Lyon Normotensive Research Articles

Body Composition and Metabolic Changes in a Lyon Hypertensive Congenic Rat and Identification of Ercc6l2 as a Positional Candidate Gene.

Central obesity is genetically complex, and its exponential increase in the last decades have made it a critical public health issue. The Lyon Hypertensive (LH) rat is a well-characterized hypertensive model that also exhibits spontaneous and profound differences in body weight and adiposity, relative to its metabolically healthy control, the Lyon Normotensive (LN) rat. The mechanisms underlying the body weight differences between these strains are not well-understood, thus a congenic model (LH17LNa) was developed where a portion of the proximal arm of LN chromosome 17 is introgressed on the LH genomic background to assess the contribution of LN alleles on obesity features. Male and female LH17LNa rats were studied, but male congenics did not significantly differ from LH in this study. Female LH17LNa rats exhibited decreases in total body growth, as well as major alterations to their body composition and adiposity. The LH17LNa female rats also showed decreases in metabolic rate, and a reduction in food intake. The increased adiposity in the female LH17LNa rats was specific to abdominal white adipose tissue, and this phenomenon was further explained by significant hypertrophy in those adipocytes, with no evidence of adipocyte hyperplasia. Sequencing of the parental strains identified a novel frameshift mutation in the candidate gene Ercc6l2, which is involved in transcription-coupled DNA repair, and is implicated in premature aging. The discovery of the significance of Ercc6l2 in the context of female-specific adipocyte biology could represent a novel role of DNA repair failure syndromes in obesity pathogenesis.

Female‐Specific Features of Metabolic Syndrome in an LH Congenic Rat

Central obesity, high blood pressure, dyslipidemia, and insulin resistance are a collection of cardiovascular and metabolic risk factors that form the basis of the Metabolic Syndrome (MetS) and represent a major public health burden worldwide. The phenotypes that define MetS are all highly heritable, but their genetic complexity necessitates the use of animal models to tease apart novel pathways.The Lyon Hypertensive (LH) rat is a well‐characterized model of MetS, exhibiting spontaneous and profound differences in features of MetS compared to its metabolically healthy control, the Lyon Normotensive (LN) rat. To understand the genomic causes of MetS in the LH rat, we developed a congenic rat model, where a portion of LN chromosome 17 is introgressed on the LH genomic background. Our hypothesis was that differences in metabolic features between the LH controls and the congenic rats (LH17LNa) would be due to the replaced region of the genome. Male and female LH17LNa rats and LH controls were phenotyped for a variety of MetS characteristics, including weekly body growth until tissue collection at 15 weeks of age, body composition by nuclear magnetic resonance (NMR) at 8, 11, and 14 weeks of age, metabolic rate (Promethion system) between 10 and 12 weeks of age, and adipose tissue collection and histological examination.We observed a significant decrease in total body growth in female congenics, beginning at 12 weeks of age and persisting throughout the rest of the study. Prior to the divergence in body weight, female congenics showed significant increases in fat mass and significant decreases in fat free mass that remained consistent at all timepoints measured. We also found significant female‐specific decreases in metabolic rate. Tissue collection revealed the source of the increased adiposity in the female LH17LNa rats was specific to abdominal white adipose tissue, and this phenomenon was further explained by significant hypertrophy in those adipocytes, with no evidence of adipocyte hyperplasia.Genome resequencing of the parental strains identified a nonsense mutation in Ercc6l2, implicating this gene as a strong contender underlying the phenotype differences in the congenics. Ercc6l2 is a helicase involved in transcription‐coupled DNA repair, which is specifically mobilized to repair oxidative stress‐induced DNA damage in post‐mitotic cells. Truncating mutations in Ercc6l2 have previously been implicated in genome instability and premature aging in hematopoetic stem cells in patients with bone marrow failure. The discovery of the significance of Ercc6l2 in the context of female‐specific adipocyte biology could represent a novel role of DNA repair syndromes in MetS pathogenesis.

