Abstract-Using congenic strains of the Dahl salt-sensitive (S) rat introgressed with genomic segments from the normotensive Lewis rat, a blood pressure quantitative trait locus was previously mapped within 104 kb on chromosome 10. The goal of the current study was to conduct extensive phenotypic studies and to further fine-map this locus. At 14 weeks of age, the blood pressure of the congenic rats fed a low-salt diet was significantly higher by 47 mm Hg (PϽ0.001) compared with that of the S rat. A time-course study showed that the blood pressure effect was significant from very young ages of 50 to 52 days (13 mm Hg; PϽ0.01). The congenic strain implanted with electrocardiography transmitters demonstrated shorter-QT intervals and increased heart rate compared with S rats (PϽ0.01). The average survival of the congenic strain was shorter (134 days) compared with the S rat (175 days; PϽ0.0007). The critical region was narrowed to Ͻ42.5 kb containing 171 variants and a single gene, rififylin. Both the mRNA and protein levels of rififylin were significantly higher in the hearts of the congenic strain. Overexpression of rififylin is known to delay endocytic recycling. Endocytic recycling of fluorescently labeled holotransferrin from cardiomyocytes of the congenic strain was slower than that of S rats (PϽ0.01). Frequency of cardiomyocyte beats in the congenic strain (62Ϯ9 bpm) was significantly higher than that of the S rat (24Ϯ6 bpm; PϽ0.001). Taken together, our study provides evidence to suggest that early perturbations in endocytic recycling caused by the overexpression of Rffl is a novel physiological mechanism potentially underlying the development of hypertension.

A disintegrin-like metalloproteinase with thrombospondin motifs-16 (Adamts16) is an important candidate gene for hypertension. The goal of the present study was to further assess the candidacy of Adamts16 by targeted disruption of this gene in a rat genetic model of hypertension. A rat model was generated by manipulating the genome of the Dahl Salt-sensitive (S) rat using zinc-finger nucleases, wherein the mutant rat had a 17 bp deletion in the first exon of Adamts16, introducing a stop codon in the transcript. Systolic blood pressure (BP) of the homozygous Adamts16 mutant rats was lower by 36 mmHg compared with the BP of the S rats. The Adamts16 mutant rats exhibited significantly lower aortic pulse wave velocity and vascular media thickness compared with S rats. Scanning electron and fluorescence microscopic studies indicated that the mechanosensory cilia of vascular endothelial cells from the Adamts16 mutant rats were longer than that of the S rats. Furthermore, Adamts16 mutant rats showed splitting and thickening of glomerular capillaries and had a longer survival rate, compared with the S rats. Taken together, these physiological observations functionally link Adamts16 to BP regulation and suggest the vasculature as the potential site of action of Adamts16 to lower BP.espite strong evidence that susceptibility or resistance to the development of hypertension is heritable, the identification of genetic variants that cause blood pressure (BP) to rise into a hypertensive state has remained difficult (1, 2). Classic genetic mapping and association studies in both humans and in rats point to several genetic elements as potential candidates causing hypertension (3, 4). Most of the prioritized candidate genes for hypertension await functional assessments.Linkage analysis in the Quebec Family Study identified a quantitative trait locus (QTL) for systolic BP on human chromosome 5p15 (5). The corresponding comparative segment of human chromosome 5p15 on rat chromosome 1 is also linked to a BP QTL in rats (6). Improved resolutions of this locus in rats were obtained through iterative substitution mapping using strains differentially susceptible to the development of hypertension (6-10). A disintegrin-like metalloproteinase with thrombospondin motifs-16 (Adamts16), which was the only known gene with exonic variants within the highly resolved congenic interval, was prioritized as a candidate BP quantitative trait gene (QTG) (8). More importantly, following the congenic mapping study in rats, human allelic variants of Adamts16 were confirmed as being associated with BP in two independent cohorts, one of which was the Quebec Family Study (8). Taken together, all these studies point to Adamts16 as a prominent candidate locus linked to BP control across two species. However, due to the limitations of recombination frequencies, both the linkage and substitution mapping studies in rats cannot validate Adamts16 as the BP QTG because of the presence of other candidate variants within the linked or introgressed flanking genomic ...

Long noncoding RNAs (lncRNAs) are an emerging class of genomic regulatory molecules reported in various species. In the rat, which is one of the major mammalian model organisms, discovery of lncRNAs on a genome-wide scale is lagging. Renal LncRNA sequencing and lncRNA transcriptome analysis was conducted in three rat strains that are widely used in cardiovascular and renal research, the Dahl salt-sensitive (S) rat, the Spontaneously Hypertensive Rat (SHR) and the Dahl salt-resistant (R) rat. Through the RNA sequencing approach, 3,273 transcripts were identified as rat lncRNAs. A majority of lncRNAs were without predicted target genes. Differential expression of 273 and 749 lncRNAs was detected between S versus R and S versus SHR comparisons respectively. To couple the observed differential expression of lncRNAs with the status of mRNAs, an mRNA transcriptome analysis was conducted. Several cis mRNA genes were co-regulated with lncRNAs. Of these, the protein expression status of four target genes, Asb3, Chac2, Pex11b and Sp5, were differentially expressed between the relevant strain comparisons thereby suggesting that the differentially expressed lncRNAs associated with these genes are candidate genetic determinants of blood pressure. This study serves as a first-generation catalog of rat lncRNAs and illustrates the prioritization of lncRNAs as positional candidates for complex polygenic traits.

