In addition to sperm-related genes, the male-specific chromosome Y (chrY) contains a class of ubiquitously expressed and evolutionary conserved dosage-sensitive regulator genes that include the neighboring Uty, Ddx3y and (in mice) Eif2s3y genes. However, no study to date has investigated the functional impact of targeted mutations of any of these genes within adult non-reproductive somatic cells. We thus compared adult male mice carrying a gene trap within their Uty gene (UtyGT) to their wild-type (WT) isogenic controls, and performed deep sequencing of RNA and genome-wide profiling of chromatin features in extracts from either cardiac tissue, cardiomyocyte-specific nuclei or purified cardiomyocytes. The apparent impact of UtyGT on gene transcription concentrated mostly on chrY genes surrounding the locus of insertion, i.e. Uty, Ddx3y, long non-coding RNAs (lncRNAs) contained within their introns and Eif2s3y, in addition to possible effects on the autosomal Malat1 lncRNA. Notwithstanding, UtyGT also caused coordinate changes in the abundance of hundreds of mRNA transcripts related to coherent cell functions, including RNA processing and translation. The results altogether indicated that tightly co-regulated chrY genes had nonetheless more widespread effects on the autosomal transcriptome in adult somatic cells, most likely due to mechanisms other than just transcriptional regulation of corresponding protein-coding genes.

Angiotensinogen (AOG) is the glycoprotein precursor of the angiotensin peptides. It is cleaved by renin to yield the decapeptide angiotensinI (AngI); angiotensin-converting enzyme then removes a dipeptide from the C-terminal end of AngI to form the physiologically active octapeptide angiotensin II (AngII). Circulating AngII increases vascular tone and promotes salt and water retention by a combined action on many target organs, including vascular smooth muscles, kidneys, adrenals, brain, and sympathetic nervous system. The stimulatory effects of estrogens on circulating levels of AOG were first reported by Helmer and Griffith. This has been confirmed in many subsequent reports; in addition, synthetic steroids were found to be more effective than naturally occurring steroids. AOG is produced in tissues other than liver as well. The importance of extrahepatic production of AOG has not been determined, but it is conceivable that the concentration of AOG is higher in tissues producing this protein than in those that receive the protein only from the circulation.

Genetic reference panels are widely used to map complex, quantitative traits in model organisms. We have generated new high-resolution genetic maps of 259 mouse inbred strains from recombinant inbred strain panels (C57BL/6J × DBA/2J, ILS/IbgTejJ × ISS/IbgTejJ, and C57BL/6J × A/J) and chromosome substitution strain panels (C57BL/6J-Chr#, C57BL/6J-Chr#, and C57BL/6J-Chr#). We genotyped all samples using the Affymetrix Mouse Diversity Array with an average intermarker spacing of 4.3 kb. The new genetic maps provide increased precision in the localization of recombination breakpoints compared to the previous maps. Although the strains were presumed to be fully inbred, we found residual heterozygosity in 40% of individual mice from five of the six panels. We also identified de novo deletions and duplications, in homozygous or heterozygous state, ranging in size from 21 kb to 8.4 Mb. Almost two-thirds (46 out of 76) of these deletions overlap exons of protein coding genes and may have phenotypic consequences. Twenty-nine putative gene conversions were identified in the chromosome substitution strains. We find that gene conversions are more likely to occur in regions where the homologous chromosomes are more similar. The raw genotyping data and genetic maps of these strain panels are available at http://churchill-lab.jax.org/website/MDA.

Several complex disorders (including auto-immune, inflammatory and cardiovascular diseases) display a strong sex bias that cannot be explained solely on the basis of differences in sex hormone production.1 While it then becomes logical to consider the potential contributions of sex chromosomes, the latter have long constituted sort of a ‘blind spot’ in genetic association studies. For reasons that include lack of coverage, difficulties in genotype calling and the fact that chromosome X is not fully inactivated in females, the vast majority of genome-wide association studies (GWAS) have either not analyzed chromosome X properly or (most frequently) ignored it altogether. New computational methods are beginning to emerge to overcome these shortcomings.2, 3 The situation is even more complicated for the male-specific portion of chromosome Y (MSY). Because MSY is transmitted in its entirety from father to son, polymorphisms on this chromosome are not useful in linkage-disequilibrium-based studies. One additional hurdle might in fact have been a ‘cultural one’: because of MSY losing most of its genes in the course of evolution, it has often been considered a ‘degenerative’ chromosome and a ‘genetic wasteland’ that plays no role beyond testis determination and sperm production,1 and therefore not worthy of attention.

