BACKGROUND
it is crucial for the human body to maintain iron homeostasis.
since there is no adjustable mechanism to influence iron loss from the body, tight regulation of iron absorption at the intestinal level is vital  <cit> .
in order to maintain iron balance, iron export from enterocytes, reticuloendothelial macrophages and hepatocytes into the blood stream has to be controlled as well.
functional derangement of proteins involved in these regulatory mechanisms can cause hereditary hemochromatosis .
this genetic disorder of iron overload is characterized by high transferrin saturation, low iron content in macrophages, and deposition of iron in several organs including the liver, heart, and pancreas.
causative mutations for hh have been described in several genes, namely hfe, tfr <dig> , hjv , and hamp   <cit> .
it has been proposed that these mutations cause deficient hepcidin synthesis  <cit> .
the antimicrobial peptide hepcidin is the central regulator of iron metabolism.
it is produced mainly in the liver and exerts its function by binding to the iron export protein, ferroportin, inducing its internalization and degradation  <cit> .
ferroportin is located in the cellular membranes of enterocytes, reticuloendothelial cells, hepatocytes and placental cells  <cit> .
therefore, hepcidin acts to decrease the export of iron from these cells into the circulation.
hemojuvelin is a glycosyl phosphatidylinositol-anchored protein which belongs to the repulsive guidance molecule  protein family  <cit> .
recent studies suggest that hemojuvelin exists in two forms.
one is a rarer full-length protein shed to the extracellular fluid, where it has a long half-life.
the other is a smaller, membrane-associated disulfide-linked heterodimer, which is a more abundant but shorter-lived form composed of n- and c-terminal fragments  <cit> .
according to latest studies the most common mutation in hemojuvelin  affects the targeting of the membrane-associated form and reduces the amount of the soluble form  <cit> .
interestingly, studies in cultured cells suggest that the two forms regulate hepcidin expression reciprocally by competing for a receptor binding site  <cit> .
evidence shows that hemojuvelin is a bone morphogenetic protein co-receptor, and its interaction with bmp initiates a signaling cascade that leads to regulation of hepcidin expression  <cit> .
on the other hand, it has been observed that overexpressed hemojuvelin binds to the membrane receptor neogenin and that this interaction is required for the accumulation of iron in cultured cells  <cit> .
zhang et al.
also showed that the g320v mutated hemojuvelin overexpressed in vitro was not able to bind neogenin, and that iron did not accumulate in the cells under these conditions.
furthermore, a recent study in cultured cells suggested that neogenin may mediate inhibition of hemojuvelin shedding in response to iron  <cit> .
we have previously determined the sites of simultaneous expression of hemojuvelin and neogenin  <cit> .
the highest expression of hemojuvelin transcript is found in the skeletal muscle and heart.
although in vivo evidence of a combined role of hemojuvelin and neogenin in iron homeostasis has not been provided yet, it has been suggested that hemojuvelin shed from skeletal muscle and heart by neogenin-dependent mechanism could reach the liver to influence hepcidin expression  <cit> .
cardiomyopathy develops in some hh patients  <cit> .
in order to better understand the mechanisms behind pathological effects of iron overload in muscle cells, we have performed a genome-wide expression analysis of genes in skeletal muscle and heart of mice with or without dietary iron loading.
microarray data analysis identified several genes whose expression was either down- or up-regulated due to iron overload.
these results may reveal novel links between iron overload and pathological manifestations of hh.
RESULTS
documentation of iron overload in the liver and heart of iron-fed mice
the mice were fed either standard  or high-iron  diet for  <dig> weeks.
iron concentrations of liver and heart specimens were determined to confirm the validity of the animal model.
figure 1a demonstrates that the livers of mice of all three strains were highly iron-loaded when fed an iron-rich diet.
a much smaller increment in cardiac iron content after a high-iron diet was observed also in all three strains and in both genders , although statistical significance was not reached in all the cases.
basal cardiac iron levels were lower than basal hepatic contents.
in general, female mice showed slightly higher hepatic and cardiac iron levels than male mice.
identification and validation of changes in gene expression induced by dietary iron overload in skeletal muscle and heart
we obtained a list of  <dig> genes with iron-induced up-regulated expression in skeletal muscle  and forty with down-regulated expression .
in the heart, iron loading resulted in the up-regulation of  <dig> genes , while forty genes had down-regulated expression after iron overload .
there were seven genes which were up-regulated in both the heart and skeletal muscle, while nine genes were down-regulated in both tissues.
data obtained with  <dig> high-iron and  <dig> control samples.
data obtained with  <dig> high-iron and  <dig> control samples.
*data obtained with  <dig> high-iron and  <dig> control samples.
