Published online before print April 3, 2006, doi: 10.1161/01.HYP.0000217364.45622.f0
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(Hypertension. 2006;47:937.)
© 2006 American Heart Association, Inc.
Original Articles |
Rho Kinase Polymorphism Influences Blood Pressure and Systemic Vascular Resistance in Human Twins
Role of Heredity
From the Departments of Biology (D.W.S.), Medicine (F.R., S.K., B.P.K., E.O.L., M.G.Z., D.T.O.), Pharmacology (T.M.S., J.H.B., D.T.O.), and Psychiatry (J.W., B.K.R., N.J.S., J.H.B.), Polymorphism Research Laboratory (N.J.S.), Center for Molecular Genetics (D.T.O.), San Diego, Ca; and Center for Human Genetics and Genomics (N.J.S., D.T.O.), University of California at San Diego, San Diego, Ca; and VA San Diego Healthcare System (D.T.O.), San Diego, Ca.
Correspondence to Daniel T. O’Connor or Joan Heller Brown, Departments of Medicine and Pharmacology, University of California at San Diego School of Medicine and VA San Diego Healthcare System, 9500 Gilman Dr, La Jolla, CA 92093-0838. E-mail doconnor{at}ucsd.edu or jhbrown@ucsd.edu
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Abstract |
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The Rho/Rho kinase (ROCK) pathway is implicated in experimental hypertension. We, therefore, explored the role of ROCK2 genetic variation in human blood pressure (BP) regulation, exploiting the advantages of a human twin sample to probe heritability. The focus of this work is the common nonsynonymous variant at ROCK2: Thr431Asn. Cardiovascular and autonomic traits displayed substantial heritability (from
33% to 71%; P<0.05). The Asn/Asn genotype (compared with Asn/Thr or Thr/Thr) was associated with greater resting systolic (P<0.001), diastolic (P<0.0001), and mean BP (P<0.0001); allelic variation at ROCK2 accounted for up to 5% of BP variation (P<0.0001). Systemic vascular resistance was higher in Asn/Asn individuals (P=0.049), whereas cardiac output, large artery compliance, and vasoactive hormone secretion were not different. Coupling of the renin-angiotensin system to systemic resistance and BP was diminished in Asn/Asn homozygotes, suggesting genetic pleiotropy of Thr431Asn, confirmed by bivariate genetic analyses. The Asn/Asn genotype also predicted higher BP after environmental (cold) stress. The rise in heart rate after cold was less pronounced in Asn/Asn individuals, consistent with intact baroreceptor function, and baroreceptor slope was not influenced by genotype. Common genetic variation (Thr431Asn) at ROCK2 predicts increased BP, systemic vascular resistance (although not large artery compliance), and resistance in response to the endogenous renin-angiotensin system, indicating a resistance vessel-based effect on elevated BP. The results suggest that common variation in ROCK2 exerts systemic resistance-mediated changes in BP, documenting a novel mechanism for human circulatory control, and suggesting new possibilities for diagnostic profiling and treatment of subjects at risk of developing hypertension.
Key Words: vascular resistance • blood pressure • hypertension, renal • renin
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Introduction |
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Vasoconstrictor amines, such as norepinephrine and epinephrine, and peptides, such as angiotensin II and endothelin, act as agonists at 7-membrane–spanning or heterotrimeric G protein–coupled receptors (GPCRs). Signaling through these GPCR pathways augments systemic vascular resistance (SVR) or cardiac output (CO), which, in turn, result in increased mean arterial pressure (MAP). In the case of vascular contraction, these agonists bind to their cognate receptors on vascular smooth muscle cells (VSMCs), activating heterotrimeric G proteins of the Gq and Gi families. Activation of phospholipase C leads to generation of inositol triphosphate and an increase in the release of Ca2+ from the sarcoplasmic reticulum, as well as Ca2+ entry through receptoroperated Ca2+ channels.
