Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes

Abstract

The differential availability of iron during hypoxia is presumed to affect the functioning of cardiac and skeletal myocytes. Rat H9C2 cardiomyocytes and L6G8C5 myocytes were cultured for 48 h in normoxic or hypoxic conditions at the optimal, reduced or increased iron concentration. The mRNA expression levels of markers of apoptosis [B‑cell lymphoma‑2 (Bcl2; inhibition) and Bcl‑2‑activated X protein (Bax; induction)], atrophy (Atrogin), glycolysis (pyruvate kinase 2; PKM2) and iron metabolism [transferrin receptor 1 (TfR1; iron importer), ferroportin 1 (FPN1; iron exporter), ferritin heavy chain (FTH; iron storage protein) and hepcidin (HAMP; iron regulator)] were determined using reverse transcription‑quantitative polymerase chain reaction, and cell viability was measured using an tetrazolium reduction assay. Cardiomyocytes and myocytes, when exposed to hypoxia, demonstrated an increased Bax/Bcl‑2 gene expression ratio (P<0.05). Additional deferoxamine (DFO) treatment resulted in further increases in Bax/Bcl‑2 in each cell type (P<0.001 each) and this was associated with the 15% loss in viability. The analogous alterations were observed in both cell types upon ammonium ferric citrate (AFC) treatment during hypoxia; however, the increased Bax/Bcl‑2 ratio and associated viability loss was lower compared with that in case of DFO treatment (P<0.05 each). Under hypoxic conditions, myocytes demonstrated an increased expression of PKM2 (P<0.01). Additional DFO treatment caused an increase in the mRNA expression levels of PKM2 and Atrogin‑1 (P<0.001 and P<0.05, respectively), whereas AFC treatment caused an increased mRNA expression of PKM2 (P<0.01) and accompanied decreased mRNA expression of Atrogin‑1 (P<0.05). The expression augmentation of PKM2 during hypoxia was greater upon low iron compared with that of ferric salt treatment (P<0.01). Both cell types upon DFO during hypoxia demonstrated the increased expression of TfR1 and HAMP (all P<0.05), which was associated with the increased Bax/Bcl‑2 ratio (all R>0.6 and P<0.05). In conclusion, during hypoxia iron deficiency impairs the viability of cardiomyocytes and myocytes more severely compared with iron excess. In myocytes, during hypoxia iron may act in a protective manner, since the level of atrophy is decreased in the iron‑salt‑treated cells.

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

“…It may be hypothesized that the disturbed iron metabolism observed in HF affects skeletal muscles, which leads to their dysfunction and exercise intolerance. The present authors have recently demonstrated that, during hypoxia, reduced iron concentration had a greater negative impact on the viability and functioning of myocytes, compared with augmented iron availability ( 24 ).…”

“…Rat L6G8C5 skeletal myocytes (L6; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were cultured in normoxia (18% O 2 , 5% CO 2 ) or hypoxia (1% O 2 , 5% CO 2 , 94% N 2 ), for 48 h ( 24 ), supplemented with 100 µ M deferoxamine (DFO; Sigma-Aldrich; Merck KGaA) or 200 µ M ammonium ferric citrate (AFC; Sigma-Aldrich; Merck KGaA) in order to change iron accessibility ( Fig. 1 ).…”

“…The following parameters were measured in the L6 cell line using differing states of iron availability in the culture medium: Atrogin-1 and muscle-specific RING-finger 1 ( MuRF-1 ), which are muscle-specific ubiquitin E3-ligases that are a part of the ubiquitin proteasome pathway associated with protein degradation during muscle atrophy, and are markedly induced in almost all types of atrophy ( 25 – 27 ); mothers against decapentaplegic homolog 4 ( SMAD4 ), which is a transcriptional factor that serves a central role in the balance between atrophy and hypertrophy ( 28 – 30 ); and Desmin , which is a structural protein that builds class-III intermediate filaments found in muscle cells. B cell lymphoma-2 (Bcl-2) associated protein X (Bax)/Bcl-2 gene expression ratio ( 31 ) and expression of transferrin receptor 1 ( TfR1 ) ( 32 ) were evaluated as previously described ( 24 ), to evaluate apoptotic activity and iron influx, respectively.…”

Adverse Outcome Prediction of Iron Deficiency in Patients with Acute Coronary Syndrome

“…There are three main issues that need to be considered in relation to the ACS pathophysiology. First, accumulated experimental data clearly show that during hypoxic conditions, mainly caused by underlying ischaemic disease, iron impairs the viability of cardiomyocytes [ 20 ]. Iron seems to act as a protective agent, since the levels of cellular atrophy decreases in hypoxic rat cardiomyocytes treated with iron salt [ 19 ].…”

Intrinsic Iron Release Is Associated with Lower Mortality in Patients with Stable Coronary Artery Disease—First Report on the Prospective Relevance of Intrinsic Iron Release

“…This might enhance ischemic stress and apoptosis of cardiomyocytes [ 32 ]. Moreover, experimental data clearly show that during hypoxic conditions, mainly caused by underlying ischaemic disease, iron deficiency impairs the viability of cardiomyocytes [ 33 ]. In contrast, the availability of iron during hypoxic conditions is beneficial on genes associated with apoptotic activity in rat cardiomyocytes and also on skeletal myocytes.…”