** data obtained with  <dig> high-iron and  <dig> control samples.
*data obtained with  <dig> high-iron and  <dig> control samples.
** data obtained with  <dig> high-iron and  <dig> control samples.
from the lists of microarray results we selected  <dig> genes which presented the highest fold change values.
the expression levels of these genes were then analyzed in the same mouse strain  by q-rt-pcr .
certainly, the results from these analyses showed a good correlation between the two methods; the expression of all the genes was regulated and displayed the same direction of change.
the fold change values obtained from pcr experiments were all over  <dig>  except for the tfrc gene whose downregulation in the skeletal muscle reached the value of - <dig> .
representations of these q-rt-pcr results are depicted in figures  <dig> and  <dig>  in general, the fold-change values obtained by microarray analysis were smaller than those determined by q-rt-pcr.
this phenomenon has been described previously and is probably due to the fact that array analyses are less quantitative than q-pcr  <cit> .
the hepatic mrna levels of these  <dig> genes were also analyzed by q-rt-pcr.
the results for genes whose expression varied in the same direction in both skeletal muscle and heart after iron loading are shown together with their expression in the liver in figure  <dig>  the expression of four of the  <dig> genes  was considered negligible in the liver because of very low signal intensity.
among the  <dig> remaining genes only the hepatic expression of pdk <dig>  and cxcl <dig>  was not significantly regulated by dietary iron.
expression of genes involved in hepcidin regulatory pathway during dietary iron overload
one of the aims of this study was to explore the effect of dietary iron overload on the expression of the iron-related genes hemojuvelin  and neogenin  in skeletal muscle and heart.
we did not observe differential expression of these genes or any of the traditional iron-regulatory genes  by the microarray approach, except for the transferrin receptor gene , which was down-regulated by iron in heart, skeletal muscle and liver .
even though the microarray method we used proved to be very accurate, we wanted to verify these results and to explore the response of hepcidin expression in the studied tissues by q-rt-pcr.
the expression of hepcidin <dig> and hepcidin <dig> in the liver was greatly up-regulated by iron overload and varied according to mouse strain and gender  <cit> .
in balb/c and c57bl/ <dig> mice, hepcidin  <dig> was the predominant form expressed in the liver, while in dba <dig> mice, the hepatic expression of hepcidin  <dig> was dominant .
the expression of hepcidin <dig> in the skeletal muscle was negligible in all strains .
in the heart muscle, it showed a slight tendency towards decreased expression in most iron fed mice, although the baseline signal in control mice was already quite low .
only dba <dig> mice expressed hepcidin <dig> in the skeletal muscle and heart, and this expression was not clearly regulated by iron overload .
the results for hemojuvelin expression did not indicate any clear regulation by iron overload, strain or gender in any of the tissues studied .
this is in agreement with previous studies of hepatic expression  <cit> .
hemojuvelin expression only showed a minor trend downwards in skeletal muscle and heart of mice fed with high-iron diet.
no significant changes were observed for neogenin expression .
discussion
excess free iron participates in the formation of free radicals causing oxidative stress and cell damage, which is evidenced as a series of pathological manifestations  <cit> .
while some studies have analyzed the effects of iron on the transcriptional profiles in liver and duodenum, this is the first study reporting changes in mrna expression that may contribute to iron-induced effects on skeletal muscle and heart.
we used a genome-wide mrna expression profiling approach and validated the most substantive changes by q-rt-pcr.
expression of antioxidant enzymes is considered a protective mechanism against oxidative stress-induced damage.
however, the regulation of antioxidant enzymes in response to oxidative stress is a rather controversial issue, as the results vary greatly depending on the type and length of the stimulus and the type of cells or tissue tested.
we did not find increased expression of oxidative stress-related genes or antioxidant enzymes after iron overload, except for glutathione peroxidase  <dig>  whose expression was induced by  <dig> -fold change in heart .
the data analysis identified two genes encoding calcium- and zinc-binding proteins, s100a <dig>  and s100a <dig>  among the up-regulated genes presenting the highest fold changes.
these proteins form a rather ubiquitous heterodimer called calprotectin.
the highest amounts of this protein complex are located in neutrophil granulocytes, monocytes and keratinocytes  <cit> .
calprotectin is a pro-inflammatory cytokine that is upregulated in many inflammatory conditions, and is involved in innate immunity, leukocyte adhesion, endothelial transmigration and processes of chronic inflammation  <cit> .
in vitro studies have shown that reactive oxygen species  induce protein levels of s100a <dig>  <cit> .
additionally, s100a <dig> expression is induced in keratinocytes upon exposure to ultraviolet a  radiation, a stimulus causing oxidative stress.
