Transgenic overexpression of glutathione S-transferase μ-type 1 reduces hypertension and oxidative stress in the stroke-prone spontaneously hypertensive rat

Background: Combined congenic breeding and microarray gene expression profiling previously identified glutathione S-transferase m-type 1 (Gstm1) as a positional and functional candidate gene for blood pressure (BP) regulation in the stroke-prone spontaneously hypertensive (SHRSP) rat. Renal Gstm1 expression in SHRSP rats is significantly reduced when compared with normotensive Wistar Kyoto (WKY) rats. As Gstm1 plays an important role in the secondary defence against oxidative stress, significantly lower expression levels may be functionally relevant in the development of hypertension. The aim of this study was to investigate the role of Gstm1 in BP regulation and oxidative stress by transgenic overexpression of the Gstm1 gene. Method: Two independent Gstm1 transgenic SHRSP lines were generated by microinjecting SHRSP embryos with a linear construct controlled by the EF-1a promoter encoding WKY Gstm1 cDNA [SHRSP-Tg(Gstm1)1WKY and SHRSPTg(Gstm1)2WKY]. Results: Transgenic rats exhibit significantly reduced BP and pulse pressure when compared with SHRSP [systolic: SHRSP 205.2 3.7 mmHg vs. SHRSP-Tg(Gstm1)1WKY 175.5 1.6 mmHg and SHRSP-Tg(Gstm1)2WKY 172 3.2 mmHg, P< 0.001; pulse pressure: SHRSP 58.4 0.73 mmHg vs. SHRSP-Tg(Gstm1)1WKY 52.7 0.19 mmHg and SHRSP-Tg(Gstm1)2WKY 40.75 0.53 mmHg, P< 0.001]. Total renal and aortic Gstm1 expression in transgenic animals was significantly increased compared with SHRSP [renal relative quantification (RQ): SHRSP-Tg(Gstm1)1WKY 1.95 vs. SHRSP 1.0, P< 0.01; aorta RQ: SHRSP-Tg(Gstm1)1WKY 2.8 vs. SHRSP 1.0, P< 0.05]. Renal lipid peroxidation (malondialdehyde: protein) and oxidized : reduced glutathione ratio levels were significantly reduced in both transgenic lines when compared with SHRSP [malondialdehyde: SHRSP 0.04 0.009mmol/l vs. SHRSP-Tg(Gstm1)1WKY 0.024 0.002mmol/l and SHRSPTg(Gstm1)2WKY 0.021 0.002mmol/l; (oxidized : reduced glutathione ratio): SHRSP 5.19 2.26mmol/l vs. SHRSPTg(Gstm1)1WKY 0.17 0.111mmol/l and SHRSPTg(Gstm1)2WKY 0.471 0.223mmol/l]. Transgenic SHRSP rats containing the WKY Gstm1 gene demonstrate significantly lower BP, reduced oxidative stress and improved levels of renal Gstm1 expression. Conclusion: These data support the hypothesis that reduced renal Gstm1 plays a role in the development of hypertension

Contribution of Autosomal Loci and the Y Chromosome to the Stress Response in Rats

Stress is a critical contributor to cardiovascular diseases through its impact on blood pressure variability and cardiac function. Familial clustering of reactivity to stress has been demonstrated in human subjects, and some rodent models of hypertension are hyperresponsive to stress. Therefore, the present study was designed to uncover the genetic determinants of the stress response. We performed a total genome linkage search to identify the loci of the body temperature response to immobilization stress in a set of recombinant inbred strains (RIS) originating from reciprocal crosses of spontaneously hypertensive rats (SHR) with a normotensive Brown Norway Lx strain. Two quantitative trait loci (QTLs) were revealed on chromosomes (Chrs) 10 and 12 (logarithm of odds scores, 2.2 and 1.3, respectively). The effects of these QTLs were enhanced by a high sodium diet (logarithm of odds scores, 4.0 and 3.3 for Chrs 10 and 12, respectively), which is suggestive of a salt-sensitive component for the phenotype, Congenics for Chr 10 confirmed both the QTL and the salt effect in RIS. Negatively associated loci were also identified on Chrs 8 and 11. Interaction between the loci of Chrs 10 and 12 was demonstrated, with the rat strains bearing SHR alleles at both loci having the highest thermal response to stress. Furthermore, the Y Chr of SHR origin enhanced the response to immobilization stress, as demonstrated in 2 independent models, RIS and Y Chr consomics. However, its full effect requires autosomes of the SHR strain. These findings provide the first evidence for the genetic determination of reactivity to stress with interactions between autosomal loci and between the Y and autosomal Chrs that contribute to the explanation of the 46% of variance in the stress response