Abstract 60: Perirenal Adipose Hypertrophy In A Congenic LH Rat: A Role For C17h6orf52

Central obesity, high blood pressure, dyslipidemia, and insulin resistance are a collection of cardiovascular and metabolic risk factors that form the basis of the Metabolic Syndrome (MetS) and represent a major public health burden worldwide. The phenotypes that define MetS are all highly heritable, but their genetic complexity necessitates the use of animal models to tease apart novel pathways. The Lyon Hypertensive (LH) rat is a well-characterized model of MetS, exhibiting profound differences in features of MetS compared to its metabolically healthy control, the Lyon Normotensive (LN) rat. To understand the genomic causes of MetS, we developed a congenic rat model, where a portion of LN chromosome 17 is introgressed on the LH genomic background. Male and female LH congenic (CON) rats and LH controls were phenotyped for a variety of MetS characteristics, including body growth and composition by nuclear magnetic resonance (NMR), metabolic rate (Promethion system), and adipose tissue collection and histological examination. There were significant decreases in body weight in the CON rats of both sexes compared to LH. We also found significant female-specific increases in body fat and decreases in metabolic rate. Tissue collection revealed the source of the increased adiposity in the female CON rats was specific to perirenal white adipose tissue (PWAT) and was further explained by significant hypertrophy in those adipocytes. Genome resequencing of the parental strains identified a gene, C17h6orf52 , as a strong contender underlying the phenotype differences in the congenics, with predicted amino acid changes and the loss of Nr2f2 transcription factor binding sites.

CRISPR‐Cas9 Gene Editing Yields a Novel Rat Model of Cardiometabolic Disease

Despite strong evidence that the Metabolic Syndrome (MetS) and its defining features (central obesity, dyslipidemia, hypertension, hyperglycemia) are highly heritable, the genetic etiology is complex and relatively few causative genes are known. For this reason, we employ the Lyon Hypertensive (LH) rat, a well‐characterized polygenic inbred MetS model. Previous genetic and computational approaches identified a novel gene (C17h6orf52) as a putative master regulator of gene expression and oxidative phosphorylation with pleiotropic effects on body weight and blood pressure regulation.In this study, two different frameshift mutations were generated independently by CRISPR‐Cas9 gene editing, producing either a 5 base pair deletion or a 10 base pair insertion in exon 2 of C17h6orf52, and females from the resulting mutant rat strains, designated M1 and M2, respectively, were characterized (Table 1). Herein, mutations in C17h6orf52 are shown to significantly increase blood pressure and serum cholesterol in vivo, despite no significant changes in body composition or body weight in LH‐derived female rats.Either mutation appears to cause modest, but significant or nearly significant, increases in blood pressure on a 0.3% NaCl diet. M1−/− females exhibit significantly increased systolic and diastolic blood pressure, while M2−/− females' systolic and diastolic blood pressure is more moderately elevated. This effect is potentiated when animals are administered a high salt diet. Finally, although neither total nor low‐density lipoprotein (LDL) cholesterol is changed in the M1 female rats with respect to their wildtype control, M2 females develop higher serum cholesterol, as well as significantly higher LDL cholesterol.The differences in phenotype between the M1 and M2 mutations were unexpected. Preliminary data indicates that 5 base pair deletion present in the M1−/− rats results in two distinct splicing variants that may be contributing to the observed phenotype differences. Nevertheless, these data suggest that C17h6orf52 is protective against cardiometabolic risk factors, given that C17h6orf52 mRNA is more highly expressed in MetS‐susceptible LH rats than MetS resistant Lyon Normotensive (LN) rats, and the mutation confers a greater burden of MetS features on an already obese and hypertensive rat. The continued study of this rat model of Metabolic Syndrome has the potential to functionally validate an uncharacterized regulatory gene, and provide novel targets for pharmacological intervention in the treatment of obesity.Support or Funding InformationR01HL089895, R21DK089417, T32GM008629, 14GRNT20410043This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genome variation and conserved regulation identify genomic regions responsible for strain specific phenotypes in rat

BackgroundThe genomes of laboratory rat strains are characterised by a mosaic haplotype structure caused by their unique breeding history. These mosaic haplotypes have been recently mapped by extensive sequencing of key strains. Comparison of genomic variation between two closely related rat strains with different phenotypes has been proposed as an effective strategy for the discovery of candidate strain-specific regions involved in phenotypic differences. We developed a method to prioritise strain-specific haplotypes by integrating genomic variation and genomic regulatory data predicted to be involved in specific phenotypes. Specifically, we aimed to identify genomic regions associated with Metabolic Syndrome (MetS), a disorder of energy utilization and storage affecting several organ systems.ResultsWe compared two Lyon rat strains, Lyon Hypertensive (LH) which is susceptible to MetS, and Lyon Low pressure (LL), which is susceptible to obesity as an intermediate MetS phenotype, with a third strain (Lyon Normotensive, LN) that is resistant to both MetS and obesity. Applying a novel metric, we ranked the identified strain-specific haplotypes using evolutionary conservation of the occupancy three liver-specific transcription factors (HNF4A, CEBPA, and FOXA1) in five rodents including rat. Consideration of regulatory information effectively identified regions with liver-associated genes and rat orthologues of human GWAS variants related to obesity and metabolic traits. We attempted to find possible causative variants and compared them with the candidate genes proposed by previous studies. In strain-specific regions with conserved regulation, we found a significant enrichment for published evidence to obesity—one of the metabolic symptoms shown by the Lyon strains—amongst the genes assigned to promoters with strain-specific variation.ConclusionsOur results show that the use of functional regulatory conservation is a potentially effective approach to select strain-specific genomic regions associated with phenotypic differences among Lyon rats and could be extended to other systems.