Background: The C57BL/6J. Y-A/J mouse strain is a chromosome-substituted line where the original male-specific portion of chromosome Y (MSY) from C57BL/6J mice was substituted for that from A/J mice. In hearts from male C57BL/6J. Y-A/J and C57BL/6J mice, orchidectomy (ORX) affected in a strictly strain-specific fashion the expression a subset of genes showing enrichment for functional categories, including that of circadian rhythms and cardiac contractility. We further tested whether: (1) there were strain-specific differences in cardiac circadian rhythms; (2) strain-dependent differences in the effects of ORX on contractility genes translated into differences in cardiac functions; and (3) differential contractility responses occurred preferentially at times when circadian rhythms also showed strain-specific differences.Methods: In hearts from the two above strains, we (1) profiled the expression levels of 15 circadian genes at 4-h intervals across a 24 h period; (2) tested the effects of either ORX or androgen replacement on expression of cardiac contractility genes, and that of ORX on myocardial functional reserve; and (3) verified whether the effects of MSY variants on cardiac contractility-related responses showed synchronicity with differences in circadian rhythms.Results: Among the 15 tested circadian genes, a subset of them were affected by strain (and thus the genetic origin of MSY), which interacted with the amplitude of their peak of maximal expression at 2: 00 PM. At that same time-point, ORX decreased (and androgen supplementation increased) the expression of three contractility-related genes, and decreased myocardial relaxation reserve in C57BL/6J. Y-A/J, but not in C57BL/6J mice. These effects were not detected at 10: 00 AM, i. e., at another time-point when circadian genes showed no strain-specific differences.Conclusions: The results indicate that in mice, androgens have activational effects on cardiac circadian rhythms, contractile gene expression, and myocardial functional reserve. All effects occurred preferentially at the same time of the day, but varied as a function of the genetic origin of MSY. Androgens may therefore be necessary but not sufficient to impart male-specific characteristics to some particular cardiac functions, with genetic material from MSY being one other necessary factor to fully define their range of actions.

Functional genomic analysis of gene expression in mice allowed us to identify a quantitative trait locus (QTL) linked in trans to the expression of 190 gene transcripts and in cis to the expression of only two genes, one of which was Ypel5. Most of the trans-expression QTL genes were interferon-stimulated genes (ISGs), and their expression in mouse macrophage cell lines was stimulated in an IFNB1-dependent manner by Ypel5 silencing. In human HEK293T cells, YPEL5 silencing enhanced the induction of IFNB1 by pattern recognition receptors and phosphorylation of TBK1/IKBKE kinases, whereas co-immunoprecipitation experiments revealed that YPEL5 interacted physically with IKBKE. We thus found that the Ypel5 gene (contained in a locus linked to a network of ISGs in mice) is a negative regulator of IFNB1 production and innate immune responses that interacts functionally and physically with TBK1/IKBKE kinases.

Chromosome Y (chrY) variation has been associated with many complex diseases ranging from cancer to cardiovascular disorders. Functional roles of chrY genes outside of testes are suggested by the fact that they are broadly expressed in many other tissues and correspond to regulators of basic cellular functions (such as transcription, translation, and protein stability). However, the unique genetic properties of chrY (including the lack of meiotic crossover and the presence of numerous highly repetitive sequences) have made the identification of causal variants very difficult. Despite the prior lack of reliable sequences and/or data on genetic polymorphisms, earlier studies with animal chrY consomic strains have made it possible to narrow down the phenotypic contributions of chrY. Some of the evidence so far indicates that chrY gene variants associate with regulatory changes in the expression of other autosomal genes, in part via epigenetic effects. In humans, a limited number of studies have shown associations between chrY haplotypes and disease traits. However, recent sequencing efforts have made it possible to greatly increase the identification of genetic variants on chrY, which promises that future association of chrY with disease traits will be further refined. Continuing studies (both in humans and in animal models) will be critical to help explain the many sex-biased disease states in human that are contributed to not only by the classical sex steroid hormones, but also by chrY genetics.