interestingly, the antioxidant enzymes superoxide dismutase and catalase, whose mrna expression was unaffected in our microarray, abrogate s100a <dig> induction  <cit> .
we found that both s100a <dig> and s100a <dig> transcripts were substantially up-regulated in skeletal muscle, heart and liver of iron-loaded mice, although the levels of s100a <dig> transcript in skeletal muscle were negligible and, in general, both genes were weakly expressed.
this demonstrates the high sensitivity and accuracy of both the microarray analysis and the q-rt-pcr method.
it is noteworthy that s100a <dig> and s100a <dig> transcripts showed a very similar up-regulation pattern in the tissues studied, which agrees with the concept of the two proteins forming a heterodimer.
the transcriptional regulation of the s100a <dig> and s100a <dig> genes seems to be rather complex, with promoter binding sites for transcription factors such as activator protein  <dig> , nuclear factor kappa b  and c/ebp.
consistently, at least ap- <dig> and nf-κb have been previously identified to be regulated by the intracellular redox state  <cit> .
pyruvate dehydrogenase kinase  <dig>  phosphorylates and inactivates the pyruvate dehydrogenase complex, decreasing the rate of glucose oxidation and thus increasing blood glucose levels.
increased pdk <dig> expression and activity has been observed in both skeletal muscle  <cit>  and heart  <cit>  of insulin-resistant mouse models.
the question arises of whether pdk <dig> overexpression causes insulin resistance or vice versa.
insulin suppresses pdk <dig> expression in skeletal muscle  <cit>  and, according to a recent study by kim and coworkers  <cit> , this effect is impaired in insulin resistance, suggesting that insulin resistance may indeed induce pdk <dig> expression.
however, several studies using high-fat dietary models of insulin resistance indicate that pdk <dig> overexpression occurs before the development of insulin resistance  <cit> .
although it has not been documented directly that increased pdk <dig> mrna levels can indeed cause insulin resistance, it seems possible that a vicious cycle may exist between these two phenomena.
in the present study, we show an up-regulation of pdk <dig> mrna levels in skeletal muscle and heart but not in the liver of iron-loaded mice.
diabetes mellitus is the major endocrine disorder associated with hh.
the mechanisms responsible for this clinical manifestation are still obscure, but two processes have been proposed: the pancreatic β-cell iron accumulation results in cell damage and diminished insulin secretion, and liver iron overload leads to insulin resistance  <cit> .
the herein reported induction of pdk <dig> expression in the skeletal and heart muscle might represent a novel mechanism involved in the development of diabetes mellitus in hh.
angiopoietin-like  <dig>  is a secreted protein produced mainly in fat tissue, and to a lesser extent in liver, placenta, skeletal muscle and heart.
it is directly involved in regulating glucose homeostasis, lipid metabolism, and insulin sensitivity.
angptl <dig> decreases the activity of lipoprotein lipase , thus inhibiting lipoprotein metabolism and increasing plasma triglyceride levels.
transgenic mice with angptl <dig> overexpression directed to heart muscle  show reduced cardiac lpl activity, decreased triglyceride utilization and impaired cardiac function resulting in cardiomyopathy  <cit> .
transgenic overexpression of angptl <dig> from a liver-specific promoter causes hypertriglyceridemia similar to that induced by adenoviral over-expression  <cit> .
these results support the hypothesis that angptl <dig> has lpl-dependent actions  <cit> .
accordingly, in lpl-expressing tissues  angptl <dig> may bind directly and inactivate lpl, restricting acquisition of free fatty acids to these sites: it is not shed into plasma from these tissues but rather acts in an autocrine/paracrine fashion.
on the other hand, in the liver, which has low lpl expression, angptl <dig> is shed to plasma and inhibits lpl in other locations, causing a general reduction of triglyceride utilization and acting as an endocrine factor.
interestingly, we showed a 5-fold increase in the level of angptl <dig> transcript in the heart of iron-loaded mice, raising the possibility that early induction of angptl <dig> expression could contribute to the pathogenesis of cardiomyopathy in hh.
the increased expression of pdk <dig> and angptl <dig> observed in skeletal muscle and heart of iron-loaded mice might have its origin in a common mechanism.
the forkhead transcription factor foxo <dig> is a major regulator of insulin action in insulin-sensitive tissues  and it is involved in insulin's action to suppress pdk <dig> and angptl <dig>  <cit> .
myosin light polypeptide  <dig>  ) and myosin light polypeptide  <dig>  ) show a 10-fold up-regulation in the cardiac muscle of iron-loaded mice.
both genes belong to the ef-hand family of ca2+ binding proteins and are part of the myosin molecular complex.
they appear to be involved in force development during muscle contraction.
