BACKGROUND
there is growing evidence that sexual dimorphism exists in the physiological response to diet and other environmental factors  <cit> , including differences in insulin and leptin sensitivity  <cit> , particularly in response to high fat diets  <cit> .
the heart requires high levels of atp in order to maintain contractility, and this essential energy requirement is usually met by the oxidation of fatty acids , which are the most important source of energy for the adult heart.
fas have also been shown to be involved in signal transduction and the regulation of gene transcription  <cit> ; however excessive fa uptake may cause detrimental lipotoxicity, resulting in deregulated cardiac function  <cit> .
several key studies have recently shown that considerable sex-dimorphism exists in both rodents and humans  <cit> ; and have established sex-specific pathways in cardiac transcription in response to both pressure overload  <cit>  and dilated cardiomyopathy  <cit> .however there is a relative scarcity of information concerning sex-specific effects of diet on cardiac gene expression.
dietary intake of added fats and oils has increased by over 60% during the past  <dig> years  <cit> , and use of hydrogenated oils and shortenings in order to prolong shelf-life has resulted in an increase in trans fatty acid  consumption, which now accounts for between  <dig>  - 8% of the global dietary fat intake   <cit> .
tfa consumption has been linked to the increased risk of obesity  <cit> , inflammation  <cit> , coronary heart disease   <cit>  and type  <dig> diabetes  <cit> .
unlike saturated fat, food nutrition labeling does not set a recommended intake limit for trans-fat, however one meta-analysis study found that a 2% increase in energy intake from tfa was associated with a 23% increase in the incidence of chd  <cit> .
concern over the deleterious effects of dietary trans-fat has prompted several countries to regulate the industrial production  <cit> ; and retrospective meta-analysis of observational controlled dietary trials and prospective cohort studies suggests that replacement of trans-fat with nonhydrogenated oils would result in significant reduction of the incidence of chd  <cit> .
gender differences in the prevalence of chd are well documented  <cit> , and many factors appear to be responsible including lipid profile  <cit>  and genetic susceptibility  <cit> .
neonatal administration of high doses of the food flavor enhancer monosodium glutamate  has also been shown to result in obesity  <cit>  together with stunted heart growth and hypoplasticity in rodents  <cit> .
furthermore, the cardiovascular system of these animals have been shown to respond deficiently to certain challenges, including an attenuated blood pressure response to the systemic injection of nitric oxide synthase inhibitors, angiotensin ii and other vasoactive compounds  <cit> .
injections of high doses of msg  cause ablation of the arcuate nucleus via glutamate-induced degeneration of those areas of the immature brain which are insufficiently protected by a mature blood-brain barrier, including the hypothalamic area  <cit> .
this suggested that the hypothalamus may play an important role in the growth of the heart.
it is now apparent that maternal administration of msg can penetrate the placental barrier and distribute to the embryonic tissues of the fetus.
oral administration of msg and 3h-labeled glutamate  as a tracer to pregnant mice resulted in marked elevations of 3h- glu in the placenta and in fetal brain, liver and kidney  <cit> .
msg consumption has increased globally in recent years, with recent estimations of the current average daily intake believed to be up to  <dig> g/day  <cit> .
despite the widespread consumption of this common food flavor enhancer, which often occurs in processed and packaged foods in combination with trans-fat, very few studies have addressed the effect of dietary trans-fat and/or msg consumption on cardiac gene expression.
furthermore, although sexual dimorphism at the growth hormone axis has been demonstrated in msg-treated rodents  <cit> , little is known about sex-specific gene expression in response to exposure to msg.
the aim of this work was to establish the effect of dietary tfa and/or msg on cardiac gene expression in an in vivo animal model, and to examine sex-specific differences as well as commonalities of gene expression in response to diet.
the amount of oral msg used in this study reflects current consumption levels  <cit>  and is  <dig> -  <dig> times less that the level previously reported to induce neuronal damage when injected neonatally  <cit> .
since it has previously been ascertained that msg excitotoxicity occurs only when the blood brain barrier is vulnerable, for example neonatally  <cit> , and because developmental programming of cardiac gene expression may be affected by maternal nutritional balance  <cit> , we bred our study animals from females previously established on these diets for  <dig> weeks prior to mating, in accordance with our previous studies  <cit> .
exposure to these diets occurred throughout the study, in order to mimic as closely as possible the situation that occurs in humans.
the results of our microarray analysis revealed considerable sexual dimorphism in response to the dietary interventions that we tested, with males appearing to exhibit more differential gene expression compared to females.
