Induction of K(Ca)3.1 (IKCa) potassium channel plays an important role in vascular smooth muscle cell proliferation. Here, we report that the gene encoding K(Ca)3.1 (KCNN4) contains a functional repressor element 1-silencing transcription factor (REST or NRSF) binding site and is repressed by REST. Although not previously associated with vascular smooth muscle cells, REST is present and recruited to the KCNN4 gene in situ. Significantly, expression of REST declines when there is cellular proliferation, showing an inverse relationship with functional K(Ca)3.1. Downregulated REST and upregulated K(Ca)3.1 are also evident in smooth muscle cells of human neointimal hyperplasia grown in organ culture. Furthermore, inhibition of K(Ca)3.1 suppresses neointimal formation, and exogenous REST reduces the functional impact of K(Ca)3.1. Here, we show REST plays a previously unrecognized role as a switch regulating potassium channel expression and consequently the phenotype of vascular smooth muscle cells and human vascular disease.

Cardiac hypertrophy is associated with a dramatic change in the gene expression profile of cardiac myocytes. Many genes important during development of the fetal heart but repressed in the adult tissue are reexpressed, resulting in gross physiological changes that lead to arrhythmias, cardiac failure, and sudden death. One transcription factor thought to be important in repressing the expression of fetal genes in the adult heart is the transcriptional repressor REST (repressor element 1-silencing transcription factor). Although REST has been shown to repress several fetal cardiac genes and inhibition of REST function is sufficient to induce cardiac hypertrophy, the molecular mechanisms employed in this repression are not known. Here we show that continued REST expression prevents increases in the levels of the BNP (Nppb) and ANP (Nppa) genes, encoding brain and atrial natriuretic peptides, in adult rat ventricular myocytes in response to endothelin-1 and that inhibition of REST results in increased expression of these genes in H9c2 cells. Increased expression of Nppb and Nppa correlates with increased histone H4 acetylation and histone H3 lysine 4 methylation of promoter-proximal regions of these genes. Furthermore, using deletions of individual REST repression domains, we show that the combined activities of two domains of REST are required to efficiently repress transcription of the Nppb gene; however, a single repression domain is sufficient to repress the Nppa gene. These data provide some of the first insights into the molecular mechanism that may be important for the changes in gene expression profile seen in cardiac hypertrophy.The repressor element 1-silencing transcription factor (REST) was originally identified as an important transcription factor regulating the expression of neuron-specific genes (12, 53) but has since been shown to be a key transcriptional regulator in heart development (28) and vascular smooth muscle growth (11). Disruption of REST function by expression of a dominant-negative form specifically in the heart results in cardiomyopathy, arrhythmias, and sudden death (28). These effects are thought to result from the reexpression of fetal cardiac genes and have led to the proposition that REST represses the fetal cardiac gene program in the adult heart (28). In vascular smooth muscle, loss of REST has been implicated in neointimal hyperplasia, and inhibition of REST results in increased smooth muscle proliferation (11). Several genes that are repressed by REST in myocytes have been identified, including the genes encoding the brain and atrial natriuretic peptides (Nppb and Nppa, encoding BNP and ANP, respectively), ␣-skeletal actin (Acta1), potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels 2 and 4 (Hcn2 and Hcn4), and voltage-gated calcium channel subunit alpha Cav3.2 (Cacna1h) (26, 43). Levels of BNP and ANP are particularly important, since increased levels of these peptides in circulation are clinical indicators of the severity of hypertrophy (8,29,38,61)....