GPCRs can also signal through small (low molecular weight) G proteins, such as RhoA. RhoA is a member of the Rho family of small G proteins that also includes Rac1 and Cdc42. RhoA is best known for its effects on cell morphology, including stress fiber formation in fibroblasts and process retraction and cell rounding of neurons and astrocytoma cells.1–4 A subset of GPCR agonists, particularly those for receptors that couple to the G12/13 family, is capable of activating RhoA and, thus, signaling through this pathway.5,6 Rho, in turn, has been shown to signal through its effector Rho kinase to phosphorylate and inhibit the myosin binding subunit of myosin phosphate, thereby promoting increased accumulation of phosphorylated myosin light chain and vascular smooth muscle contraction.7,8 The addition of activated Rho/Rho kinase (ROCK) to permeabilized blood vessels has been shown to elicit vascular contraction,9 and inhibition of ROCK with the pharmacological antagonist Y-27632 was reported to reduce blood pressure (BP) in 3 different experimental models,10 suggesting an important role for ROCK in BP regulation. Our group has shown that RhoA is also required for VSMC proliferation and migration11 and that RhoA expression and activation, as well as 
-dependent proliferation, are upregulated in experimental hypertension.12
Two isoforms of Rho kinase have been identified: ROCK1 and ROCK2. ROCK1 is also known as p160ROCK or ROCKß, whereas ROCK2 is also as ROK
or Rho kinase. The serine-threonine kinase domain is located at the amino terminus of the protein. This is followed by a potential coiled-coil domain wherein lies the Rho binding region (Figure 1) followed by a C-terminal pleckstrin homology domain. ROCK1 and ROCK2 appear to be widely expressed, with ROCK2 most abundant in smooth muscle, suggesting an important role of this isoform in contraction (reviewed in Reference 13). In addition to upregulation of vascular Rho activity in vessels and smooth muscle cells from hypertensive rats,12 Rho kinase activity is also augmented in spontaneously hypertensive rat (SHR) vessels (reviewed in References 14,15). For example, Y-27632 produces greater cerebral vessel (basilar artery) dilation in the SHR versus the Wistar-Kyoto (WKY) rat,16 and SHR cerebral vessels exhibit greater Rho kinase-dependent myogenic tone,17 suggesting enhanced Rho kinase activity. Phenylephrine- and serotonin-stimulated coronary and carotid arteries from SHR rats responded with greater relaxation to Y-27632,18 whereas mesenteric arteries from mineralocorticoid hypertensive and SHR rats also showed greater dilation after Y-27632.19,20
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Given the clear influence of ROCK on BP and their involvement in experimental hypertension, interindividual polymorphisms at ROCK might reasonably be hypothesized to influence human pressor responses. We tested this hypothesis in twin pairs, evaluating whether polymorphism at ROCK contributes to heritability of BP or heart rate responses (thus contributing to interindividual variability in response: h2=VG/VP, where h2 is heritability, VG is additive genetic variance, and VP is total phenotypic variance). Although we found that a number of ROCK2 polymorphisms were associated with pressor responses, 1 nonsynonymous SNP (Asn431Thr) was of particular interest, because it encodes an amino acid substitution in the predicted coiled-coil domain of the protein (available online at http://us. expasy.org/cgi-bin/niceprot.pl?O75116; Figure 1), which is associated with ROCK dimerization and Rho binding.
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Methods |
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Polymorphisms at ROCK2
A sample of EDTA-anticoagulated whole blood was obtained from each participant and stored at –70°C before leukocyte DNA extraction (PureGene; GentraSystems) and single nucleotide polymorphism (SNP) genotyping. ROCK2 SNPs were selected based on the following criteria: available from the public NCBI Single Nucleotide Polymorphism database (available online at http://www.ncbi.nlm.nih.gov/SNP), minor allele frequencies >20% in white populations (to augment statistical power for association tests), spanning a large portion (
36 kb) of the gene (to capture the possibility of multiple blocks of linkage disequilibrium; LD), and (if possible) location in putative functional regions of the gene. DNA was extracted from leukocytes in EDTA-anticoagulated whole blood, and SNP genotypes were established by bp extension and mass spectrometry on the extension product in a 2-stage base-extension assay.19,20 During stage 1, PCR primers flanking the polymorphism were used to amplify the target region from 5 ng of genomic DNA. In stage 2, an oligonucleotide primer flanking the variant was annealed to the amplified template and extended across the variant base. The mass of the extension product (wild-type versus variant) was scored by matrix-assisted laser desorption ionization-time of flight mass spectrometry (low mass allele versus high mass allele).