elc is important in the interaction between myosin and actin  <cit> .
there are two forms of elc in the cardiac muscle, elca and elcv .
elca has a higher performance than elcv and its elevated accumulation in diseased heart is considered a compensatory response in heart failure  <cit> .
furthermore, transgenic rats overexpressing elca in the heart show an improvement in contractile parameters  <cit> .
these observations open the possibility that the induction of cardiac myl <dig> and myl <dig> expression observed in our experiments is a compensatory response to early damage produced by iron accumulation.
additionally, according to our microarray results, other myosin genes were induced by iron in mouse heart  and skeletal muscle .
actin filaments play an essential role, along with myosin, in muscle contraction.
curiously, in the present work, iron suppressed the expression of skeletal muscle and smooth muscle isoforms of actin  in the heart, as well as the cardiac isoform  in skeletal muscle.
the present microarray data analysis identified one gene , which showed marked upregulation  in the skeletal muscle and downregulation  in the heart after iron overload.
this finding was also confirmed by q-rt-pcr.
scd <dig> is an iron-containing enzyme with a central lipogenic role.
it catalyzes the insertion of a double bond into fatty acyl-coa substrates, the preferred one being stearoyl-coa, and yielding oleoyl-coa.
oleic acid is the major monounsaturated fatty acid of membrane phospholipids, triglycerides, cholesterol esters, wax esters and alkyl- <dig> -diacylglicerol.
the  ratio has important effects on cell membrane fluidity and signal transduction.
the overexpression of scd <dig> has been shown to be associated with genetic predisposition to hepatocarcinogenesis  <cit> .
scd <dig> mrna levels were induced  <dig>  times in mouse liver during iron overload , an effect that was previously shown in both enteral and parenteral models of iron overload  <cit> .
as pigeon and coworkers have discussed, most likely the effect of iron on scd <dig> expression in the liver is not direct, but a compensatory mechanism in response to the need to renew unsaturated fatty acids.
the fbj osteosarcoma oncogene  is a major component of activator-protein- <dig> , a redox-sensitive transcription factor complex, which also includes members of the jun , maf and atf subfamilies.
fos is thought to have an important role in signal transduction, cell proliferation and differentiation.
expression of c-fos and c-jun can be induced by many stimuli and compounds, including some metals such as iron  <cit> .
accordingly, the present work shows increased expression of c-fos in the heart and liver of iron-loaded mice.
however, in skeletal muscle, c-fos was down-regulated and, according to the microarray results, the same is true for c-jun.
probably other mechanisms are influencing the transcription of c-fos and c-jun in skeletal muscle.
interestingly, a recent study suggested that c-jun and junb negatively regulate the transcription of s100a <dig> and s100a <dig>  <cit> .
furthermore, ap- <dig> activity had been previously connected to iron metabolism in several ways.
for example, ap- <dig> regulates transcription of ceruloplasmin   <cit> , and the promoter region of hfe contains an ap- <dig> transcription element  <cit> .
heat shock proteins, or stress proteins, are expressed in response to heat shock and a variety of other stress stimuli including oxidative free radicals and toxic metal ions.
the members of the 70-kda heat shock protein family  assist cells in maintaining functional proteins under stressful conditions  <cit> .
hsp <dig> proteins stimulate the atpase activity of hsp <dig> proteins and stabilize the interaction of these chaperons with their substrate proteins  <cit> .
in the present study, dietary iron overload decreased the expression of hspa1b  and dnajb <dig>  in skeletal muscle, heart and liver of mice as validated by q-rt-pcr.
in accordance with these findings, our microarray results also showed decreased expression of several other heat shock protein genes in skeletal muscle  and heart  of iron-loaded mice.
this unexpected result might represent novel regulatory mechanisms specific to these concrete experimental conditions.
the post-transcriptional regulation of transferrin receptor  <dig> and divalent metal transporter  <dig> by iron is mediated through iron-responsive elements located in the 3'-untranslated region of their mrnas  <cit> .
as expected, we found decreased tfrc mrna expression in skeletal muscle, heart and liver of iron-loaded mice but, surprisingly, the expression of divalent metal transporter  <dig> was not changed substantially.
the expression levels of hepcidin <dig> and hepcidin <dig> transcripts in the liver are markedly influenced by strain and gender, in accordance with previous reports  <cit> .
dba <dig> mice differ markedly in the expression levels of their hepcidin genes when compared with balb/c and c57bl/ <dig> mice.
for dba <dig>  the difference in hepcidin <dig> expression was evident not only in liver, but also in the heart and skeletal muscle.
these results further demonstrate that iron responses can vary between different mouse strains.