Contribution of independent and pleiotropic genetic effects in the metabolic syndrome in a hypertensive rat.

Hypertension is a major risk factor for cardiovascular disease, Type 2 diabetes, and end organ failure, and is often found concomitant with disorders characteristic of the Metabolic Syndrome (MetS), including obesity, dyslipidemia, and insulin resistance. While the associated features often occur together, the pathway(s) or mechanism(s) linking hypertension in MetS are not well understood. Previous work determined that genetic variation on rat chromosome 17 (RNO17) contributes to several MetS-defining traits (including hypertension, obesity, and dyslipidemia) in the Lyon Hypertensive (LH) rat, a genetically determined MetS model. We hypothesized that at least some of the traits on RNO17 are controlled by a single gene with pleiotropic effects. To address this hypothesis, consomic and congenic strains were developed, whereby a defined fragment of RNO17 from the LH rat was substituted with the control Lyon Normotensive (LN) rat, and MetS phenotypes were measured in the resultant progeny. Compared to LH rats, LH-17LN consomic rats have significantly reduced body weight, blood pressure, and lipid profiles. A congenic strain (LH-17LNc), with a substituted fragment at the distal end of RNO17 (17q12.3; 74–97 Mb; rn4 assembly), showed differences from the LH rat in blood pressure and serum total cholesterol and triglycerides. Interestingly, there was no difference in body weight between the LH-17LNc and the parental LH rat. These data indicate that blood pressure and serum lipids are regulated by a gene(s) in the distal congenic interval, and could be due to pleiotropy. The data also indicate that body weight is not determined by the same gene(s) at this locus. Interestingly, only two small haplotypes spanning a total of approximately 0.5 Mb differ between the LH and LN genomes in the congenic interval. Genes in these haplotypes are strong candidate genes for causing dyslipidemia in the LH rat. Overall, MetS, even in a simplified genetic model such as the LH-17LN rat, is likely due to both independent and pleiotropic gene effects.

Abstract P638: Investigating Gene Pleiotropy in the Metabolic Syndrome in Lyon Hypertensive Rats

The metabolic syndrome (MetS) - hypertension, obesity, dyslipidemia, and insulin resistance - is a major risk factor for cardiovascular disease and stroke. Our overall goal is to identify novel genes and pathways causing MetS. Our previous work determined that rat chromosome 17 (RNO17) contributes to several MetS-defining traits (including high blood pressure, obesity, and dyslipidemia) in the Lyon Hypertensive (LH) rat, a genetically determined MetS rat model. We hypothesized that at least some of the traits on RNO17 are controlled by a single gene with pleiotropic effects. To address this hypothesis, we generated congenic strains where a defined fragment of RNO17 from the LH rat was substituted by that of the control Lyon Normotensive (LN) rat, and measured MetS phenotypes. One congenic (LH-17 LN a), with the proximal 30 Mb of RNO17 from the LH genome substituted with that of the LN genome, did not show significant differences from the LH parental strain. However, another congenic strain (LH-17 LN c), with a substituted fragment at the distal end of RNO17 (84-97 Mb), showed significant differences from the LH rat in serum total cholesterol (3.15 ± 0.15 vs. 4.29 ± 0.17 mMol; p<0.01) and triglycerides (0.47 ± 0.06 vs. 1.27± 0.13 mMol; p<0.001), and a trend for reduced blood pressure (SBP 150.8 ± 3.4 vs. 157.1 ± 1.7 mmHg; p=0.1). Interestingly, there was no difference in body weight between the LH-17 LN c and the parental LH rat (440 + 7.2 vs. 435 + 9.1 g). These data indicate that serum cholesterol and triglycerides, and possibly blood pressure are regulated by a gene(s) in the distal congenic interval, and could be due to pleiotropy. The data also indicate body weight is not determined by the same gene(s). Interestingly, only two small haplotypes spanning a total of 1 Mb differ between the LH and LN genomes in the congenic interval. Genes in these haplotypes are being studied as candidate genes for causing dyslipidemia in the LH rat. Overall MetS, even in a simplified genetic model such as the LH-17 LN rat, is likely due to both independent and pleiotropic gene effects.