AIMSAssessment of cardiac anatomy and function by cardiovascular magnetic resonance (CMR) is accurate and reproducible and is commonly performed to clarify borderline results obtained by other techniques. Normal reference values are lacking in a large sample of young healthy adults. As CMR is increasingly solicited to discriminate normality from equivocal disease in this population, we sought to determine reliable reference values.METHODS AND RESULTSA sample of 434 Caucasian adults aged 26 ± 4 years (45% male) without cardiovascular disease or risk factors (including obesity and smoking) underwent CMR. Blood pressure, electrocardiogram, and plasma markers (lipid profile, fasting glucose, troponin, and Nt-pro-BNP) were within normal limits and typical of a low-cardiometabolic-risk profile. End-diastolic (ED), end-systolic (ES), and stroke volumes were greater in men for left and right atria and ventricles. Left ventricular (LV) mass was higher in men. ED wall thickness of all segments was greater in men, whereas ES wall thickening (segmental function) was similar in both genders. After normalization to body surface area, all gender differences remained. Left and right ventricular volumes were lower, and left atrial volumes were higher in older individuals. In contrast, LV mass was not associated with age.CONCLUSIONThis is the first large database of reference ranges for ventricular and atrial functions, volumes, and mass in young Caucasian men and women devoid of cardiovascular disease and risk factors. These data will contribute to improving the accuracy of CMR interpretation for clinical and research applications.

Despite indications that hearts from the C57BL/6N and C57BL/6J mouse substrains differ in terms of their contractility and their responses to stress-induced overload, no information is available about the underlying molecular and cellular mechanisms. We tested whether subacute (48 hours) and chronic (14 days) administration of angiotensin II (500 ng/kg per day) had different effects on the left ventricles of male C57BL/6J and C57BL/6N mice. Despite higher blood pressure in C57BL/6J mice, chronic angiotensin II induced fibrosis and increased the left ventricular weight/body weight ratio and cardiac expression of markers of left ventricular hypertrophy to a greater extent in C57BL/6N mice. Subacute angiotensin II affected a greater number of cardiac genes in C57BL/6N than in C57BL/6J mice. Some of the most prominent differences were observed for markers of (1) macrophage activation and M2 polarization, including 2 genes (osteopontin and galectin-3) whose inactivation was reported as sufficient to prevent angiotensin II–induced myocardial fibrosis; and (2) fibroblast activation. These differences were confirmed in macrophage- and fibroblast-enriched populations of cells isolated from the hearts of experimental mice. When testing F2 animals, the amount of connective tissue present after chronic angiotensin II administration did not cosegregate with the inactivation mutation of the nicotinamide nucleotide transhydrogenase gene from C57BL/6J mice, thus discounting its possible contribution to differences in cardiac remodeling. However, expression levels of osteopontin and galectin-3 were cosegregated in hearts from angiotensin II–treated F2 animals and may represent endophenotypes that could facilitate the identification of genetic regulators of the cardiac fibrogenic response to angiotensin II.

We have previously reported Lvm1 as a quantitative trait locus (QTL) on chromosome 13 that links to cardiac left ventricular mass (LVM) in a panel of AxB/BxA mouse recombinant inbred strains (RIS). When performing a gene expression QTL (eQTL) analysis, we detected 33 cis-eQTLs that correlated with LVM. Among the latter, a group of eight cis-eQTLs clustered in a genomic region smaller than 6 Mb and surrounding the Lvm1 peak on chr13. Co-variant analysis indicated that all eight genes correlated with the phenotype in a causal rather than a reactive fashion, a finding that (despite its functional interest) did not provide grounds to prioritize any of these candidate genes. As a complementary approach, we performed weighted gene co-expression network analysis, which allowed us to detect 49 modules of highly connected genes. The module that correlated best with LVM: (1) showed linkage to a module QTL whose boundaries matched closely those of the phenotypic Lvm1 QTL on chr13; (2) harbored a disproportionately high proportion of genes originating from a small genomic region on chromosome 13 (including the 8 previously detected cis-eQTL genes); (3) contained genes that, beyond their individual level of expression, correlated with LVM as a function of their inter-connectivity; and (4) showed increased abundance of polymorphic insertion–deletion elements in the same region. Taken together, these data suggest that a domain on chromosome 13 constitutes the biologic principle responsible for the organization and linkage of the gene co-expression module, and indicate a mechanism whereby genetic variants within chromosome domains may associate to phenotypic changes via coordinate changes in the expression of several genes. One other possible implication of these findings is that candidate genes to consider as contributors to a particular phenotype should extend further than those that are closest to the QTL peak.