CONCLUSIONS
to conclude, we have identified genes whose expression is altered in skeletal muscle and heart during iron overload.
the number of the affected genes and the magnitude of the changes were relatively low, which is probably due to the fact that skeletal muscle and heart are not the primary targets of iron loading.
interestingly, some of the regulated genes identified in this study are involved in modulation of glucose and lipid metabolism, transcription and cellular stress responses.
these might represent novel links between iron overload and the pathogenesis of cardiomyopathy and diabetes in hh.
further investigation of these genes may help to understand how iron excess leads to these common hh manifestations.

 it is crucial for the human body to maintain iron homeostasis. since there is no adjustable mechanism to influence iron loss from the body, tight regulation of iron absorption at the intestinal level is vital. in order to maintain iron balance, iron export from enterocytes, reticuloendothelial macrophages and hepatocytes into the blood stream has to be controlled as well. functional derangement of proteins involved in these regulatory mechanisms can cause hereditary hemochromatosis. this genetic disorder of iron overload is characterized by high transferrin saturation, low iron content in macrophages, and deposition of iron in several organs including the liver, heart, and pancreas. causative mutations for hh have been described in several genes, namely hfe, tfr, hjv, and hamp. it has been proposed that these mutations cause deficient hepcidin synthesis. the antimicrobial peptide hepcidin is the central regulator of iron metabolism. it is produced mainly in the liver and exerts its function by binding to the iron export protein, ferroportin, inducing its internalization and degradation. ferroportin is located in the cellular membranes of enterocytes, reticuloendothelial cells, hepatocytes and placental cells. therefore, hepcidin acts to decrease the export of iron from these cells into the circulation. hemojuvelin is a glycosyl phosphatidylinositol-anchored protein which belongs to the repulsive guidance molecule protein family. recent studies suggest that hemojuvelin exists in two forms. one is a rarer full-length protein shed to the extracellular fluid, where it has a long half-life. the other is a smaller, membrane-associated disulfide-linked heterodimer, which is a more abundant but shorter-lived form composed of n- and c-terminal fragments. according to latest studies the most common mutation in hemojuvelin affects the targeting of the membrane-associated form and reduces the amount of the soluble form. interestingly, studies in cultured cells suggest that the two forms regulate hepcidin expression reciprocally by competing for a receptor binding site. evidence shows that hemojuvelin is a bone morphogenetic protein co-receptor, and its interaction with bmp initiates a signaling cascade that leads to regulation of hepcidin expression. on the other hand, it has been observed that overexpressed hemojuvelin binds to the membrane receptor neogenin and that this interaction is required for the accumulation of iron in cultured cells. zhang et al. also showed that the g320v mutated hemojuvelin overexpressed in vitro was not able to bind neogenin, and that iron did not accumulate in the cells under these conditions. furthermore, a recent study in cultured cells suggested that neogenin may mediate inhibition of hemojuvelin shedding in response to iron. we have previously determined the sites of simultaneous expression of hemojuvelin and neogenin. the highest expression of hemojuvelin transcript is found in the skeletal muscle and heart. although in vivo evidence of a combined role of hemojuvelin and neogenin in iron homeostasis has not been provided yet, it has been suggested that hemojuvelin shed from skeletal muscle and heart by neogenin-dependent mechanism could reach the liver to influence hepcidin expression. cardiomyopathy develops in some hh patients. in order to better understand the mechanisms behind pathological effects of iron overload in muscle cells, we have performed a genome-wide expression analysis of genes in skeletal muscle and heart of mice with or without dietary iron loading. microarray data analysis identified several genes whose expression was either down- or up-regulated due to iron overload. these results may reveal novel links between iron overload and pathological manifestations of hh.  documentation of iron overload in the liver and heart of iron-fed mice the mice were fed either standard or high-iron diet for weeks. iron concentrations of liver and heart specimens were determined to confirm the validity of the animal model. figure 1a demonstrates that the livers of mice of all three strains were highly iron-loaded when fed an iron-rich diet. a much smaller increment in cardiac iron content after a high-iron diet was observed also in all three strains and in both genders, although statistical significance was not reached in all the cases. basal cardiac iron levels were lower than basal hepatic contents. in general, female mice showed slightly higher hepatic and cardiac iron levels than male mice. identification and validation of changes in gene expression induced by dietary iron overload in skeletal muscle and heart we obtained a list of genes with iron-induced up-regulated expression in skeletal muscle and forty with down-regulated expression. in the heart, iron loading resulted in the up-regulation of genes, while forty genes had down-regulated expression after iron overload. there were seven genes which were up-regulated in both the heart and skeletal muscle, while nine genes were down-regulated in both tissues. data obtained with high-iron and control