Abstract 617: The Consomic LH-17 LN Rat Is A Single Chromosome Model Of Metabolic Syndrome

The Lyon Hypertensive (LH) rat is an inbred model of Metabolic Syndrome, exhibiting spontaneous hypertension, high body weight, high plasma lipids, altered insulin:glucose ratio. The Lyon Normotensive (LN) control strain, selectively bred for normal blood pressure from the same Sprague Dawley (SD) colony, is genetically quite similar to the LH, but displays no features of MetS. Genetic mapping in an LHxLN cross determined that all of the MetS phenotypes mapped to rat chromosome (RNO 17). In order to identify the gene(s) causing MetS on RNO17 we generated a consomic strain (LH-17 LN ) where the LH RNO17 was replaced with that of the LN strain. Substitution of the single chromosome was sufficient to significantly lower blood pressure lower systolic blood pressure (145 + 2 vs. 161 + 2 mmHg; p<0.05), body weight (408 + 6 vs 453 + 5 g; p<0.05), and plasma cholesterol (2.5 ± 0.1 vs 2.9 ± 0.08; p<0.05) compared to the LH parental strain. To identify putative causal genes for MetS, we fine-mapped RNO17 in silico utilizing the genome sequence of the LH and LN rat strains. The genomes of the LH and LN strains are highly similar, differing by <0.02% at the sequence level (in comparison, humans typically differ by about 1%). Moreover, we have shown that the variants cluster into <500 divergent haplotype blocks with an average length of 880 kb, which are presumed to contain the genetic variation causing the phenotypic variation between the LH and LN strains. On RNO17 there are just 14 divergent haplotypes, containing 11 genes with coding sequence differences in the LH. The combined in vivo and in silico approaches allowed for the identification of candidate causal variants for the phenotypes on RNO17 and will be studied further to determine their role(s) in MetS.

Genomic structure of nucleotide diversity among Lyon rat models of metabolic syndrome

BackgroundThe metabolic syndrome (MetS), a complex disorder involving hypertension, obesity, dyslipidemia and insulin resistance, is a major risk factor for heart disease, stroke, and diabetes. The Lyon Hypertensive (LH), Lyon Normotensive (LN) and Lyon Low-pressure (LL) rats are inbred strains simultaneously derived from a common outbred Sprague Dawley colony by selection for high, normal, and low blood pressure, respectively. Further studies found that LH is a MetS susceptible strain, while LN is resistant and LL has an intermediate phenotype. Whole genome sequencing determined that, while the strains are phenotypically divergent, they are nearly 98% similar at the nucleotide level. Using the sequence of the three strains, we applied an approach that harnesses the distribution of Observed Strain Differences (OSD), or nucleotide diversity, to distinguish genomic regions of identity-by-descent (IBD) from those with divergent ancestry between the three strains. This information was then used to fine-map QTL identified in a cross between LH and LN rats in order to identify candidate genes causing the phenotypes.ResultsWe identified haplotypes that, in total, contain at least 95% of the identifiable polymorphisms between the Lyon strains that are likely of differing ancestral origin. By intersecting the identified haplotype blocks with Quantitative Trait Loci (QTL) previously identified in a cross between LH and LN strains, the candidate QTL regions have been narrowed by 78%. Because the genome sequence has been determined, we were further able to identify putative functional variants in genes that are candidates for causing the QTL.ConclusionsWhole genome sequence analysis between the LH, LN, and LL strains identified the haplotype structure of these three strains and identified candidate genes with sequence variants predicted to affect gene function. This approach, merged with additional integrative genetics approaches, will likely lead to novel mechanisms underlying complex disease and provide new drug targets and therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-197) contains supplementary material, which is available to authorized users.