Abstract-A region with a major effect on blood pressure (BP) is located on rat chromosome 1. We have previously isolated this region in reciprocal congenic strains (WKY.SHR-Sa and SHR.WKY-Sa) derived from a cross of the spontaneously hypertensive rat (SHR) with the Wistar-Kyoto rat (WKY) and shown that there are 2 distinct BP quantitative trait loci, BP1 and BP2, in this region. Sisa1, a congenic substrain from the SHR.WKY-Sa animals carrying an introgressed segment of 4.3Mb, contains BP1. Here, we report further dissection of BP1 by the creation of 2 new mutually exclusive congenic substrains (Sisa1a and Sisa1b) and interrogation of candidate genes by expression profiling and targeted transcript sequencing. Only 1 of the substrains (Sisa1a) continued to demonstrate a BP difference but with a reduced introgressed segment of 3Mb. Exonic sequencing of the 20 genes located in the Sisa1a region did not identify any major differences between SHR and WKY. However, microarray expression profiling of whole kidney samples and subsequent quantitative RT-PCR identified a single gene, Spon1 that exhibited significant differential expression between the WKY and SHR genotypes at both 6 and 24 weeks of age. Western blot analysis confirmed an increased level of the Spon1 gene product in SHR kidneys. Spon1 belongs to a family of genes with antiangiogenic properties. These findings justify further investigation of this novel positional candidate gene in BP control in hypertensive rat models and humans. (Circ Res. 2007;100:992-999.)Key Words: hypertension Ⅲ genetics Ⅲ rats Ⅲ gene expression Ⅲ quantitative trait locus B lood pressure (BP) and hypertension exhibit a significant degree of genetic heritability. 1 In the past 15 years, many genomic locations contributing to hypertension in rodent models have been mapped. 2 Most of these regions, known as quantitative trait loci (QTLs), are quite large, typically 10 to Ͼ80 million base pairs (Mb). This has hampered the ability to identify the gene(s) responsible for the blood pressure effects underlying these QTLs. Constructing congenic strains has proved pivotal in reducing the genomic size of QTLs: the strategy creates animal strains where the native genomic background (eg, from a normotensive rat strain) remains unchanged except for an introgressed (replaced) specific genomic region of interest which is transferred from a contrasting strain (eg, from a hypertensive rat strain). 2 The integration of genetic strategies with expression profiling and targeted sequencing 3-6 has also proved a successful approach in accelerating the search for genes underlying various phenotypes related to cardiovascular disease. This integration strategy is based on the assumption that genes that are both differentially expressed or show structural variation and map to a disease-related QTL are likely to be involved in the pathophysiology of that disease.In a previous study, we mapped a BP QTL region to rat chromosome 1 in F 2 rats derived from a cross of the spontaneously hypertensive rat (SHR) with t...

Abstract-Copy number variation has emerged recently as an important genetic mechanism leading to phenotypic heterogeneity.The aim of our study was to determine whether copy number variants (CNVs) exist between the spontaneously hypertensive rat (SHR) and its control strain, the Wistar-Kyoto rat, whether these map to quantitative trait loci in the rat and whether CNVs associate with gene expression or blood pressure differences between the 2 strains. We performed a comparative genomic hybridization assay between SHR and Wistar-Kyoto strains using a whole-genome array. In total, 16 CNVs were identified and validated (6 because of a relative loss of copy number in the SHR and 10 because of a relative gain). CNVs were present on rat autosomes 1, 3, 4, 6, 7, 10, 14, and 17 and varied in size from 10.0 kb to 1.6 Mb. Most of these CNVs mapped to chromosomal regions within previously identified quantitative trait loci, including those for blood pressure in the SHR. Transcriptomic experiments confirmed differences in the renal expression of several genes (including Ms4a6a, Ndrg3, Egln1, Cd36, Sema3a, Ugt2b, and Idi21) located in some of the CNVs between SHR and Wistar-Kyoto rats. In F 2 animals derived from an SHRϫWistar-Kyoto cross, we also found a significant increase in blood pressure associated with an increase in copy number in the Egln1 gene. Our findings suggest that CNVs may play a role in the susceptibility to hypertension and related traits in the SHR. (Hypertension. 2010;55:1231-1238.)Key Words: DNA copy number variations Ⅲ hypertension Ⅲ inbred SHR Ⅲ genetics Ⅲ gene expression Ⅲ microarray analysis Ⅲ blood pressure H igh blood pressure (BP; hypertension) is a major risk factor for coronary, cerebrovascular, and renal disease. Most cases of hypertension have unknown etiology and are, thus, classified as essential hypertension. Hypertension has a significant genetic contribution. Despite the progress made toward the understanding of rare monogenic forms of hypertension in humans, the genetic background of essential hypertension remains poorly understood. 1 The spontaneously hypertensive rat (SHR) is one of the most widely used genetic models for hypertension. The SHR model is characterized by hypertension, insulin resistance, hypertriglyceridemia, and hypercholesterolemia. Genetically, the SHR was derived in 1963 from inbreeding Wistar rats with the highest BPs. 2 Using linkage analysis, there have been multiple efforts to map genes influencing BP and related phenotypes in the SHR. These efforts have resulted in the successful identification of several chromosome regions containing quantitative trait loci (QTLs) regulating BP or related cardiovascular and metabolic phenotypes in the SHR. 3 Yet, despite many experiments, very few genes that underlie these QTLs have been unambiguously identified. 4 Copy number variations (CNVs), defined as gains and losses of DNA typically Ͼ1 kb and up to several megabases, are being increasingly recognized as a source of differences in genomic sequence 5-8 and have been proposed...