samples. data obtained with high-iron and control samples. *data obtained with high-iron and control samples. ** data obtained with high-iron and control samples. *data obtained with high-iron and control samples. ** data obtained with high-iron and control samples. from the lists of microarray results we selected genes which presented the highest fold change values. the expression levels of these genes were then analyzed in the same mouse strain by q-rt-pcr. certainly, the results from these analyses showed a good correlation between the two methods; the expression of all the genes was regulated and displayed the same direction of change. the fold change values obtained from pcr experiments were all over except for the tfrc gene whose downregulation in the skeletal muscle reached the value of -. representations of these q-rt-pcr results are depicted in figures and in general, the fold-change values obtained by microarray analysis were smaller than those determined by q-rt-pcr. this phenomenon has been described previously and is probably due to the fact that array analyses are less quantitative than q-pcr. the hepatic mrna levels of these genes were also analyzed by q-rt-pcr. the results for genes whose expression varied in the same direction in both skeletal muscle and heart after iron loading are shown together with their expression in the liver in figure the expression of four of the genes was considered negligible in the liver because of very low signal intensity. among the remaining genes only the hepatic expression of pdk and cxcl was not significantly regulated by dietary iron. expression of genes involved in hepcidin regulatory pathway during dietary iron overload one of the aims of this study was to explore the effect of dietary iron overload on the expression of the iron-related genes hemojuvelin and neogenin in skeletal muscle and heart. we did not observe differential expression of these genes or any of the traditional iron-regulatory genes by the microarray approach, except for the transferrin receptor gene, which was down-regulated by iron in heart, skeletal muscle and liver. even though the microarray method we used proved to be very accurate, we wanted to verify these results and to explore the response of hepcidin expression in the studied tissues by q-rt-pcr. the expression of hepcidin and hepcidin in the liver was greatly up-regulated by iron overload and varied according to mouse strain and gender. in balb/c and c57bl/ mice, hepcidin was the predominant form expressed in the liver, while in dba mice, the hepatic expression of hepcidin was dominant. the expression of hepcidin in the skeletal muscle was negligible in all strains. in the heart muscle, it showed a slight tendency towards decreased expression in most iron fed mice, although the baseline signal in control mice was already quite low. only dba mice expressed hepcidin in the skeletal muscle and heart, and this expression was not clearly regulated by iron overload. the results for hemojuvelin expression did not indicate any clear regulation by iron overload, strain or gender in any of the tissues studied. this is in agreement with previous studies of hepatic expression. hemojuvelin expression only showed a minor trend downwards in skeletal muscle and heart of mice fed with high-iron diet. no significant changes were observed for neogenin expression. discussion excess free iron participates in the formation of free radicals causing oxidative stress and cell damage, which is evidenced as a series of pathological manifestations. while some studies have analyzed the effects of iron on the transcriptional profiles in liver and duodenum, this is the first study reporting changes in mrna expression that may contribute to iron-induced effects on skeletal muscle and heart. we used a genome-wide mrna expression profiling approach and validated the most substantive changes by q-rt-pcr. expression of antioxidant enzymes is considered a protective mechanism against oxidative stress-induced damage. however, the regulation of antioxidant enzymes in response to oxidative stress is a rather controversial issue, as the results vary greatly depending on the type and length of the stimulus and the type of cells or tissue tested. we did not find increased expression of oxidative stress-related genes or antioxidant enzymes after iron overload, except for glutathione peroxidase whose expression was induced by -fold change in heart. the data analysis identified two genes encoding calcium- and zinc-binding proteins, s100a and s100a among the up-regulated genes presenting the highest fold changes. these proteins form a rather ubiquitous heterodimer called calprotectin. the highest amounts of this protein complex are located in neutrophil granulocytes, monocytes and keratinocytes. calprotectin is a pro-inflammatory cytokine that is upregulated in many inflammatory conditions, and is involved in innate immunity, leukocyte adhesion, endothelial transmigration and processes of chronic inflammation. in vitro studies have shown that reactive oxygen species induce protein levels of s100a. additionally, s100a expression is induced in keratinocytes upon exposure to ultraviolet a radiation, a stimulus causing oxidative stress. interestingly, the antioxidant enzymes superoxide dismutase and catalase, whose mrna expression was unaffected in our microarray, abrogate s100a induction. we found that both s100a and s100a transcripts were substantially up-regulated in skeletal muscle, heart and liver of iron-loaded mice, although the levels of s100a transcript in skeletal muscle were negligible and, in general, both genes were weakly expressed. this demonstrates the high sensitivity and accuracy of both the microarray analysis and the q-rt-pcr method. it is noteworthy that