Abstract 18436: Systems Biology Identifies a Gene Driving Expression of Metabolic Syndrome in Lyon Rats

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including obesity, hypertension, dyslipidemia, and insulin resistance. The Lyon Hypertensive (LH) and Lyon Normotensive (LN) rats are models of MetS sensitivity and resistance, respectively. To identify genetic loci influencing phenotypes underlying MetS, an F2 intercross between LH and LN was studied to identify physiological quantitative trait loci (pQTL) contributing to plasma lipid and leptin levels, blood pressure, and body weight/adiposity. We performed mRNA-seq in F2 rats to identify the loci regulating gene expression, or expression (e)QTL, followed by gene expression network analyses. Systems biology methods were applied to identify candidate MetS genes, including genes previously shown to be MetS-related. We found an eQTL hotspot on RNO17, which was also located within pQTLs for MetS-related traits. The genes regulated by this eQTL hotspot were mainly in two co-expression network modules (a mitochondria related module and a gene regulation related module) and causally affect many MetS-related traits. Multiple evidences strongly and consistently support RGD1562963, a gene regulated in cis by this eQTL hotspot as the eQTL driver gene directly affected by genetic variation between LH and LN rats. Our study sheds light on the intricate pathogenesis of MetS and that systems biology with high-throughput sequencing is a powerful method to study the etiology of complex multifactorial diseases.

Abstract 518: Systems Biology Approach to Identify Genetic Mechanisms Underlying the Metabolic Syndrome in the LH Rat

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including hypertension, obesity, dyslipidemia, and insulin resistance. The Lyon Hypertensive (LH) and Lyon Normotensive (LN) rats are models of MetS sensitivity and resistance, respectively. To identify genetic determinants and mechanisms underlying MetS, rats from an F2 intercross between LH and LN were studied. Multi-dimensional data were obtained including SNP genotypes, physiological traits including blood pressure, plasma lipid and leptin levels, and body weight/adiposity, and more than 150 billion nucleotides of RNA-Seq reads from the livers of F2 (n=36), LH (n=6), and LN (n=6) individuals. We identified 17 pQTLs (physiological quantitative trait loci) and 1200 eQTLs (gene expression quantitative trait loci) at genome-wide p<0.05. Systems biology methods were applied to prioritize candidate MetS genes, including genes previously shown to be MetS-related. We identified an eQTL peak on rat chromosome 17 (RNO17) shared by large number of genes; RNO17 also has pQTL for MetS-related traits. Network analysis in these genes identified two major co-regulation modules involving mitochondria and gene regulation; genes in the two modules causally affect multiple MetS traits. A gene, RGD1562963, closest to the eQTL peak was identified as the eQTL driver gene. It is directly affected by genetic variation between LH and LN rats, affects downstream genes in the eQTL cluster, and is correlated with MetS phenotypes. Our study sheds light on the intricate pathogenesis of MetS and proved that systems biology with high-throughput sequencing is a powerful method to study the etiology of complicated diseases.

Abstract 651: Genetic Mapping Of Traits And Transcriptome In The Lyon Hypertensive Rat

The metabolic syndrome (MetS) is characterized by obesity, dyslipidemia, hypertension, and insulin resistance and is a major risk factor for cardiovascular disease. Using the Lyon Hypertensive and Lyon Normotensive rats as models of the metabolic syndrome, we aim to determine both physiological and transcriptional regulation underlying the syndrome. To identify genetic loci influencing phenotypes underlying MetS, an F2 intercross between LH and LN with 150 offspring was studied to identify physiological quantitative trait loci (pQTL) contributing to plasma lipid levels, blood pressure, and body weight/adiposity. We performed RNA-seq in livers from 36 selected rats from the intercross to identify the mapping of gene expression, or expression (e)QTL. Linkage analyses identified pQTL and eQTL with significant logarithm of odds (lod) scores, whose genome-wide significance was determined by permutation testing. We identified 17 pQTL on 5 rat chromosomes and 75 on 16 chromosomes. It has been determined that LH chromosome 17 contributes to multiple features of MetS; furthermore, an eQTL on rat chromosome 17 was identified that regulates the expression of multiple genes in trans, meaning the regulated gene falls outside the QTL interval or on another chromosome. We hypothesize these genes share a common transcriptional regulator which is affected by genetic variation on chromosome 17. eQTLs are likely to influence precursor pathways and cofactors of the metabolic syndrome. Identifying these in the LH rat could lead to translational studies to determine their role in the human MetS.