RESULTS
consumption of trans-fat and/or msg results in sex-specific changes in physiological characteristics
abc uncommon letters indicate difference in means between diet groups within a row .
differences between genders within a diet group for each variable are denoted by *,** and *** for p-values < <dig> , < <dig>  & < <dig>
sexual dimorphism in cardiac gene expression
we used affymetrix mouse gene  <dig>  st expression arrays to determine differences in global cardiac gene/expressed sequence tags  in response to diet, in both males and females.
using a false discovery rate with a significance level set at  <dig> , we identified two subsets of genes/ests that were differentially expressed between males  and females  at various stringency levels .
to better understand the relevance of these sex-dimorphic genes we used a program based on gene ontology  categories of biological processes, molecular function and cellular component.
additional file  <dig>  table s <dig> lists the main functional go categories of transcripts that were differentially expressed between the sexes with a stringency of ±  <dig> -fold change in expression .
degs were further characterized as being relatively up-regulated either in males or in females.
increased transcription of male genes in the "biological processes" category included genes involved in cellular macromolecule metabolism, anatomical structure development and blood vessel development.
these genes included notch <dig>  interstitial matrix gene col3a <dig> , fibulin  <dig>  and integrin αv .
the predominant male-specific kegg pathways included the regulation of actin cytoskeleton and antigen processing pathways .
conversely female-specific transcription were enriched in the categories of cellular and metabolic processes, including mitochondrial carbonic anhydrase 5b , glutathione s-transferase  <dig>  and magnesium transporter  <dig> .
one kegg-pathway for cysteine and methionine metabolism was also upregulated.
chromosomal localization of differentially expressed cardiac genes
to investigate a possible non-random chromosomal distribution of the degs, we used the fisher exact test to compare the frequencies of significant genes on each chromosome against the total number of genes interrogated on each chromosome.
chromosomes  <dig>  and y were enriched for male-biased genes whereas chromosomes  <dig> , <dig> and x contained more female-biased genes .
notably we found four genes encoded on the y chromosome which were male-specific , and  <dig> female-biased x-linked genes including ace <dig>  dystrophin, and several transcription factors such as tceal <dig>  tceal <dig> and eif2s3x.
trans-fat feeding induces sex-specific alterations in cardiac gene expression
in order to identify the subset of genes and ests with a fold difference of ±  <dig>  or more within male or female comparisons for mice fed the tfa diet versus control, we used the results from a 2-way anova model described in the methods section.
we identified  <dig> degs  that were responsive to diet in males; and approximately half as many diet-responsive degs in females:  <dig> .
specifically, the trans-fat diet significantly downregulated the expression of many male cardiac genes involved in the regulation of biological processes, transcription and phosphorylation.
conversely females upregulated the expression of genes with functional ontologies relating to binding, nitrogen compound metabolism and ketone metabolism .
significant male degs downregulated by tfa consumption included key transcription modulator sterol regulatory element binding factor  <dig> , transcription factors mef2d  and gata <dig> ; vasoconstrictive alpha-2b adrenergic receptor ; major cardiac na+ channel scn5a , heat shock transcription factor  <dig> , calcium-regulating enzyme phospholipase c δ , and circadian clock gene per <dig> .
signaling molecules calcium/calmodulin-dependent protein kinase kinase 1α  and mitogen-acrivated protein kinase kinase  <dig>  expression were also halved in male tfa mice.
conversely pro-hypertrophic hdac <dig> , which represses mef <dig> activity, was upregulated in males and downregulated in females.
other genes with this pattern of sex-dimorphic expression included plastin  <dig>  and tachykinin  <dig> .
in both sexes, the tfa diet led to increased expression of fatty acid oxidizing 3-hydroxy-3-methylglutaryl coa synthase  and fructose bisphosphatase  <dig> ; however expression of tnf superfamily member light/tnfsf <dig> was increased 7-fold in male trans-fat hearts compared to control, whereas it was only increase 2-fold in female trans-fat hearts.
diet-induced changes in female hearts were less significant, with only  <dig> degs significantly up-regulated, including progestin adipoq receptor family member v  and ras guanyl releasing protein  <dig> .
enrichment analysis of functional go pathways within this subset revealed that females exhibited a four-fold less down-regulation of cardiac genes with classified biological function than males.
we next investigated the relationship between these genes using ingenuity pathways analysis .
a network of  <dig> degs relating to cardiovascular disease and lipid metabolism among others was identified as being significantly affected by the tfa diet in cardiac tissue from male mice , whereas a network of  <dig> functionally related genes involved in female lipid metabolism and cardiovascular development is shown in figure 4b .
monosodium glutamate consumption alters cardiac gene expression with sex-specific differences
a different pattern of gene expression was induced by msg feeding, which again showed significant sex-specific differences in cardiac gene expression.
2-way anova analysis revealed a subset of  <dig> male cardiac degs  which were responsive to dietary msg compared to control diet, and  <dig> female degs .
.
unlike trans-fat, msg upregulated more cardiac degs and down-regulated less.
genes/ests upregulated in males but not females in response to msg included ing <dig> , glutathione s-transferase  <dig> and pancreatic lipase .
transcripts enriched for female-biased expression included myosin 7a , pdzd <dig>  and aldehyde dehydrogenase gene aldh1a <dig>  functional analysis of gene ontologies of these subsets showed increased expression of male cellular and metabolic processes, whilst decreasing expression of phosphorylation-related degs .
conversely females upregulated more genes/ests with developmental and anatomical function ontologies.
genes/ests that were upregulated in both sexes included ionotropic glutamate receptor grik <dig> ; nebulin which regulates actin filament length .
msg alters trans-fat induced cardiac gene expression
in order to address the question as to whether the addition of msg to a trans-fat diet could affect cardiac gene expression, we next identified a subset of  <dig> degs which showed significant sex-dimorphism in response to these two diets.