s100a and s100a transcripts showed a very similar up-regulation pattern in the tissues studied, which agrees with the concept of the two proteins forming a heterodimer. the transcriptional regulation of the s100a and s100a genes seems to be rather complex, with promoter binding sites for transcription factors such as activator protein, nuclear factor kappa b and c/ebp. consistently, at least ap- and nf-κb have been previously identified to be regulated by the intracellular redox state. pyruvate dehydrogenase kinase phosphorylates and inactivates the pyruvate dehydrogenase complex, decreasing the rate of glucose oxidation and thus increasing blood glucose levels. increased pdk expression and activity has been observed in both skeletal muscle and heart of insulin-resistant mouse models. the question arises of whether pdk overexpression causes insulin resistance or vice versa. insulin suppresses pdk expression in skeletal muscle and, according to a recent study by kim and coworkers, this effect is impaired in insulin resistance, suggesting that insulin resistance may indeed induce pdk expression. however, several studies using high-fat dietary models of insulin resistance indicate that pdk overexpression occurs before the development of insulin resistance. although it has not been documented directly that increased pdk mrna levels can indeed cause insulin resistance, it seems possible that a vicious cycle may exist between these two phenomena. in the present study, we show an up-regulation of pdk mrna levels in skeletal muscle and heart but not in the liver of iron-loaded mice. diabetes mellitus is the major endocrine disorder associated with hh. the mechanisms responsible for this clinical manifestation are still obscure, but two processes have been proposed: the pancreatic β-cell iron accumulation results in cell damage and diminished insulin secretion, and liver iron overload leads to insulin resistance. the herein reported induction of pdk expression in the skeletal and heart muscle might represent a novel mechanism involved in the development of diabetes mellitus in hh. angiopoietin-like is a secreted protein produced mainly in fat tissue, and to a lesser extent in liver, placenta, skeletal muscle and heart. it is directly involved in regulating glucose homeostasis, lipid metabolism, and insulin sensitivity. angptl decreases the activity of lipoprotein lipase, thus inhibiting lipoprotein metabolism and increasing plasma triglyceride levels. transgenic mice with angptl overexpression directed to heart muscle show reduced cardiac lpl activity, decreased triglyceride utilization and impaired cardiac function resulting in cardiomyopathy. transgenic overexpression of angptl from a liver-specific promoter causes hypertriglyceridemia similar to that induced by adenoviral over-expression. these results support the hypothesis that angptl has lpl-dependent actions. accordingly, in lpl-expressing tissues angptl may bind directly and inactivate lpl, restricting acquisition of free fatty acids to these sites: it is not shed into plasma from these tissues but rather acts in an autocrine/paracrine fashion. on the other hand, in the liver, which has low lpl expression, angptl is shed to plasma and inhibits lpl in other locations, causing a general reduction of triglyceride utilization and acting as an endocrine factor. interestingly, we showed a 5-fold increase in the level of angptl transcript in the heart of iron-loaded mice, raising the possibility that early induction of angptl expression could contribute to the pathogenesis of cardiomyopathy in hh. the increased expression of pdk and angptl observed in skeletal muscle and heart of iron-loaded mice might have its origin in a common mechanism. the forkhead transcription factor foxo is a major regulator of insulin action in insulin-sensitive tissues and it is involved in insulin's action to suppress pdk and angptl. myosin light polypeptide ) and myosin light polypeptide ) show a 10-fold up-regulation in the cardiac muscle of iron-loaded mice. both genes belong to the ef-hand family of ca2+ binding proteins and are part of the myosin molecular complex. they appear to be involved in force development during muscle contraction. elc is important in the interaction between myosin and actin. there are two forms of elc in the cardiac muscle, elca and elcv. elca has a higher performance than elcv and its elevated accumulation in diseased heart is considered a compensatory response in heart failure. furthermore, transgenic rats overexpressing elca in the heart show an improvement in contractile parameters. these observations open the possibility that the induction of cardiac myl and myl expression observed in our experiments is a compensatory response to early damage produced by iron accumulation. additionally, according to our microarray results, other myosin genes were induced by iron in mouse heart and skeletal muscle. actin filaments play an essential role, along with myosin, in muscle contraction. curiously, in the present work, iron suppressed the expression of skeletal muscle and smooth muscle isoforms of actin in the heart, as well as the cardiac isoform in skeletal muscle. the present microarray data analysis identified one gene, which showed marked upregulation in the skeletal muscle and downregulation in the heart after iron overload. this finding was also confirmed by q-rt-pcr. scd is an iron-containing enzyme with a central lipogenic role. it catalyzes the insertion of a double bond into fatty acyl-coa substrates, the preferred one being stearoyl-coa, and yielding oleoyl-coa. oleic acid is the major monounsaturated fatty acid of membrane phospholipids, triglycerides, cholesterol esters, wax esters and alkyl- -diacylglicerol. the ratio has important effects on