Visfatin is associated with lipid metabolic abnormalities in Lyon hypertensive rats

1. Visfatin (also known as nicotinamide phosphoribosyltransferase or Nampt) is a novel adipokine associated with metabolic abnormalities in obesity and diabetes. The aim of the present study was to determine whether visfatin levels in the circulation and visfatin expression in fat tissues are altered in Lyon hypertensive (LH) rats, a hypertensive strain with numerous metabolic abnormalities, and, if so, to explore the possible correlations between blood visfatin levels and the metabolic abnormalities in LH rats. 2. Male 18-20-week-old LH rats and their control, Lyon normotensive (LN) rats, were used. Blood pressure was recorded in conscious, unrestrained rats. Fat tissue weight was determined. Serum visfatin concentrations were determined by enzyme immunoassay, whereas visfatin expression in fat tissues was determined by western blotting. Relationships between two parameters were evaluated by linear regression analysis. 3. The LH rats had significantly higher bodyweight and fat tissue weight compared with LN rats. Serum lipid levels of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) were all significantly higher in LH rats than in LN rats. Moreover, serum visfatin levels were higher in LH rats than in LN rats and were positively correlated with bodyweight, fat tissue weight and serum lipid levels (i.e. TC, HDL-C, LDL-C, TG and the TG:HDL-C ratio). There were no significant differences in serum glucose and insulin concentrations, or in the whole-body insulin resistance index, between LN and LH rats, and serum visfatin levels were not correlated with any of these parameters. Visfatin expression in visceral fat tissue, but not in subcutaneous fat tissue, was markedly upregulated in LH compared with LN rats. 4. In conclusion, the results of the present study demonstrate that serum visfatin levels in LH rats are elevated and are associated with lipid metabolic abnormalities.

Y27632, a Rho‐activated kinase inhibitor, normalizes dysregulation in alpha1‐adrenergic receptor‐induced contraction of Lyon hypertensive rat artery smooth muscle

RhoA-activated kinase (ROK) is involved in the disorders of smooth muscle contraction found in hypertension model animals and patients. We examined whether the alpha1-adrenergic receptor agonist-induced ROK signal is perturbed in resistance small mesentery artery (SMA) of Lyon genetically hypertensive (LH) rats, using a ROK antagonist, Y27632. Smooth muscle strips of SMA and aorta were isolated from LH and Lyon normotensive (LN) rats. After Ca(2+)-depletion and pre-treatment with phenylephrine (PE), smooth muscle contraction was induced by serial additions of CaCl(2). In LH SMA Ca(2+) permeated cells to a lesser extent as compared with LN SMA, while CaCl(2)-induced contraction of LH SMA was greater than that of LN SMA, indicating a higher ratio of force to Ca(2+) in LH SMA contraction (Ca(2+) sensitization). No hyper-contraction was observed in LH aorta tissues. Treatment of LH SMA with Y27632 restored both Ca(2+) permeability and Ca(2+)-force relationship to levels seen for LN SMA. In response to PE stimulation, phosphorylation of CPI-17, a phosphorylation-dependent myosin phosphatase inhibitor protein, and MYPT1 at Thr853, the inhibitory phosphorylation site of the myosin phosphatase regulatory subunit, was increased in LN SMA, but remained unchanged in LH SMA. These results suggest that the disorder in ROK-dependent Ca(2+) permeability and Ca(2+)-force relationship is responsible for LH SMA hyper-contraction. Unlike other hypertensive models, the ROK-induced hyper-contractility of LH SMA is independent of MYPT1 and CPI-17 phosphorylation, which suggests that ROK-mediated inhibition of myosin phosphatase does not affect SMA hyper-contractility in LH SMA cells.

Renin-angiotensin system in two genetically normotensive strains of lyon rats

Compared to the Lyon normotensive (LN) controls, adult Lyon hypertensive rats (LH) exhibit a renin-angiotensin system (RAS) dependent hypertension despite a low renin secretion. This discrepancy could be explained by the elevated slow pressor response to angiotensin II (AII) found in LH rats compared to LN controls. To evaluate more precisely the pathophysiological importance of this increased response, the present work aimed at determining whether the characteristics of the RAS were identical in LN and low blood pressure (LL) rats, the other normotensive control strain simultaneously selected with LH rats. Plasma and kidney renin and prorenin were measured in 11-week-old LN and LL rats. Aortic blood pressure (BP) was recorded at 15 weeks of age in freely moving rats of both strains either untreated or having received an angiotensin converting enzyme inhibitor, perindopril (3 mg/kg/day orally) since the age of 3 weeks. Acute dose-response curves were constructed for AII and norepinephrine (NE). The long-term pressor effects of AII (200 ng/kg/min) and NE (1000 ng/kg/min) were measured after chronic infusions in perindopril-treated LN and LL rats. LN and LL rats exhibited similar mean BP level before (114 ± 2 and 117 ± 2 mm Hg, respectively) and after perindopril treatment (91 ± 3 and 93 ± 1 mm Hg, respectively). Plasma and kidney renin and prorenin were decreased in LL rats. In acute conditions, LL rats exhibited an unspecific hypersensitivity to AII and NE. Chronically given AII exerted a greater pressor effect in LL than in LN rats after 4 weeks (113 ± 3 v 97 ± 5 mm Hg in LL and LN rats respectively, P < .05) and, even more, after 8 weeks of infusion (144 ± 9 v 124 ± 4 mm Hg in LL and LN rats respectively, P < .05). The NE was devoid of chronic pressor effects. In conclusion, 1) the increased slow pressor response to AII may not be a critical pathogenetic factor in the development of hypertension, as it also exists in normotensive LL rats; 2) LN and LL rats have the same normal BP despite marked differences in their RAS, thus suggesting that there could be several forms of normotension as known for hypertension; and 3) the simple comparison between one genetically hypertensive strain and one single normotensive control strain does not allow one to conclude that a phenotypic difference is of pathophysiological significance.

Major cardiovascular risk factors in Lyon hypertensive rats. A correlation analysis in a segregating population

To investigate the association of blood pressure with the other major cardiovascular risk factors in a large population of back-cross to Lyon hypertensive (LH) rats. Mean arterial pressure was recorded in male freely moving Lyon normotensive (LN), LH, F1 and backcross to LH rats aged 30 weeks. Plasma cholesterol, triglycerides, insulin, creatinine, urea, fibrinogen and haematocrit levels, and the insulin: glucose ratio were measured in 31-week-old rats and 24h albuminuria in 6-week-old rats. Adult LH rats exhibited a significant increase in plasma lipids, insulin, fibrinogen, creatinine, urea and haematocrit levels compared with LN rats. In young LH rats, at an age at which blood pressure is slightly increased, albuminuria was increased to a greater extent than expected from their blood pressure levels. In the adult back-cross to LH rats, only the plasma cholesterol level was associated with blood pressure. Moreover, the plasma cholesterol level was related to fibrinogen and haematocrit levels. Finally, in the same rats, albuminuria developed early in life was positively related to hypercholinesterolaemia measured later in life. Plasma cholesterol, fibrinogen, haematocrit levels and early albuminuria could act synergistically in the enhancement or the development, or both, of vascular and kidney damage in the LH rat. Most interestingly, the association between plasma cholesterol level and blood pressure indicates that, as in essential hypertension, hypercholesterolaemia is a major phenotype associated with hypertension in the LH rat.

Left ventricular weight but not blood pressure is associated with sex chromosomes in Lyon rats

To investigate, by studying reciprocal back-crosses to Lyon hypertensive (LH) rats, the involvement of sex chromosome loci in high blood pressure and heart weight in LH rats. Reciprocal F1 and F'1 generation male rats obtained from male LH x female Lyon normotensive (LN) and male LN x female LH crosses respectively, male back-cross rats obtained from male LH x female F1 or F'1 and from male F1 or F'1 x female LH crosses, and parental LH and LN male rats were studied at 29-31 weeks of age. Systolic, diastolic, mean and pulse pressures were measured beat to beat in unrestrained rats for 1 h. In addition, relative left and right ventricular weights were measured. The average blood pressures did not differ between F1 and F'1 rats or between the four populations of back-cross rats. On the contrary, the relative left ventricular weight was higher in F'1 than in F1 rats. As a role for loci on the Y chromosome could be discarded by comparison of the two populations of back-cross rats, which differ only in the origin of their Y chromosome, this increase in the relative left ventricular weight of F'1 rats was consistent with an effect of a locus on the LH X chromosome. This was supported by findings in the back-cross populations, those populations in which 100% of the rats carried an LH X chromosome having a significantly higher left ventricular weight than those in which only 50% of the rats carried such a chromosome. The estimate of the genetic determination was higher for left ventricular weight (41%) than for mean arterial pressure (19%). Although these two parameters were associated in the back-cross population, the origin of the X chromosome had no influence on this association. These findings indicate that, against the genetic background provided by the cross studied and at the age studied, the sex chromosomes of LH and LN rats do not contain loci at which alleles differentially influence blood pressure. They do, however, suggest that relative left ventricular weight in this model has a specific genetic determination, partly contributed by a locus on the X chromosome.