<dig> degs  in males showed significant response to the inclusion of msg in the trans fat diet, and  <dig>  in females .
of note in both sexes, expression of lipogenic stearoyl-coa desaturase  <dig>  was upregulated in tfa+msg diet group hearts compared to tfa alone  and was accompanied by an increase in myosin h <dig>  magnesium transporter slc41a <dig>  and apoptosis-inducing bcl2-interacting killer .
downregulated degs included fibrulin, sclerostin and dystrophin, together with fatty acid-oxidizing enzymes phospholipase a <dig>  and aldehyde dehydrogenase .
significant differentially expressed cardiac transcripts in tfa + msg-fed versus tfa-fed mice were examined for enrichment of go and kegg classifications .
in males, major functional go categories included metabolism, biosynthesis and transcription, whereas enrichment of female degs upregulated by msg feeding again fell into the categories of developmental and anatomical structural function.
cellular positional mapping reveals sex-specific differences in diet-deregulated gene expression
a final analysis of significant diet-induced deregulation of cardiac gene expression between all diet combinations co-analyzed yielded a cohort of  <dig> mapable genes which were deregulated by all dietary manipulations only in males , and half that number of genes which were only deregulated in females .
interestingly in this male subset, expression of genes previously demonstrated to be upregulated by tfa feeding  were downregulated in animals consuming the msg-enriched diets .
male-specific genes in this subset included stress-related proteins dnaja <dig> and pdia <dig>  together with phosphodiesterase 6a and histone h1e .
females displayed a slightly divergent pattern .
additionally  <dig> transcripts were identified as being significantly differentially expressed in both sexes in response to all dietary manipulations.
figure  <dig> shows a heatmap of a selection based on functional relevance, together with a number of x-linked and y-linked diet-induced degs, p <  <dig>  with a stringency of ±  <dig> -fold change in expression.
eighteen genes were randomly chosen for further analyzed by qrt pcr based on biological relevance.
pearson correlation coefficients between the microarray analysis and qrt pcr were calculated.
ratios of expressions between the diet comparisons calculated from the microarray data set correlated well with the ratio calculated from the real-time pcr data .
a complete list of these genes and pcr primers is given in additional file  <dig>  table s <dig>
discussion
our results indicate that dietary manipulation prenatally, and over the first  <dig> months of life can markedly influence cardiac gene expression in a sex-specific manner.
sexual dimorphism in gene expression exists even in the healthy heart  <cit> , and chromosome  <dig> has been shown to have more female-biased differential cardiac gene expression  <cit> , whereas male-biased enrichment was found in chromosomes  <dig> and  <dig>  in addition to the x and y chromosomes which segregated as anticipated, we found female-biased chromosomal enrichment on mouse chromosomes  <dig>   <dig> and 12; and male-biased expression on chromosomes  <dig> and  <dig>  interestingly, a locus on murine chromosome  <dig> has been associated with markers of the metabolic syndrome, including body weight, insulin, leptin and triglyceride  <cit> .
the main outcome from our study was that trans-fat induced more than twice as many degs in males compared to females, and downregulated four times as many genes, including a number that have been implicated in cellular pathways leading to hypertrophy such as gata <dig> and mef2d  <cit> .
expression of gata <dig>  previously identified as being of importance in stress responsiveness  <cit> , was halved in males in response to the tfa diet.
gata <dig> regulates transcription of a large number of cardiac genes via binding to the consensus 5'-wgatar-3' sequence, and is implicated in cardiac muscle apoptosis, where under conditions of oxidative stress gata <dig> downregulation is preceded by mek/erk-dependent phosphorylation  <cit> .
cardiac hypertrophy and chd are mediated by transcription factor integration of upstream stress signals, and mef2d-deficient mice have an attenuated hypertrophic response to cardiac stress  <cit> .
mef2d activity is repressed by the histone deacetylase hdac <dig>  which we found to be increased in male tfa mice.
histone deacetylases are also regulated by calmodulin and calmodulin kinase, and are among the negative regulators of cardiac hypertrophy which attenuates mef <dig> transcriptional activity  <cit> .
in the present study, we found a reduction of expression of camkk <dig> and an increase in expression of camk2b, suggesting that trans-fat may deregulate both the calmodulin kinase/mef2/hdac pathway and the pkc/mapk/gata <dig> pathways of cardiac gene transcription.
trans-fat feeding deregulated other stress-associated genes in males including phospholipase c δ <dig>  the main cardiac isoform implicated in oxidative stress-induced redox signaling  <cit> , and light/tnfsf <dig>  demonstrated to be upregulated in clinical and experimental heart failure  <cit> .
additionally, key circadian clock gene per <dig> was decreased by more than half in the hearts of male tfa-fed mice.
downregulation of per <dig> by glucose has been shown to be accompanied by changes in the expression of genes involved in lipid metabolism, transcription and cell cycle  <cit> , such as sterol regulatory element binding factor , hydroxy-3-methylglutaryl coa synthase  and stearoyl-coa desaturase , all of which were differentially regulated by trans-fat in our study.
whilst it is known that trans-fat consumption is linked to cardiovascular disease  <cit> , the mechanism behind this is incompletely understood.
elaidic acid, the main tfa in partially hydrogenated vegetable shortening, incorporates into cardiac tissues  <cit> ; and it has been suggested that tfas are oxidized at a slower rate than cis-fatty acids  <cit> , resulting in the accumulation of reactive oxidation intermediates  <cit> , which could lead to oxidative stress and the activation of the gata <dig>  mefd <dig> and hdac-associated transcriptional pathways.
taken together, our results suggest that trans-fat feeding results in significant impairment of transcriptional pathways relating to cellular stress, particularly in males.
it has recently been shown that male c57bl/6j mice are more vulnerable than females to the impact of a high-fat diet in terms of ensuing weight gain and deleterious metabolic peturbations  <cit> ; and it is therefore possible that the sex-differences in trans fat -induced cardiac gene expression seen in our study may have contributed to the resultant increase in male weight gain and heart: body ratio compared to females.
the msg diet resulted in a different pattern of sexual dimorphism in cardiac transcription, with fairly equal numbers of degs being up- and down-regulated.
however there were a number of genes which were overexpressed in males but not in females, and vice versa.
the functional categories of overexpressed genes differed too, with females showing more enrichment of genes associated with developmental processes for example, whereas male upregulated transcripts were enriched for cellular and metabolic function.
neonatal injections of msg result in smaller hearts with less dna synthesizing activity  <cit> ; and hypotension has been observed in female, but not male rats treated neonatally with msg  <cit> .
in our study, female hearts from msg-diet group mice weighed significantly less than male hearts, however an examination of genes deregulated in our msg mice did not provide many clues as to the mechanism responsible for this apparent difference.
we have previously shown that msg alters trans fat -induced hepatic and adipose tissue gene expression with the result that several key transcripts involved in ß-oxidation of lipids were downregulated in tfa+msg treated mice  <cit> ; and in our present study we found that the combination of tfa+msg decreased the expression of several fatty acid-oxidizing enzymes such as phospholipase a <dig> and aldehyde dehydrogenase,whilst increasing the expression of lipogenic cardiac-specific stearoyl-coa desaturase  <dig> , previously shown to be regulated by leptin and dietary factors  <cit> .
when we compared cardiac gene transcription between all dietary combinations co-analyzed, we again found that more significant degs were induced in males compared to females.
our analysis identified a subset of over  <dig> transcripts which significantly changed only in male mice in response to diet, and approximately half this number of transcripts differentially expressed in females only, and a similar number of shared degs were common to both sexes.
this suggests that males may have a higher cardiac transcriptional response to dietary manipulation, and indeed greater transcriptional response to exercise has previously been demonstrated in males but not females  <cit> .
several theories have been put forward as to this disparity, the cardioprotective effect of estrogen being one possibility  <cit> , especially in light of epidemiological data indicating a lower incidence of female chd, at least until menopause, after which this dissimilarity declines.
however, this is unlikely to account for all the sex-specific differences observed, since after correcting for lifestyle factors, the discrepancy in chd rates was reduced  <cit> .
by using microarrays to analyze diet-induced cardiac gene expression patterns, we have identified genes which are differentially regulated and may play important roles in the development of chd associated with trans-fat consumption.
however there are some issues to consider when interpreting the results from the present study.
for the microarray experiments, we employed a pooling design in order to reduce biological variation, and to lower the costs associated with sample size and chip availability  <cit> .
in order to address a possible loss of sensitivity and an increase in the incidence of false positives, we employed stringent criteria for the identification of the diet-regulated degs.
one limitation of this study is the fact that these experiments were only performed on c57bl/6j mice of both sexes.
it would be advantageous but beyond the scope of the present study, to ascertain whether similar results could be obtained using a different genetic background, or possibly a higher animal model.
CONCLUSIONS
we have found substantial evidence for sexual dimorphism in response to dietary manipulation, with males apparently showing the greater cardiac transcriptional response.
in particular, we found that trans-fat feeding in male mice resulted in deregulation of the expression of genes involved in cellular stress pathways.
excessive tfa consumption is believed to be a risk factor in human heart disease, the incidence of which appears to be higher in male patients.
our present observations may provoke further studies which could result in sex-specific medical intervention in the future.