cell membrane fluidity and signal transduction. the overexpression of scd has been shown to be associated with genetic predisposition to hepatocarcinogenesis. scd mrna levels were induced times in mouse liver during iron overload, an effect that was previously shown in both enteral and parenteral models of iron overload. as pigeon and coworkers have discussed, most likely the effect of iron on scd expression in the liver is not direct, but a compensatory mechanism in response to the need to renew unsaturated fatty acids. the fbj osteosarcoma oncogene is a major component of activator-protein-, a redox-sensitive transcription factor complex, which also includes members of the jun, maf and atf subfamilies. fos is thought to have an important role in signal transduction, cell proliferation and differentiation. expression of c-fos and c-jun can be induced by many stimuli and compounds, including some metals such as iron. accordingly, the present work shows increased expression of c-fos in the heart and liver of iron-loaded mice. however, in skeletal muscle, c-fos was down-regulated and, according to the microarray results, the same is true for c-jun. probably other mechanisms are influencing the transcription of c-fos and c-jun in skeletal muscle. interestingly, a recent study suggested that c-jun and junb negatively regulate the transcription of s100a and s100a. furthermore, ap- activity had been previously connected to iron metabolism in several ways. for example, ap- regulates transcription of ceruloplasmin, and the promoter region of hfe contains an ap- transcription element. heat shock proteins, or stress proteins, are expressed in response to heat shock and a variety of other stress stimuli including oxidative free radicals and toxic metal ions. the members of the 70-kda heat shock protein family assist cells in maintaining functional proteins under stressful conditions. hsp proteins stimulate the atpase activity of hsp proteins and stabilize the interaction of these chaperons with their substrate proteins. in the present study, dietary iron overload decreased the expression of hspa1b and dnajb in skeletal muscle, heart and liver of mice as validated by q-rt-pcr. in accordance with these findings, our microarray results also showed decreased expression of several other heat shock protein genes in skeletal muscle and heart of iron-loaded mice. this unexpected result might represent novel regulatory mechanisms specific to these concrete experimental conditions. the post-transcriptional regulation of transferrin receptor and divalent metal transporter by iron is mediated through iron-responsive elements located in the 3'-untranslated region of their mrnas. as expected, we found decreased tfrc mrna expression in skeletal muscle, heart and liver of iron-loaded mice but, surprisingly, the expression of divalent metal transporter was not changed substantially. the expression levels of hepcidin and hepcidin transcripts in the liver are markedly influenced by strain and gender, in accordance with previous reports. dba mice differ markedly in the expression levels of their hepcidin genes when compared with balb/c and c57bl/ mice. for dba the difference in hepcidin expression was evident not only in liver, but also in the heart and skeletal muscle. these results further demonstrate that iron responses can vary between different mouse strains.  to conclude, we have identified genes whose expression is altered in skeletal muscle and heart during iron overload. the number of the affected genes and the magnitude of the changes were relatively low, which is probably due to the fact that skeletal muscle and heart are not the primary targets of iron loading. interestingly, some of the regulated genes identified in this study are involved in modulation of glucose and lipid metabolism, transcription and cellular stress responses. these might represent novel links between iron overload and the pathogenesis of cardiomyopathy and diabetes in hh. further investigation of these genes may help to understand how iron excess leads to these common hh manifestations. 
these may represent novel connections between iron overload and pathological manifestations of hh such as cardiomyopathy and diabetes. 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. 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. therefore, we investigated the effect of iron overload on gene expression in skeletal muscle and heart using illumina™ arrays containing over probes. heart muscle is one of the target tissues affected by iron overload, with resultant cardiomyopathy in some hh patients. genes with up-regulated expression after iron overload in both skeletal and heart muscle included angiopoietin-like pyruvate dehydrogenase kinase and calgranulin a and b. the expression of transferrin receptor, heat shock protein 1b and dnaj homolog b were down-regulated by iron in both muscle types. the most apparent changes in gene expression were confirmed using real-time rt-pcr.
 hereditary hemochromatosis encompasses genetic disorders of iron overload characterized by deficient expression or function of the iron-regulatory hormone hepcidin. mutations in genes have been linked to this disease: hfe, tfr, hamp, slc40a and hjv. 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 illumina™ arrays containing over probes. the most apparent changes in gene expression were confirmed using real-time rt-pcr.  genes with up-regulated expression after iron overload in both skeletal and heart muscle included angiopoietin-like pyruvate dehydrogenase kinase and calgranulin a and b. the expression of transferrin receptor, heat shock protein 1b and dnaj homolog b 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.  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. 