The transcriptional regulation of pathological cardiac hypertrophy involves the interplay of transcription factors and chromatin remodeling enzymes. The Microphthalmia-associated transcription factor (MITF) is highly expressed in cardiomyocytes and is required for cardiac hypertrophy. However, the transcriptional mechanisms by which MITF promotes cardiac hypertrophy have not been elucidated. In this study, we tested the hypothesis that MITF promotes cardiac hypertrophy by activating transcription of pro-hypertrophy genes through interactions with the SWI/SNF chromatin remodeling complex. In an in vivo model of cardiac hypertrophy, expression of MITF and the BRG1 subunit of the SWI/SNF complex increased coordinately in response to pressure overload. Expression of MITF and BRG1 also increased in vitro when cardiomyocytes were stimulated with angiotensin II or a β-adrenergic agonist. Both MITF and BRG1 were required to increase cardiomyocyte size and activate expression of hypertrophy markers in response to β-adrenergic stimulation. We detected physical interactions between MITF and BRG1 in cardiomyocytes and found that they cooperate to regulate expression of a pro-hypertrophic transcription factor, GATA4. Our data show that MITF binds to the E box element in the GATA4 promoter and facilitates recruitment of BRG1. This is associated with enhanced expression of the GATA4 gene as evidenced by increased Histone3 lysine4 tri-methylation (H3K4me3) on the GATA4 promoter. Thus, in hypertrophic cardiomyoctes, MITF is a key transcriptional activator of a pro-hypertrophic gene, GATA4, and this regulation is dependent upon the BRG1 component of the SWI/SNF complex.

The present study describes the synthesis of a series of 22 chalcone analogs. These compounds were evaluated as potential human MAO-A and MAO-B inhibitors. The compounds showed varied selectivity against the two isoforms. The IC 50 values were found to be in the micromolar to submicromolar range. The K i values of compound 16 were determined to be 0.047 and 0.020 μM for the inhibition of MAO-A and MAO-B, respectively. Dialysis of enzyme-inhibitor mixtures indicated a reversible competitive mode of inhibition. Most of the synthesized chalcone analogs showed a better selectivity toward MAO-B. However, introducing of 2,4,6-trimethoxy substituents on ring B shifted the selectivity toward MAO-A. In addition, we investigated the molecular mechanism of MAO-B inhibition by selected chalcone analogs. Our results revealed that these selected chalcone analogs increased dopamine levels in the rat hepatoma (H4IIE) cells and decreased the relative mRNA expression of the MAO-B enzyme.

Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) is an orphan member of the nuclear receptor family of transcriptional regulators. Although hormonal activation of COUP-TFII has not yet been identified, rodent genetic models have uncovered vital and diverse roles for COUP-TFII in biological processes. These include control of cardiac function and angiogenesis, reproduction, neuronal development, cell fate and organogenesis. Recently, an emerging body of evidence has demonstrated COUP-TFII involvement in various metabolic systems such as adipogenesis, lipid metabolism, hepatic gluconeogenesis, insulin secretion, and regulation of blood pressure. The potential relevance of these observations to human pathology has been corroborated by the identification of single nucleotide polymorphism in the human COUP-TFII promoter controlling insulin sensitivity. Of particular interest to metabolism is the ability of COUP-TFII to interact with the Glucocorticoid Receptor (GR). This interaction is known to control gluconeogenesis, principally through direct binding of COUP-TFII/GR complexes to the promoters of gluconeogenic enzyme genes. However, it is likely that this interaction is critical to other metabolic processes, since GR, like COUP-TFII, is an essential regulator of adipogenesis, insulin sensitivity, and blood pressure. This review will highlight these unique roles of COUP-TFII in metabolic gene regulation.

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Because of the lack of appropriate animal models, the potentially causal contributions of inherited mitochondrial genomic factors to complex traits are less well studied compared with inherited nuclear genomic factors. We previously detected variations between the mitochondrial DNA (mtDNA) of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR). Specifically, multiple variations were detected in mitochondrial genes coding for subunits of proteins essential for electron transport, in mitochondrial reactive oxygen species production, and within the D-loop region. To evaluate the effects of these mtDNA variations in the absence of the corresponding nuclear genomic factors as confounding variables, novel reciprocal strains of S and SHR were constructed and characterized. When compared with that of the S rat, the heart tissue from the S.SHR(mt) conplastic strain wherein the mtDNA of the S rat was substituted with that of the SHR had a significant increase in mtDNA copy number and decrease in mitochondrial reactive oxygen species production. A corresponding increase in aerobic treadmill running capacity and a significant increase in survival that was not related to changes in blood pressure were observed in the S.SHR(mt) rats compared with the S rat. The reciprocal SHR.S(mt) rats did not differ from the SHR in any phenotype tested, suggesting lower penetrance of the S mtDNA on the nuclear genomic background of the SHR. These novel conplastic strains serve as invaluable tools to further dissect the relationship between heart function, aerobic fitness, cardiovascular disease progression, and mortality.