 there is growing evidence that sexual dimorphism exists in the physiological response to diet and other environmental factors, including differences in insulin and leptin sensitivity, particularly in response to high fat diets. the heart requires high levels of atp in order to maintain contractility, and this essential energy requirement is usually met by the oxidation of fatty acids, which are the most important source of energy for the adult heart. fas have also been shown to be involved in signal transduction and the regulation of gene transcription; however excessive fa uptake may cause detrimental lipotoxicity, resulting in deregulated cardiac function. several key studies have recently shown that considerable sex-dimorphism exists in both rodents and humans; and have established sex-specific pathways in cardiac transcription in response to both pressure overload and dilated cardiomyopathy.however there is a relative scarcity of information concerning sex-specific effects of diet on cardiac gene expression. dietary intake of added fats and oils has increased by over 60% during the past years, and use of hydrogenated oils and shortenings in order to prolong shelf-life has resulted in an increase in trans fatty acid consumption, which now accounts for between - 8% of the global dietary fat intake. tfa consumption has been linked to the increased risk of obesity, inflammation, coronary heart disease and type diabetes. unlike saturated fat, food nutrition labeling does not set a recommended intake limit for trans-fat, however one meta-analysis study found that a 2% increase in energy intake from tfa was associated with a 23% increase in the incidence of chd. concern over the deleterious effects of dietary trans-fat has prompted several countries to regulate the industrial production; and retrospective meta-analysis of observational controlled dietary trials and prospective cohort studies suggests that replacement of trans-fat with nonhydrogenated oils would result in significant reduction of the incidence of chd. gender differences in the prevalence of chd are well documented, and many factors appear to be responsible including lipid profile and genetic susceptibility. neonatal administration of high doses of the food flavor enhancer monosodium glutamate has also been shown to result in obesity together with stunted heart growth and hypoplasticity in rodents. furthermore, the cardiovascular system of these animals have been shown to respond deficiently to certain challenges, including an attenuated blood pressure response to the systemic injection of nitric oxide synthase inhibitors, angiotensin ii and other vasoactive compounds. injections of high doses of msg cause ablation of the arcuate nucleus via glutamate-induced degeneration of those areas of the immature brain which are insufficiently protected by a mature blood-brain barrier, including the hypothalamic area. this suggested that the hypothalamus may play an important role in the growth of the heart. it is now apparent that maternal administration of msg can penetrate the placental barrier and distribute to the embryonic tissues of the fetus. oral administration of msg and 3h-labeled glutamate as a tracer to pregnant mice resulted in marked elevations of 3h- glu in the placenta and in fetal brain, liver and kidney. msg consumption has increased globally in recent years, with recent estimations of the current average daily intake believed to be up to g/day. despite the widespread consumption of this common food flavor enhancer, which often occurs in processed and packaged foods in combination with trans-fat, very few studies have addressed the effect of dietary trans-fat and/or msg consumption on cardiac gene expression. furthermore, although sexual dimorphism at the growth hormone axis has been demonstrated in msg-treated rodents, little is known about sex-specific gene expression in response to exposure to msg. the aim of this work was to establish the effect of dietary tfa and/or msg on cardiac gene expression in an in vivo animal model, and to examine sex-specific differences as well as commonalities of gene expression in response to diet. the amount of oral msg used in this study reflects current consumption levels and is - times less that the level previously reported to induce neuronal damage when injected neonatally. since it has previously been ascertained that msg excitotoxicity occurs only when the blood brain barrier is vulnerable, for example neonatally, and because developmental programming of cardiac gene expression may be affected by maternal nutritional balance, we bred our study animals from females previously established on these diets for weeks prior to mating, in accordance with our previous studies. exposure to these diets occurred throughout the study, in order to mimic as closely as possible the situation that occurs in humans. the results of our microarray analysis revealed considerable sexual dimorphism in response to the dietary interventions that we tested, with males appearing to exhibit more differential gene expression compared to females.  consumption of trans-fat and/or msg results in sex-specific changes in physiological characteristics abc uncommon letters indicate difference in means between diet groups within a row. differences between genders within a diet group for each variable are denoted by *,** and *** for p-values <, < & < sexual dimorphism in cardiac gene expression we used affymetrix mouse gene st expression arrays to determine differences in global cardiac gene/expressed sequence tags in response to diet, in both males and females. using a false discovery rate with a significance level set at, we identified two subsets of genes/ests that were differentially expressed between males and females at various stringency levels. to better understand the relevance of these sex-dimorphic genes we used a program based on gene ontology categories of biological processes, molecular function and cellular component. additional file table s lists the main functional go categories of transcripts that were differentially expressed between the sexes with a stringency of ± -fold change in expression. degs were further characterized as being relatively up-regulated either in males or in females. increased transcription of male genes in the "biological processes" category included genes involved in cellular macromolecule metabolism, anatomical structure development and blood vessel development. these genes included notch interstitial matrix gene col3a, fibulin and integrin αv. the predominant male-specific kegg pathways included the regulation of actin cytoskeleton and antigen processing pathways. conversely female-specific transcription were enriched in the categories of cellular and metabolic processes, including mitochondrial carbonic anhydrase 5b, glutathione s-transferase and magnesium transporter. one kegg-pathway for cysteine and methionine metabolism was also upregulated. chromosomal localization of differentially expressed cardiac genes to investigate a possible non-random chromosomal distribution of the degs, we used the fisher exact test to compare the frequencies of significant genes on each chromosome against the total number of genes interrogated on each chromosome. chromosomes and y were enriched for male-biased genes whereas chromosomes, and x contained more female-biased genes. notably we found four genes encoded on the y chromosome which were male-specific, and female-biased x-linked genes including ace dystrophin, and several transcription factors such as tceal tceal and eif2s3x. trans-fat feeding induces sex-specific alterations in cardiac gene expression in order to identify the subset of genes and ests with a fold difference of ± or more within male or female comparisons for mice fed the tfa diet versus control, we used the results from a 2-way anova model described in the methods section. we identified degs that were responsive to diet in males; and approximately half as many diet-responsive degs in females:. specifically, the trans-fat diet significantly downregulated the expression of many male cardiac genes involved in the regulation of biological processes, transcription and phosphorylation. conversely females upregulated the expression of genes with functional ontologies relating to binding, nitrogen compound metabolism and ketone metabolism. significant male degs downregulated by tfa consumption included key transcription modulator sterol regulatory element binding factor, transcription factors mef2d and gata; vasoconstrictive alpha-2b adrenergic receptor; major cardiac na+ channel scn5a, heat shock transcription factor, calcium-regulating enzyme phospholipase c δ, and circadian clock gene per. signaling molecules calcium/calmodulin-dependent protein kinase kinase 1α and mitogen-acrivated protein kinase kinase expression were also halved in male tfa mice. conversely pro-hypertrophic hdac, which represses mef activity, was upregulated in males and downregulated in females. other genes with this pattern of sex-dimorphic expression included plastin and tachykinin. in both sexes, the tfa diet led to increased expression of fatty acid oxidizing 3-hydroxy-3-methylglutaryl coa synthase and fructose bisphosphatase; however expression of tnf superfamily member light/tnfsf was increased 7-fold in male trans-fat hearts compared to control, whereas it was only increase 2-fold in female trans-fat hearts. diet-induced changes in female hearts were less significant, with only degs significantly up-regulated, including progestin adipoq receptor family member v and ras guanyl releasing protein. enrichment analysis of functional go pathways within this subset revealed that females exhibited a four-fold less down-regulation of cardiac genes with classified biological function than males. we next investigated the relationship between these genes using ingenuity pathways analysis. a network of degs relating to cardiovascular disease and lipid metabolism among others was identified as being significantly affected by the tfa diet in cardiac tissue from male mice, whereas a network of functionally related genes involved in female lipid metabolism and cardiovascular development is shown in figure 4b. monosodium glutamate consumption alters cardiac gene expression with sex-specific differences a different pattern of gene expression was induced by msg feeding, which again showed significant sex-specific differences in cardiac gene expression. 2-way anova analysis revealed a subset of male cardiac degs which were responsive to dietary msg compared to control diet, and female degs.. unlike trans-fat, msg upregulated more cardiac degs and down-regulated less. genes/ests upregulated in males but not females in response to msg included ing, glutathione s-transferase and pancreatic lipase. transcripts enriched for female-biased expression included myosin 7a, pdzd and aldehyde dehydrogenase gene aldh1a functional analysis of gene ontologies of these subsets showed increased expression of male cellular and metabolic processes, whilst decreasing expression of phosphorylation-related degs. conversely females upregulated more genes/ests with developmental and anatomical function ontologies. genes/ests that were upregulated in both sexes included ionotropic glutamate receptor grik; nebulin which regulates actin filament length. msg alters trans-fat induced cardiac gene expression in order to address the question as to whether the addition of msg to a trans-fat diet could affect cardiac gene expression, we next identified a subset of degs which showed significant sex-dimorphism in response to these two diets. degs in males showed significant response to the inclusion of msg in the trans fat diet, and in females. of note in both sexes, expression of lipogenic stearoyl-coa desaturase was upregulated in tfa+msg diet group hearts compared to tfa alone and was accompanied by an increase in myosin h magnesium transporter slc41a and apoptosis-inducing bcl2-interacting killer. downregulated degs included fibrulin, sclerostin and dystrophin, together with fatty acid-oxidizing enzymes phospholipase a and aldehyde dehydrogenase. significant differentially expressed cardiac transcripts in tfa + msg-fed versus tfa-fed mice were examined for enrichment of go and kegg classifications. in males, major functional go categories included metabolism, biosynthesis and transcription, whereas enrichment of female degs upregulated by msg feeding again fell into the categories of developmental and anatomical structural function. cellular positional mapping reveals sex-specific differences in diet-deregulated gene expression a final analysis of significant diet-induced deregulation of cardiac gene expression between all diet combinations co-analyzed yielded a cohort of mapable genes which were deregulated by all dietary manipulations only in males, and half that number of genes which were only deregulated in females. interestingly in this male subset, expression of genes previously demonstrated to be upregulated by tfa feeding were downregulated in animals consuming the msg-enriched diets. male-specific genes in this subset included stress-related proteins dnaja and pdia together with phosphodiesterase 6a and histone h1e. females displayed a slightly divergent