Increased myocardial trabeculations define noncompaction cardiomyopathy (NCC). Imaging advancements have led to increasingly common identification of prominent trabeculations with unknown implications. We quantified and determined the impact of trabeculations' burden on cardiac function and stretch in a population of healthy young adults. One hundred adults aged 18 to 35 years (28±4 years, 55% women) without known cardiovascular disease were prospectively studied by cardiovascular magnetic resonance. Left ventricular (LV) volumes, segmental function, and ejection fraction (EF) and left atrial volumes were determined. Thickness and area of trabeculated (T) and dense (D) myocardium were measured for each standardized LV segment. N-terminal pro-brain natriuretic peptide (Nt-pro-BNP) was measured. Eighteen percent of the subjects had ≥1 positive traditional criteria for NCC, and 11% meet new proposed NCC cardiovascular magnetic resonance criteria. Trabeculated over dense myocardium ratio (T/D) ratios were uniformly greater at end-diastole versus end-systole (0.90±0.25 vs 0.42±0.13, p<0.0001), in women versus men (0.85±0.24 vs 0.72±0.19, p=0.006), at anterior versus nonanterior segments (1.41±0.59 vs 0.88±0.35, p<0.0001), and at apical versus nonapical segments (1.31±0.56 vs 0.87±0.38, p<0.0001). The largest T/D ratios were associated with lower LVEF (57.0±5.3 vs 62±5.5, p=0.0001) and greater Nt-pro-BNP (203±98 vs 155±103, p=0.04). Multivariable regression identified greater end-systolic T/D ratios as the strongest independent predictor of lower LVEF, beyond age and gender, left atrial or LV volumes, and Nt-pro-BNP (β=-9.9, 95% CI -15 to 4.9, p<0.001). In conclusion, healthy adults possess variable amounts of trabeculations that regularly meet criteria for NCC. Greater trabeculations are associated with decreased LV function. Apparently healthy young adults with increased trabecular burden possess evidence of mildly impaired cardiac function.

In biology, networks are used in different contexts as ways to represent relationships between entities, such as for instance interactions between genes, proteins or metabolites. Despite progress in the analysis of such networks and their potential to better understand the collective impact of genes on complex traits, one remaining challenge is to establish the biologic validity of gene co-expression networks and to determine what governs their organization. We used WGCNA to construct and analyze seven gene expression datasets from several tissues of mouse recombinant inbred strains (RIS). For six out of the 7 networks, we found that linkage to “module QTLs” (mQTLs) could be established for 29.3% of gene co-expression modules detected in the several mouse RIS. For about 74.6% of such genetically-linked modules, the mQTL was on the same chromosome as the one contributing most genes to the module, with genes originating from that chromosome showing higher connectivity than other genes in the modules. Such modules (that we considered as “genetically-driven”) had network statistic properties (density and centralization) that set them apart from other modules in the network. Altogether, a sizeable portion of gene co-expression modules detected in mouse RIS panels had genetic determinants as their main organizing principle. In addition to providing a biologic interpretation validation for these modules, these genetic determinants imparted on them particular properties that set them apart from other modules in the network, to the point that they can be predicted to a large extent on the basis of their network statistics.

Little is known about the functions of chromosome Y (chrY) genes beyond their effects on sex and reproduction. In hearts, postpubertal testosterone affects the size of cells and the expression of genes differently in male C57BL/6J than in their C57.Y(A) counterparts, where the original chrY has been substituted with that from A/J mice. We further compared the 2 strains to better understand how chrY polymorphisms may affect cardiac properties, the latter being sexually dimorphic but unrelated to sex and reproduction. Genomic regions showing occupancy with androgen receptors (ARs) were identified in adult male hearts from both strains by chromatin immunoprecipitation. AR chromatin immunoprecipitation peaks (showing significant enrichment for consensus AR binding sites) were mostly strain specific. Measurements of anogenital distances in male pups showed that the biologic effects of perinatal androgens were greater in C57BL/6J than in C57.Y(A). Although perinatal endocrine manipulations showed that these differences contributed to the strain-specific differences in the response of adult cardiac cells to testosterone, the amounts of androgens produced by fetal testes were not different in each strain. Nonetheless, chrY polymorphisms associated in newborn pups' hearts with strain-specific differences in genomic regions showing either AR occupancy, accessible chromatin sites, or trimethylation of histone H3 Lysine 4 marks, as well as with differential expression of 2 chrY-encoded histone demethylases. In conclusion, the effects of chrY on adult cardiac phenotypes appeared to result from an interaction of this chromosome with the organizational programming effects exerted by the neonatal testosterone surge and show several characteristics of being mediated by an epigenetic remodeling of chromatin.

Background: Although C57Bl/6 mice are widely used in cardiovascular research, little is known about possible substrain differences. We compared the left ventricular (LV) remodeling induced by angiotensin II (angII) in C57Bl/6J and C57Bl/6N mice, these 2 substrains corresponding to two main branches having diverged since 1951. Methods and Results: Male C57Bl/6J and C57Bl/6N mice were treated with angII (350ng/kg/min) or vehicle via mini-osmotic pumps for either 48h (for microarray profiling of gene expression and macrophage cytofluorometry counting) or 15 days (for both gene expression and histology). AngII (15d) induced fibrosis in LV from C57BL/6N (as shown by histology and col1a expression), but not in C57BL/6J. After 48h of treatment, about 100 genes responded in a strain-specific fashion, most responses being specific for C57Bl/6N mice. Among genes showing greater than 2-fold induction by angII (48h) in C57Bl/6N mice, there was enrichment for markers of macrophages activation and M2 polarization (including osteopontin, arginase1 and galectin3). These strain-specific differences were confirmed (both in LV tissues and macrophages isolated from LVs) by Q-RT-PCR, and occurred despite that fact that AngII increased the abundance of Cd11b+ macrophages to the same extent in both strains. Moreover, AngII (48h) increased expression of several markers of fibroblast activation (including Timp1, Lox and tenascin) in C57Bl/6N (both in LV tissue and fibroblasts isolated from LVs), but not in C57Bl/6J. Although one of the best known genetic differences in both substrains is the inactivation of the Nnt gene in C57Bl6/J mice, experiments performed in F2 mice do not indicate that the fibrotic response co-segregates with the Nnt mutation. Conclusions: Although angII-treatment induces macrophage recruitment in the LVs of both C57Bl/6J and C57Bl/6N mice, activation of cardiac macrophages and their M2 pro-reparative polarization (with subsequent activation of fibroblasts and synthesis of collagen) occurs only in the genetic C57Bl/6N background. The absence of macrophage activation and cardiac fibrosis in C57BL/6J may possibly explain some differences in experimental results obtained by various investigators using different C57BL/6 substrains.

MOTIVATION:Recently, mapping studies of expression quantitative loci (eQTL) (where gene expression levels are viewed as quantitative traits) have provided insight into the biology of gene regulation. Bayesian methods provide natural modeling frameworks for analyzing eQTL studies, where information shared across markers and/or genes can increase the power to detect eQTLs. Bayesian approaches tend to be computationally demanding and require specialized software. As a result, most eQTL studies use univariate methods treating each gene independently, leading to suboptimal results.RESULTS:We present a powerful, computationally optimized and free open-source R package, iBMQ. Our package implements a joint hierarchical Bayesian model where all genes and SNPs are modeled concurrently. Model parameters are estimated using a Markov chain Monte Carlo algorithm. The free and widely used openMP parallel library speeds up computation. Using a mouse cardiac dataset, we show that iBMQ improves the detection of large trans-eQTL hotspots compared with other state-of-the-art packages for eQTL analysis.AVAILABILITY:The R-package iBMQ is available from the Bioconductor Web site at http://bioconductor.org and runs on Linux, Windows and MAC OS X. It is distributed under the Artistic Licence-2.0 terms.CONTACT:christian.deschepper@ircm.qc.ca or rgottard@fhcrc.org.SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

