Our results indicate that dietary manipulation prenatally, and over the first 8 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 4 has been shown to have more female-biased differential cardiac gene expression , whereas male-biased enrichment was found in chromosomes 3 and 14. In addition to the X and Y chromosomes which segregated as anticipated, we found female-biased chromosomal enrichment on mouse chromosomes 3, 4 and 12; and male-biased expression on chromosomes 8 and 11. Interestingly, a locus on murine chromosome 4 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 Gata4 and Mef2d . Expression of Gata4, previously identified as being of importance in stress responsiveness , was halved in males in response to the TFA diet. Gata4 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 Gata4 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 Hdac2, 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 Mef2 transcriptional activity . In the present study, we found a reduction of expression of Camkk1 and an increase in expression of Camk2b, suggesting that Trans-fat may deregulate both the calmodulin kinase/Mef2/Hdac pathway and the PKC/MAPK/GATA4 pathways of cardiac gene transcription.
Trans-fat feeding deregulated other stress-associated genes in males including phospholipase C δ1, the main cardiac isoform implicated in oxidative stress-induced REDOX signaling , and LIGHT/Tnfsf14, demonstrated to be upregulated in clinical and experimental heart failure . Additionally, key circadian clock gene Per1 was decreased by more than half in the hearts of male TFA-fed mice. Downregulation of Per1 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 (Srebf2), hydroxy-3-methylglutaryl CoA synthase (Hmgcs) and stearoyl-coA desaturase (Scd), 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 Gata4, Mefd2 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 A2 and aldehyde dehydrogenase,whilst increasing the expression of lipogenic cardiac-specific stearoyl-coA desaturase 4 (Scd4), 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 100 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.