pattern. additionally transcripts were identified as being significantly differentially expressed in both sexes in response to all dietary manipulations. figure shows a heatmap of a selection based on functional relevance, together with a number of x-linked and y-linked diet-induced degs, p < with a stringency of ± -fold change in expression. eighteen genes were randomly chosen for further analyzed by qrt pcr based on biological relevance. pearson correlation coefficients between the microarray analysis and qrt pcr were calculated. ratios of expressions between the diet comparisons calculated from the microarray data set correlated well with the ratio calculated from the real-time pcr data. a complete list of these genes and pcr primers is given in additional file table s discussion our results indicate that dietary manipulation prenatally, and over the first months of life can markedly influence cardiac gene expression in a sex-specific manner. sexual dimorphism in gene expression exists even in the healthy heart, and chromosome has been shown to have more female-biased differential cardiac gene expression, whereas male-biased enrichment was found in chromosomes and in addition to the x and y chromosomes which segregated as anticipated, we found female-biased chromosomal enrichment on mouse chromosomes and 12; and male-biased expression on chromosomes and interestingly, a locus on murine chromosome has been associated with markers of the metabolic syndrome, including body weight, insulin, leptin and triglyceride. the main outcome from our study was that trans-fat induced more than twice as many degs in males compared to females, and downregulated four times as many genes, including a number that have been implicated in cellular pathways leading to hypertrophy such as gata and mef2d. expression of gata previously identified as being of importance in stress responsiveness, was halved in males in response to the tfa diet. gata regulates transcription of a large number of cardiac genes via binding to the consensus 5'-wgatar-3' sequence, and is implicated in cardiac muscle apoptosis, where under conditions of oxidative stress gata downregulation is preceded by mek/erk-dependent phosphorylation. cardiac hypertrophy and chd are mediated by transcription factor integration of upstream stress signals, and mef2d-deficient mice have an attenuated hypertrophic response to cardiac stress. mef2d activity is repressed by the histone deacetylase hdac which we found to be increased in male tfa mice. histone deacetylases are also regulated by calmodulin and calmodulin kinase, and are among the negative regulators of cardiac hypertrophy which attenuates mef transcriptional activity. in the present study, we found a reduction of expression of camkk and an increase in expression of camk2b, suggesting that trans-fat may deregulate both the calmodulin kinase/mef2/hdac pathway and the pkc/mapk/gata pathways of cardiac gene transcription. trans-fat feeding deregulated other stress-associated genes in males including phospholipase c δ the main cardiac isoform implicated in oxidative stress-induced redox signaling, and light/tnfsf demonstrated to be upregulated in clinical and experimental heart failure. additionally, key circadian clock gene per was decreased by more than half in the hearts of male tfa-fed mice. downregulation of per by glucose has been shown to be accompanied by changes in the expression of genes involved in lipid metabolism, transcription and cell cycle, such as sterol regulatory element binding factor, hydroxy-3-methylglutaryl coa synthase and stearoyl-coa desaturase, all of which were differentially regulated by trans-fat in our study. whilst it is known that trans-fat consumption is linked to cardiovascular disease, the mechanism behind this is incompletely understood. elaidic acid, the main tfa in partially hydrogenated vegetable shortening, incorporates into cardiac tissues; and it has been suggested that tfas are oxidized at a slower rate than cis-fatty acids, resulting in the accumulation of reactive oxidation intermediates, which could lead to oxidative stress and the activation of the gata mefd and hdac-associated transcriptional pathways. taken together, our results suggest that trans-fat feeding results in significant impairment of transcriptional pathways relating to cellular stress, particularly in males. it has recently been shown that male c57bl/6j mice are more vulnerable than females to the impact of a high-fat diet in terms of ensuing weight gain and deleterious metabolic peturbations; and it is therefore possible that the sex-differences in trans fat -induced cardiac gene expression seen in our study may have contributed to the resultant increase in male weight gain and heart: body ratio compared to females. the msg diet resulted in a different pattern of sexual dimorphism in cardiac transcription, with fairly equal numbers of degs being up- and down-regulated. however there were a number of genes which were overexpressed in males but not in females, and vice versa. the functional categories of overexpressed genes differed too, with females showing more enrichment of genes associated with developmental processes for example, whereas male upregulated transcripts were enriched for cellular and metabolic function. neonatal injections of msg result in smaller hearts with less dna synthesizing activity; and hypotension has been observed in female, but not male rats treated neonatally with msg. in our study, female hearts from msg-diet group mice weighed significantly less than male hearts, however an examination of genes deregulated in our msg mice did not provide many clues as to the mechanism responsible for this apparent difference. we have previously shown that msg alters trans fat -induced hepatic and adipose tissue gene expression with the result that several key transcripts involved in ß-oxidation of lipids were downregulated in tfa+msg treated mice; and in our present study we found that the combination of tfa+msg decreased the expression of several fatty acid-oxidizing enzymes such as phospholipase a and aldehyde dehydrogenase,whilst increasing the expression of lipogenic cardiac-specific stearoyl-coa desaturase, previously shown to be regulated by leptin and dietary factors. when we compared cardiac gene transcription between all dietary combinations co-analyzed, we again found that more significant degs were induced in males compared to females. our analysis identified a subset of over transcripts which significantly changed only in male mice in response to diet, and approximately half this number of transcripts differentially expressed in females