Rosuvastatin Can Block Pro-Inflammatory Actions of Transgenic Human C-Reactive Protein Without Reducing its Circulating Levels

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Heritability and Tissue Specificity of Expression Quantitative Trait Loci

Variation in gene expression is heritable and has been mapped to the genome in humans and model organisms as expression quantitative trait loci (eQTLs). We applied integrated genome-wide expression profiling and linkage analysis to the regulation of gene expression in fat, kidney, adrenal, and heart tissues using the BXH/HXB panel of rat recombinant inbred strains. Here, we report the influence of heritability and allelic effect of the quantitative trait locus on detection of cis- and trans-acting eQTLs and discuss how these factors operate in a tissue-specific context. We identified several hundred major eQTLs in each tissue and found that cis-acting eQTLs are highly heritable and easier to detect than trans-eQTLs. The proportion of heritable expression traits was similar in all tissues; however, heritability alone was not a reliable predictor of whether an eQTL will be detected. We empirically show how the use of heritability as a filter reduces the ability to discover trans-eQTLs, particularly for eQTLs with small effects. Only 3% of cis- and trans-eQTLs exhibited large allelic effects, explaining more than 40% of the phenotypic variance, suggestive of a highly polygenic control of gene expression. Power calculations indicated that, across tissues, minor differences in genetic effects are expected to have a significant impact on detection of trans-eQTLs. Trans-eQTLs generally show smaller effects than cis-eQTLs and have a higher false discovery rate, particularly in more heterogeneous tissues, suggesting that small biological variability, likely relating to tissue composition, may influence detection of trans-eQTLs in this system. We delineate the effects of genetic architecture on variation in gene expression and show the sensitivity of this experimental design to tissue sampling variability in large-scale eQTL studies

Cardioprotective Regimen of Adaptation to Chronic Hypoxia Diversely Alters Myocardial Gene Expression in SHR and SHR-mtBN Conplastic Rat Strains

Adaptation to continuous normobaric hypoxia (CNH) protects the heart against acute ischemia/reperfusion injury. Recently, we have demonstrated the infarct size-limiting effect of CNH also in hearts of spontaneously hypertensive rats (SHR) and in conplastic SHR-mtBN strain characterized by the selective replacement of the mitochondrial genome of SHR with that of more ischemia-resistant Brown Norway rats. Importantly, cardioprotective effect of CNH was more pronounced in SHR-mtBN than in SHR. Thus, here we aimed to identify candidate genes which may contribute to this difference between the strains. Rats were adapted to CNH (FiO2 0.1) for 3 weeks or kept at room air as normoxic controls. Screening of 45 transcripts was performed in left ventricles using Biomark Chip. Significant differences between the groups were analyzed by univariate analysis (ANOVA) and the genes contributing to the differences between the strains unmasked by CNH were identified by multivariate analyses (PCA, SOM). ANOVA with Bonferroni correction revealed that transcripts differently affected by CNH in SHR and SHR-mtBN belong predominantly to lipid metabolism and antioxidant defense. PCA divided four experimental groups into two main clusters corresponding to chronically hypoxic and normoxic groups, and differences between the strains were more pronounced after CNH. Subsequently, the following 14 candidate transcripts were selected by PCA, and confirmed by SOM analyses, that can contribute to the strain differences in cardioprotective phenotype afforded by CNH: Alkaline ceramidase 2 (Acer2), Fatty acid translocase (Cd36), Aconitase 1 (Aco1), Peroxisome proliferator activated receptor gamma (Pparg), Hemoxygenase 2 (Hmox2), Phospholipase A2 group IIA (Ppla2g2a), Dynamin-related protein (Drp), Protein kinase C epsilon (Pkce), Hexokinase 2 (Hk2), Sphingomyelin synthase 2 (Sgms2), Caspase 3 (Casp3), Mitofussin 1 (Mfn1), Phospholipase A2 group V (Pla2g5), and Catalase (Cat). Our data suggest that the stronger cardioprotective phenotype of conplastic SHR-mtBN strain afforded by CNH is associated with either preventing the drop or increasing the expression of transcripts related to energy metabolism, antioxidant response and mitochondrial dynamics

histoneHMM:Differential analysis of histone modifications with broad genomic footprints

