P<0.001) as compared with the cells cultured in a normal iron concentration. The exposition of cardiomyocytes and myocytes to AFC treatment in hypoxia resulted in an unchanged or a decreased expression of HAMP (P<0.01) (Fig. 3D).

In both cardiomyocytes and myocytes, the expression of genes involved in iron influx (TfR1) and regulation (HAMP) was associated with the Bax/Bcl-2 gene expression ratio in the whole spectrum of iron status during hypoxia (all R>0.6, P<0.05). In particular, in a low iron state, the observed upregulation of TfR1 and HAMP genes was associated with increased apoptotic activity of each of the cell lines. In addition, L6 cells demonstrated positive correlation between the expression of FTH and the Bax/Bcl-2 gene expression ratio (R=0.64, P<0.05) (Fig. 4).