Effects of iron loading on muscle: genome-wide mRNA expression profiling in the mouse

Abstract

Background: Hereditary hemochromatosis (HH) encompasses genetic disorders of iron overload characterized by deficient expression or function of the iron-regulatory hormone hepcidin. Mutations in 5 genes have been linked to this disease: HFE, TFR2 (encoding transferrin receptor 2), HAMP (encoding hepcidin), SLC40A1 (encoding ferroportin) and HJV (encoding hemojuvelin). Hepcidin inhibits iron export from cells into plasma. Hemojuvelin, an upstream regulator of hepcidin expression, is expressed in mice mainly in the heart and skeletal muscle. It has been suggested that soluble hemojuvelin shed by the muscle might reach the liver to influence hepcidin expression. Heart muscle is one of the target tissues affected by iron overload, with resultant cardiomyopathy in some HH patients. Therefore, we investigated the effect of iron overload on gene expression in skeletal muscle and heart using Illuminatrade mark arrays containing over 47,000 probes. The most apparent changes in gene expression were confirmed using real-time RT-PCR.

Results: Genes with up-regulated expression after iron overload in both skeletal and heart muscle included angiopoietin-like 4, pyruvate dehydrogenase kinase 4 and calgranulin A and B. The expression of transferrin receptor, heat shock protein 1B and DnaJ homolog B1 were down-regulated by iron in both muscle types. Two potential hepcidin regulatory genes, hemojuvelin and neogenin, showed no clear change in expression after iron overload.

Conclusion: Microarray analysis revealed iron-induced changes in the expression of several genes involved in the regulation of glucose and lipid metabolism, transcription and cellular stress responses. These may represent novel connections between iron overload and pathological manifestations of HH such as cardiomyopathy and diabetes.

Figure 1

Hepatic and cardiac non-heme iron concentrations. Iron contents were studied in three strains of male and female mice fed either the control or high-iron diet. The result values are expressed as mean +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. F = female; M = male.

Figure 2

Confirmation of microarray results for skeletal muscle by Q-RT-PCR. The experiments were performed on samples derived from C57BL/6 male mice. The result values are expressed as mean of triplicate runs +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. A, Q-RT-PCR analysis of 6 genes with up-regulated expression after iron overload. B, Q-RT-PCR evaluation of 5 genes with iron-induced down-regulation of expression by microarray analysis.

Figure 3

Verification of data obtained for heart samples by microarray analysis using Q-RT-PCR. Samples from C57BL/6 male mice were used in these experiments. The result values are expressed as mean of triplicate runs +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. A, Q-RT-PCR evaluation of seven genes with up-regulated expression after iron overload. B, Q-RT-PCR analysis of seven genes with iron-induced down-regulation of expression by microarray.

Figure 4

Expression analysis of genes presenting same trend in muscular tissues and comparison with hepatic expression. C57BL/6 male mice were used in this analysis. The result values are expressed as mean of triplicate runs +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. A-D, Genes with up-regulated expression in both skeletal muscle and heart after iron overload. E-G, Genes with down-regulated expression in skeletal muscle and heart after iron overload.

Figure 5

Expression of hepcidin1 in skeletal muscle (A), heart (B) and liver (C) assessed by Q-RT-PCR. The expression of hepcidin1 transcripts was assessed in control versus iron fed mice of 3 strains (Balb/c, C57BL/6, DBA2). The result values are expressed as mean of triplicate runs +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. F = female; M = male.

Figure 6

Q-RT-PCR analysis of hepcidin2 mRNA expression in skeletal muscle (A), heart (B) and liver (C). The expression of hepcidin2 in control versus iron overloaded mice was analyzed in 3 strains (Balb/c, C57BL/6, DBA2). The result values are expressed as mean of triplicate runs +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. F = female; M = male.

Figure 7

Expression of hemojuvelin (HJV) in skeletal muscle (A), heart (B) and liver (C). Q-RT-PCR analysis of HJV mRNA levels in control versus iron overloaded mice of 3 strains (Balb/c, C57BL/6, DBA2). The result values are expressed as mean of triplicate runs +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. F = female; M = male.

Figure 8

Neogenin transcript levels in skeletal muscle (A), heart (B) and liver (C). 3 mouse strains (Balb/c, C57BL/6, DBA2) were used for this Q-RT-PCR analysis. The result values are expressed as mean of triplicate runs +/- standard deviation. Statistical significant differences relative to control diet fed mice were determined. *p < 0,05; **p < 0,01; ***p < 0,001. F = female; M = male.