BACKGROUND: ChIP-seq has become a routine method for interrogating the genome-wide distribution of various histone modifications. An important experimental goal is to compare the ChIP-seq profiles between an experimental sample and a reference sample, and to identify regions that show differential enrichment. However, comparative analysis of samples remains challenging for histone modifications with broad domains, such as heterochromatin-associated H3K27me3, as most ChIP-seq algorithms are designed to detect well defined peak-like features. RESULTS: To address this limitation we introduce histoneHMM, a powerful bivariate Hidden Markov Model for the differential analysis of histone modifications with broad genomic footprints. histoneHMM aggregates short-reads over larger regions and takes the resulting bivariate read counts as inputs for an unsupervised classification procedure, requiring no further tuning parameters. histoneHMM outputs probabilistic classifications of genomic regions as being either modified in both samples, unmodified in both samples or differentially modified between samples. We extensively tested histoneHMM in the context of two broad repressive marks, H3K27me3 and H3K9me3, and evaluated region calls with follow up qPCR as well as RNA-seq data. Our results show that histoneHMM outperforms competing methods in detecting functionally relevant differentially modified regions. CONCLUSION: histoneHMM is a fast algorithm written in C++ and compiled as an R package. It runs in the popular R computing environment and thus seamlessly integrates with the extensive bioinformatic tool sets available through Bioconductor. This makeshistoneHMM an attractive choice for the differential analysis of ChIP-seq data. Software is available from http://histonehmm.molgen.mpg.de

Wars2 is a determinant of angiogenesis.

Coronary flow (CF) measured ex vivo is largely determined by capillary density that reflects angiogenic vessel formation in the heart in vivo. Here we exploit this relationship and show that CF in the rat is influenced by a locus on rat chromosome 2 that is also associated with cardiac capillary density. Mitochondrial tryptophanyl-tRNA synthetase (Wars2), encoding an L53F protein variant within the ATP-binding motif, is prioritized as the candidate at the locus by integrating genomic data sets. WARS2(L53F) has low enzyme activity and inhibition of WARS2 in endothelial cells reduces angiogenesis. In the zebrafish, inhibition of wars2 results in trunk vessel deficiencies, disordered endocardial-myocardial contact and impaired heart function. Inhibition of Wars2 in the rat causes cardiac angiogenesis defects and diminished cardiac capillary density. Our data demonstrate a pro-angiogenic function for Wars2 both within and outside the heart that may have translational relevance given the association of WARS2 with common human diseases

Author Correction: WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Dissection of chromosome 18 blood pressure and salt-sensitivity quantitative trait loci in the spontaneously hypertensive rat

Hypertension in humans and experimental models has a strong hereditary basis, but identification of causative genes remains challenging. Quantitative trait loci (QTLs) for hypertension and salt sensitivity have been reported on rat chromosome 18. We set out to genetically isolate and prioritise genes within the salt sensitivity and hypertension QTLs on the spontaneously hypertensive rat (SHR) chromosome 18, by developing and characterising a series of congenic strains derived from the SHR and normotensive Brown Norway (BN) rat strains. The SHR.BN-D18Rat113/D18Rat82 (SHR-18) congenic strain exhibits significantly lower blood pressure and is salt-resistant compared to SHR. Transplantation of kidneys from SHR-18 donors into SHR recipients is sufficient to attenuate increased blood pressure but not salt sensitivity. Derivation of congenic sublines allowed separation of salt sensitivity from hypertension QTL regions. Renal expression studies with microarray and Solexa-based sequencing in parental and congenic strains identified four differentially expressed genes within the hypertension QTL region, one of which is an unannotated transcript encoding a previously undescribed, small non-coding RNA. Sequencing selected biological candidate genes within the minimal congenic interval revealed a non-synonymous variant in SHR Transcription factor 4. The minimal congenic interval is syntenic to a region of human chromosome 18 where significant linkage to hypertension was observed in family-based linkage studies. These congenic lines provide reagents for identifying causative genes that underlie the chromosome 18 SHR QTLs for hypertension and salt sensitivity. Candidate genes identified in these studies merit further investigation as potentially causative hypertension genes in SHR and human hypertension

A Revamped Rat Reference Genome Improves the Discovery of Genetic Diversity in Laboratory Rats

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats