title: Both iron excess and iron depletion impair viability of rat H9C2 cardiomyocytes and L6G8C5 myocytes.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Laboratory for Applied Research on the Cardiovascular System, Department of Heart Diseases, Wroclaw Medical University, Wroclaw, Poland. monikakasztura@gmail.com.
authors.forename: Monika
authors.initials: M
authors.lastname: Kasztura
authors.forename: Magdalena
authors.initials: M
authors.lastname: Dzięgała
authors.forename: Kamil
authors.initials: K
authors.lastname: Kobak
authors.forename: Jacek
authors.initials: J
authors.lastname: Bania
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Mazur
authors.forename: Waldemar
authors.initials: W
authors.lastname: Banasiak
authors.forename: Piotr
authors.initials: P
authors.lastname: Ponikowski
authors.forename: Ewa Anita
authors.initials: EA
authors.lastname: Jankowska
mesh_terms: D018798:Anemia, Iron-Deficiency; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D027682:Cation Transport Proteins / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival; D005293:Ferritins / Q000235:genetics; D005786:Gene Expression Regulation; D064451:Hepcidins / Q000235:genetics; D007501:Iron / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D019190:Iron Overload; D032342:Muscle Cells / Q000378:metabolism / Q000502:physiology*; D009752:Nutritional Status; D051381:Rats; D011990:Receptors, Transferrin / Q000235:genetics; D000098742:Ferroportin
chemical_list: D015703:Antigens, CD; C068126:CD71 antigen; D027682:Cation Transport Proteins; D064451:Hepcidins; D011990:Receptors, Transferrin; D000098742:Ferroportin; D005293:Ferritins; D007501:Iron
keywords: cardiomyocyte; iron deficiency; iron excess; iron metabolism genes; skeletal myocyte