Human Subjects: Twin Samples
Twin recruitment proceeded by access to a population birth record–based twin registry,21 as well as by newspaper advertisement. We recruited 344 individuals from 172 twin pairs. There were 119 monozygotic (MZ) pairs (24 male/male, 95 female/female) and 53 dizygotic (DZ) pairs (9 male/male, 33 female/female, and 11 male/female). Zygosity was initially established by self-report and was then confirmed by use of either >100 multiallelic microsatellites (on chromosomes 1 and 2 from genome scan) for self-identified DZ twins or biallelic SNP data (median, 149 SNPs; 50 SNPs at independent loci), as well as the multiallelic TH (TCAT)n microsatellite22 on self-reported both MZ and DZ twins. For biallelic SNP genotypes, the presence of only 2 alleles per locus and the consequent relatively low degree of heterozygosity (as compared with multiallelic microsatellite genotypes) results in relatively greater identify by state versus identical by descent (IBD;identity) levels for allele sharing. For MZ twin pairs, the median degree of SNP allele sharing was 2.0/2.0 (100%), whereas for DZ twin pairs, the median degree of SNP allele sharing was 1.73/2.0 (86.5%). For IBD (ideal/theoretical, fully heterozygous, completely informative markers), expected degrees of allele sharing would, of course, be MZ at 100% and DZ at 50%. The 344 subjects were all white (European ancestry); ethnicity was established by self-identification, as well as by that for both parents and all 4 grandparents. The median age of the twins is 40.8 years (range, 15 to 84 years). Family histories for hypertension in a first-degree relative before the age of 60 years23,24 were as follows: 75 were positive (1 or both parents), 82 pairs were negative, and 15 pairs were indeterminate/unknown. Self-reported hypertension status was normotensive for 309 subjects and hypertensive for 35 subjects (26 treated with antihypertensive medications and 9 untreated). None of the subjects had a history of renal failure. Definitions of subject characteristics are according to previous reports from our laboratory.23,25 Subjects were volunteers from urban Southern California (San Diego), and each subject gave informed, written consent; the protocol was approved by the Institutional Review Board at University of California at San Diego.
Biochemical Phenotyping
Samples for the measurement of plasma and urine catecholamines were quickly frozen at –70°C before a sensitive radioenzymatic assay based on catechol-O-methylation.26 The assay uses a preconcentration step that increases sensitivity by 10-fold over other catechol-O-methyltransferase-based assays and 20-fold over many high-performance liquid chromatography assays, permitting accurate measurement of basal plasma epinephrine levels, which are at the limit of sensitivity for high-performance liquid chromatography assays. Aldosterone, renin, and cortisol were measured by immunoassay. Urine values were normalized to creatinine excretion in the same sample.
Physiological/Autonomic Phenotyping In Vivo
Environmental (Cold Stress)
BP and pulse interval (R-R interval or heart period, in ms/beat) were recorded continuously and noninvasively for 5 minutes in seated subjects with a radial artery applanation device and dedicated sensor hardware (Colin Pilot, Colin Instruments) and software (ATLAS, WR Medical, and Autonomic Nervous System/Tonometric Data Analysis, Colin Instruments), calibrated every 5 minutes against ipsilateral brachial arterial pressure with a cuff sphygmomanometer. Heart rate was recorded continuously with thoracic ECG electrodes to the Colin Pilot. Vital signs were also recorded with the same devices during environmental stress, in the form of the cold pressor test (immersion of the left hand in ice water for 60 seconds after a preceding 10 minute rest), as described previously.22,23,27 We identified 
3 beats with stable BP and heart rate (each beat within ±10% of the mean value) just before and at the end of the cold stress.