Partial transfer of genetic hypertension by lymphoid cells in Lyon rats

The involvement of immune factors in a given disease is suggested by evidence that a disease can be prevented by immunosuppression and can be transferred by lymphoid cells. Because the first type of experimental result was achieved in Lyon hypertensive (LH) rats, the present study was undertaken to determine whether hypertension can be transferred to normotensive recipients. As a control, the blood pressure effects of lymphoid cell grafts from renovascular hypertensive donors were also determined. Splenocytes and lymph node cells from LH and Lyon low-blood pressure (LL) rats with two-kidney Goldblatt hypertension were respectively injected into LH x Lyon normotensive (LN) F1 hybrids and LL rats aged 7, 8, 9 and 10 weeks. Blood pressure was measured by plethysmography from age 6 to 13 weeks and an intra-arterial recording was performed in 14-week-old conscious rats. Lymphoid cell injections from LL rat donors with two-kidney hypertension did not modify the blood pressure of LL rat recipients. In contrast, lymphoid cell grafts from LH rat donors induced a significant increase in blood pressure in F1 recipients compared with control F1 rats after the first injection. As confirmed by intra-arterial recording, this blood pressure effect lasted until age 14 weeks (145 +/- 1 versus 137 +/- 1 mmHg in grafted and ungrafted F1, respectively). It was not related to alterations in the acute role of the renin-angiotensin and sympathetic nervous systems and was not associated with increased pressor responses to the vasoconstrictor drugs tested. The present study demonstrates that genetic hypertension can be partially transferred by lymphoid cells in F1 recipients. The effect seems to be specific to genetic hypertension because lymphoid cells from renovascular hypertensive donors failed to transfer this secondary form of hypertension. The present results support the hypothesis that cellular immune reactions contribute to the pathogenesis of hypertension and LH rats.

Evaluation of the angiotensin II receptor AT1B gene as a candidate gene for blood pressure

To answer two specific questions concerning the possible role played by the angiotensin II receptor AT1B (AT1B) locus in genetic hypertension. First, do alleles at the AT1B locus cosegregate with blood pressure in F2 populations in the rat and, if so, is this due to AT1B or a closely linked quantitative trait locus (QTL)? Secondly, are there any significant nucleotide differences between the coding regions of the AT1B gene of the inbred Dahl salt-sensitive (SS/Jr) strain and various control strains, or between the Lyon hypertensive (LH) strain the Lyon normotensive (LN) strain? The first objective was achieved by studying several F2 rat populations and by analyzing DNA markers around the AT1B locus. The second objective was accomplished by amplifying the AT1B coding regions by polymerase chain reaction from the genomic DNA, and by sequencing the relevant coding regions of the AT1B genes. In two F2 populations involving the SS/Jr rat, AT1B cosegregated only weakly with systolic blood pressure. Also, nucleotide differences resulting in conservative changes in three amino acids were detected between the coding region of the SS/Jr AT1B allele and the AT1B alleles of the several control strains. No nucleotide differences were found in the coding regions of the AT1B alleles between the LH and LN strains. There appeared to be a QTL which had a minor effect on blood pressure, located near the AT1B locus on rat chromosome 2. However, the AT1B gene itself is not supported as the candidate gene for causing blood pressure differences, because no nucleotide changes in the coding region that were functionally meaningful or concordant with the cosegregation analysis were detected.

High blood pressure and metabolic disorders are associated in the Lyon hypertensive rat

A large population of F2 rats, obtained from a cross between male Lyon hypertensive (LH) rats and female Lyon normotensive (LN) rats, was studied in order to assess the relationship between increased body weight, hyperlipidaemia and high blood pressure which characterize LH rats. Mean arterial pressure (MAP) was recorded in male, conscious, freely moving LH, LN, F1 and F2 rats aged 30 weeks. Plasma total cholesterol, high-density lipoprotein-, low-density lipoprotein- and very low-density lipoprotein-cholesterol, phospholipids, triglycerides, insulin and glucose were measured. In the F2 cohort it was observed that high MAP was a recessive trait that depends on several genes and was unrelated to body weight. The left ventricular weight, corrected for tibia length, was correlated with MAP. Plasma total and high-density lipoprotein-cholesterol and phospholipids concentrations were lower in the F1 rats than in the LN rats, suggesting an overdominance of the LN alleles. In the F2 rats MAP was related to total, high-density lipoprotein- and low-density lipoprotein-cholesterol. Plasma triglycerides, insulin and the insulin:glucose ratio, which were higher in the LH rats than in the LN rats, were also correlated with MAP in the F2 cohort. Using stepwise multiple regression analysis, MAP remained correlated with plasma total cholesterol, insulin and the insulin:glucose ratio, but not with triglycerides. Hypertension in LH rats is a recessive trait that is independent of body weight. In addition, the cosegregation of blood pressure with plasma cholesterol and, to a lesser degree, with insulin levels, which was observed in the present study provides the first direct evidence that these phenotypes are associated and are not due simply to genetic drift in the Lyon model.