only, and a similar number of shared degs were common to both sexes. this suggests that males may have a higher cardiac transcriptional response to dietary manipulation, and indeed greater transcriptional response to exercise has previously been demonstrated in males but not females. several theories have been put forward as to this disparity, the cardioprotective effect of estrogen being one possibility, especially in light of epidemiological data indicating a lower incidence of female chd, at least until menopause, after which this dissimilarity declines. however, this is unlikely to account for all the sex-specific differences observed, since after correcting for lifestyle factors, the discrepancy in chd rates was reduced. by using microarrays to analyze diet-induced cardiac gene expression patterns, we have identified genes which are differentially regulated and may play important roles in the development of chd associated with trans-fat consumption. however there are some issues to consider when interpreting the results from the present study. for the microarray experiments, we employed a pooling design in order to reduce biological variation, and to lower the costs associated with sample size and chip availability. in order to address a possible loss of sensitivity and an increase in the incidence of false positives, we employed stringent criteria for the identification of the diet-regulated degs. one limitation of this study is the fact that these experiments were only performed on c57bl/6j mice of both sexes. it would be advantageous but beyond the scope of the present study, to ascertain whether similar results could be obtained using a different genetic background, or possibly a higher animal model.  we have found substantial evidence for sexual dimorphism in response to dietary manipulation, with males apparently showing the greater cardiac transcriptional response. in particular, we found that trans-fat feeding in male mice resulted in deregulation of the expression of genes involved in cellular stress pathways. excessive tfa consumption is believed to be a risk factor in human heart disease, the incidence of which appears to be higher in male patients. our present observations may provoke further studies which could result in sex-specific medical intervention in the future. 
a paucity of information on biological sex-specific differences in cardiac gene expression in response to diet has prompted this present nutrigenomics investigation. sexual dimorphism was also observed in cardiac transcription from msg-fed animals, with both sexes upregulating approximately degs exhibiting sex-specific differences in go categories. a comparison of cardiac gene expression between all diet combinations together identified a subset of degs significant only in males, degs significant in females only, and transcripts identified as differentially expressed in response to dietary manipulation in both sexes. sexual dimorphism exists in the physiological and transcriptional response to diet, particularly in response to high-fat feeding. our model identified major changes in the cardiac transcriptional profile of tfa and/or msg-fed mice compared to controls, which was reflected by significant differences in the physiological profile within the diet groups. identification of sexual dimorphism in cardiac transcription may provide the basis for sex-specific medicine in the future. impairment of the cardiovascular system has been noted in animals exposed to monosodium glutamate during the neonatal period, and sexual dimorphism in the growth axis of msg-treated animals has previously been noted. 2-factor anova indicated that the tfa diet induced over twice as many cardiac differentially expressed genes in males compared to females; and times as many male degs were downregulated including gata mef2d and srebf enrichment of functional gene ontology categories were related to transcription, phosphorylation and anatomic structure. we examined physiological differences and changes in gene expression in response to tfa and/or msg consumption compared to a control diet, in male and female c57bl/6j mice. consumption of trans-fatty acids has been linked to substantially increased risk of heart disease, in which sexual dimorphism is apparent, with males suffering a higher disease rate.
 a paucity of information on biological sex-specific differences in cardiac gene expression in response to diet has prompted this present nutrigenomics investigation. sexual dimorphism exists in the physiological and transcriptional response to diet, particularly in response to high-fat feeding. consumption of trans-fatty acids has been linked to substantially increased risk of heart disease, in which sexual dimorphism is apparent, with males suffering a higher disease rate. impairment of the cardiovascular system has been noted in animals exposed to monosodium glutamate during the neonatal period, and sexual dimorphism in the growth axis of msg-treated animals has previously been noted. processed foods may contain both tfa and msg. methods we examined physiological differences and changes in gene expression in response to tfa and/or msg consumption compared to a control diet, in male and female c57bl/6j mice.  heart and % body weight increases were greater in tfa-fed mice, who also exhibited dyslipidemia. hearts from msg-fed females weighed less than males. 2-factor anova indicated that the tfa diet induced over twice as many cardiac differentially expressed genes in males compared to females; and times as many male degs were downregulated including gata mef2d and srebf enrichment of functional gene ontology categories were related to transcription, phosphorylation and anatomic structure. a number of genes were upregulated in males and downregulated in females, including pro-apoptotic histone deacetylase-. sexual dimorphism was also observed in cardiac transcription from msg-fed animals, with both sexes upregulating approximately degs exhibiting sex-specific differences in go categories. a comparison of cardiac gene expression between all diet combinations together identified a subset of degs significant only in males, degs significant in females only, and transcripts identified as differentially expressed in response to dietary manipulation in both sexes.  our model identified major changes in the cardiac transcriptional profile of tfa and/or msg-fed mice compared to controls, which was reflected by significant differences in the physiological profile within the diet groups. identification of sexual dimorphism in cardiac transcription may provide the basis for sex-specific medicine in the future. 