Hemodynamics
BP, CO, stroke volume, contractility, SVR, and compliance were determined noninvasively with an oscillometric device, as described previously.28,29 Results containing flow terms were normalized to body surface area, as an index of body size, estimated by the Mosteller formula.
Baroreceptor Sensitivity (Slope) in the Time Domain
BP and heart rate were also continuously recorded with the same devices during spontaneous excursions of BP with reciprocal heart rate changes: upward excursions of BP with reflex bradycardia and downward excursions of BP with reflex tachycardia. In each case, baroreceptor slope in the "time domain24,27" was calculated with the Autonomic Nervous System/Tonometric Data Analysis software, with beat-by-beat regression of change in systolic BP (SBP; 
SBP, mm Hg) as a function of change in pulse interval (R-R interval, ms/beat) on the succeeding beat (phase lag=1 beat). Time windows of >4 beats were used, with SBP of >1 mm Hg and R-R of >6 ms. Baroreceptor slope (ms/mm Hg) values were recorded for regressions with target correlation coefficients of r>0.9. The slopes for 3 such regressions, if each was within ±10% of the mean value, were averaged to yield the final value for the baroreceptor slope. Baroreceptor slopes were separately determined for upward and downward spontaneous excursions of BP.
Statistical Analysis
Descriptive statistics (mean±SEM) were computed across all of the twins using generalized estimating equations in SAS (Statistical Analysis System) to take into account intra-twin–pair correlations.30 Estimates of heritability (h2=VG/VP) were obtained using the variance-component methodology implemented in the Sequential Oligogenic Linkage Analysis Routines (SOLAR) package,31 available online (http://www.sfbr.org/solar/). This method maximizes the likelihood assuming a multivariate normal distribution of phenotypes in twin pairs (MZ versus DZ) with a mean dependent on a particular set of explanatory covariates. The null hypothesis (H0) of no heritability is tested by comparing the full model, which assumes genetic variation (VG), and a reduced model, which assumes no genetic variation, using a likelihood ratio test. All of the heritability estimates were adjusted for age and sex because of the effects of these covariates on several traits. SOLAR was also used to evaluate whether allelic variation at the locus contributed to a significant fraction of the trait heritability (ie, locus-specific VG) by comparing models including or excluding the genotype as a covariate. Each SNP was defined as a 3-level variable representing the 3 possible genotypes (homozygous variant, heterozygous, and homozygous wild-type) assuming an additive effect in all of the models. Multivariable analyses were carried out to control for confounding by age and sex (as covariates) because of their effects on several of the biochemical and physiological phenotypes (Table 1). As a test of pleiotropy, bivariate analyses in SOLAR (chapter 9.2)31 were done to test whether the genotype coordinately influenced 2 dependent variables (traits), biochemical (eg, renin) and hemodynamic [eg, diastolic BP (DBP) or SVR], using nested log likelihood values for the bivariate model in the presence or absence of the genotype: –2(log likelihood)=
2 at 1 degree of freedom. Hardy-Weinberg equilibrium (HWE) was assessed with a 2 goodness-of-fit test, using 1 individual from each twin pair. Haplotypes were assigned using the HAP algorithm.32
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Results |
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Subjects and Traits
Characteristics of the 172 twin pairs are given in Table 1. Many of the traits were age dependent. In particular, older and younger subjects differed in trait means for body mass index, basal and stress-augmented SBP and DBP, MAP, baroreceptor slope, and norepinephrine excretion (Table 1).
Heritability for each trait, estimated from twin pair correlations, is recorded in Table 2. Each of the traits, physiological or biochemical, displayed significant (P<0.05) heritability.