Although gene coexpression domains have been reported in most eukaryotic organisms, data available to date suggest that coexpression rarely concerns more than doublets or triplets of adjacent genes in mammals. Using expression data from hearts of mice from the panel of AxB/BxA recombinant inbred mice, we detected (according to window sizes) 42-53 loci linked to the expression levels of clusters of three or more neighboring genes. These loci thus formed "cis-expression quantitative trait loci (eQTL) clusters" because their position matched that of the genes whose expression was linked to the loci. Compared with matching control regions, genes contained within cis-eQTL clusters showed much greater levels of coexpression. Corresponding regions showed: (1) a greater abundance of polymorphic elements (mostly short interspersed element retrotransposons), and (2) significant enrichment for the motifs of binding sites for various transcription factors, with binding sites for the chromatin-organizing CCCTC-binding factor showing the greatest levels of enrichment in polymorphic short interspersed elements. Similar cis-eQTL clusters also were detected when we used data obtained with several tissues from BxD recombinant inbred mice. In addition to strengthening the evidence for gene expression domains in mammalian genomes, our data suggest a possible mechanism whereby noncoding polymorphisms could affect the coordinate expression of several neighboring genes.

Objective. To evaluate the capacity of chronic ASA therapy to prevent cardiac alterations and increased oxidative stress in cardiomyopathic hamsters. Methods and Results. Male Syrian cardiomyopathic and age-matched inbred control hamsters received ASA orally from the age of 60 days. Animals were sacrificed at the age of 150, 250, and 350 days to evaluate the time course of cardiac hypertrophy and cardiovascular tissue superoxide anion (O2-) production. At the age of 150 days, the ventricular weight over body weight ratio, resting heart rate, and cardiovascular O2- production were much higher in cardiomyopathic hamsters than those in control. At the age of 250 days, in addition to the continual deterioration of these parameters with age, the blood pressure started to fall and the signs of heart failure appeared. In these cardiomyopathic hamsters, chronic ASA treatment (a) completely prevented elevated O2- production and the NAD(P)H oxidase activity, (b) significantly slowed down the development of the cardiac hypertrophy and fibrosis. Conclusions. Chronic ASA treatment significantly prevents the deterioration of cardiac function and structure as well as the increased oxidative stress in the cardiomyopathic hamster. Our findings suggest that ASA presents a therapeutic potential to prevent cardiac dysfunction.

Background & ObjectivesHigh fat or high sugar diets are associated with increased risk for coronary heart disease but have not been found to exacerbate cardiac dysfunction in rodent models of heart failure. We thus tested the effect of HF diet in a mouse model of diabetes and its associated cardiac dysfunction.MethodsFive‐week‐old C57Bl6J male mice received either standard (Std) or HF diet for 2 weeks, then received multiple injections of either low doses (40 mg/kg) of streptozotocin (STZ) or vehicle (Veh).ResultsTen weeks after STZ injections (and compared to Std‐Veh mice), both STZ groups displayed similar (i) fasting plasma glucose and insulin levels, which were respectively increased and decreased (p<0.001) and (ii) cardiac systolic dysfunction, as evidenced by a 8% lower ejection fraction (p<0.05) at echocardiography. However, the increase in plasma levels of ketone bodies, glycated haemoglobin and branched chain amino acids was smaller (p<0.05) in HF‐STZ mice than in Std‐STZ at both 6 and 12 weeks. Likewise, HF‐STZ mice maintained (in contrast to Std‐STZ mice) body weight and epidydymal fat mass as well as Std‐Veh.ConclusionNot only did HF not exacerbate diabetes‐induced cardiac dysfunction, but it even attenuated the systemic metabolic alterations induced by STZ, which may in the long term delay the development of heart failure. (Supported by CIHR & HSFC)