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Genomics: ROCK2 SNP Genotypes
We genotyped 4 SNPs with relatively high minor allele frequencies within the ROCK2 locus (on chromosome 2p24) in all 344 of the white twins (both MZs and DZs): C115634T, intron 5; C125592A/Thr431Asn, exon 10; C128522T, intron 12; and C151504G, intron 29. These SNPs spanned 35 870 bp of the ROCK2 locus, extending across 24 exons and introns. Two SNPs (C115634T and C128522T) were out of HWE (Table 3). To determine whether genotyping error could explain the Hardy-Weinberg disequilibrium in these 2 SNPs, we evaluated our MZ twins for discordant allele calls within pairs and found a low genotyping error rate (Thr431Asn at 2.63% and C128522T at 0.90% of MZ pairs; the other 2 SNPs at 0% discrepancy); recalculation of HWE excluding these individuals showed similar results to those in Table 3. All 4 of the ROCK2 SNPs were in substantial LD, with LD parameter D' uniformly 0.98. We confined haplotype inference to the 2 SNPs in HWE: C125592A (Thr431Asn) and C151504G; A125592
C151504 (ie, AC) was the most common haplotype (on 50.3% of chromosomes), followed by CG (28.9%), CC (20.6%), and AG (0.1%).
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Associations (Genotype Phenotype)
BP
We first noted associations of ROCK2 genotypes across the entire locus (4 SNPs spanning 36 kbp, from intron 5 intron 29; Table 3) with basal BPs (both systolic and diastolic) and SVR (data not shown). We then focused our attention on SNP C125592A because of its nonsynonymous amino acid alteration (Thr431Asn), its likely functionality based on its position in the protein domains (Figure 1), and the strong LD across all 4 of the SNPs (each D' 0.98), with general uniformity of associations (data not shown). Table 4 shows that the A (Asn) allele is associated with significantly higher resting SBP, DBP, and MAP, accounting for up to 5% of interindividual variability in BP. Because linkage disequilibrium between these 4 ROCK2 SNP pairs was almost complete (all D' 0.98), we elected not to proceed to haplotype associations (data not shown).
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Hemodynamics: CO and SVR
Alterations in BP can occur as a result of changes in either CO or SVR. We, therefore, evaluated these hemodynamic parameters for effects of Thr431Asn. Neither CO nor heart rate was different among Thr/Thr, Thr/Asn, and Asn/Asn genotype groups (Table 4). On the other hand, SVR was significantly (P=0.049) greater in individuals with the Asn amino acid change, suggesting that the Asn allele contributes to the observed higher BP by elevating peripheral vascular resistance.
Pulse Pressure and Compliance
Pulse pressure and systemic vascular compliance were not different among the Thr431Asn genotype groups (Table 4), indicating that distensibility of large vessels is similar across the groups. Thus, the 431Asn allele seemed to influence BP by affecting small vessel resistance rather than large vessel compliance.
Humoral Mediators: Catecholamines, Renin, Aldosterone, and Cortisol
Release of catecholamines from the adrenal medulla or sympathetic nerve terminals does not appear to contribute to higher BP in the Asn/Asn group, because plasma and urinary epinephrine and norepinephrine (Table 4) were not different. In addition, plasma and urinary aldosterone and cortisol levels, as well as renin, were all similar across the 3 Thr431Asn genotype groups (Table 4). Asn/Asn homozygosity seemed to change the coupling between the renin-angiotensin system and SVR, BP, or aldosterone (Figure 2). Asn/Asn homozygotes displayed greater SVR and DBP for any given level of renin. By contrast, Asn/Asn homozygotes showed lower aldosterone secretion for any level of renin. This apparent pleiotropic action of Thr431Asn was confirmed by bivariate analyses with nested log likelihoods in SOLAR, suggesting that the genotype coordinately influenced pairs of dependent variables: renin and DBP (2=18.4; P<0.0001), renin and SVR (2=6.2; P=0.0128), and renin and aldosterone (2=5.4; P=0.0201).
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Environmental Stress
Cold stress BP and heart changes were analyzed to determine whether responses to environmental stress were altered in individuals with Asn at Thr431Asn. These responses were substantially heritable in twins (Table 2). All of the groups (Thr/Thr, Thr/Asn, and Asn/Asn) responded with a characteristic increase in SBP, DBP, and MAP, as well as an increase in heart rate (Table 4). Whereas both prestress and poststress diastolic pressures were greater in the Asn/Asn group, the change (or increase) in pressure was not different among the genotype groups (Table 4). Furthermore, the increase in heart rate during cold stress was significantly lower with increasing copy number of the A (Asn) allele at Thr431Asn (Table 4).
Baroreceptor Function
Regulation of heart rate by the baroreceptor pathways is a critical compensatory mechanism in response to a rise in BP. Baroreceptor function, assessed as the heart rate response to spontaneous fluctuations of BP (either upward or downward) was intact in all of the groups and did not differ among genotype groups (Table 4). This finding is consistent with the notion that a reduced heart rate rise among Asn/Asn individuals could result from appropriate baroreceptor compensation (reflex bradycardia) in response to the higher postcold stress diastolic pressure of that genotype group.
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Discussion |
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Overview
The current study was undertaken to examine the functional role of polymorphisms in the human ROCK2 gene. This is the first work to identify a genetic variant in the Rho/ROCK pathway associated with increased BP. Here, we present evidence of 4 SNPs in ROCK2, of which 1 is a nonsynonymous alteration resulting in an amino acid change from Thr to Asn at the 431 position (Figure 1). Of particular significance is the finding that the Asn allele at Thr431Asn is associated with increased BP coupled with increased SVR (Table 4). By contrast, individuals with the Asn versus Thr alleles exhibited no differences in heart rate or CO (Table 4 and Figure 1).
These hemodynamic data indicate that increased peripheral vascular resistance accounts for the higher BP observed in the Asn groups. Further support for the conclusion that small vessel resistance is the hemodynamic culprit in genotype action on BP is that the Thr431Asn genotype did not predict either pulse pressure or large artery compliance (Table 4) nor did humoral determinants of BP (catecholamines, renin, aldosterone, and cortisol; Table 4) differ systematically among the genotypic groups.
Also consistent with a vascular action of the ROCK2 polymorphism is the observation that, for any given level of renin, SVR and BP are highest in Asn/Asn homozygotes (Table 4), suggesting increased coupling of angiotensin to vascular contraction by the Asn allele. By contrast, for any given level of renin, aldosterone secretion is lowest in Asn/Asn homozygotes (Table 4). This apparent pleiotropic effect of the Thr431Asn genotype on 2 different dependent variables, biochemical and hemodynamic (renin and SVR, renin and DBP, or renin and aldosterone), was confirmed by bivariate analyses in SOLAR (all P<0.05; Table 4). Of note, ROCK2 is highly expressed in human smooth muscle, although not in the human adrenal gland (202762_at in Human GeneAtlas GNF1H, gcRMA; available online at http://symatlas.gnf.org/SymAtlas/); thus, the involvement of the renin-angiotensin system in the BP effects of ROCK2 polymorphism seems to reside in augmented effects on blood vessels rather than increased signaling within the adrenal zona glomerulosa cells.
Our results suggest that baroreceptor function is intact in subjects with ROCK2 variants, although we did not test the baroreflex with classical pharmacological vasopressor/vasodepressor agents. However, in experimental animal hypertension resulting from NG-nitro-L-arginine methyl ester (NO deficiency) or the SHR, administration of a Rho kinase inhibitor into the nucleus of the tractus solitarius reduces arterial pressure, heart rate, and renal sympathetic nerve activity to a greater extent in hypertensive animals; such data suggest that alterations of ROCK-dependent baroreceptor pathways can contribute to increased BP.33,34
Significance of Rho Kinase Polymorphism
It is intriguing but perhaps not surprising that alterations in SVR are associated with genetic variation at ROCK2 (Table 4). Abundant evidence supports an important (if not central) role of the Rho/ROCK signaling pathway in VSMC contraction (Table 5). It is now well accepted that, in addition to increasing intracellular Ca2+, GPCR agonists can use an alternative signaling pathway involving Rho/ROCK to produce vascular contraction even in the absence of increases in intracellular Ca2+ (Ca2+ sensitization).35,36 Rho acts through its effector ROCK to phosphorylate and inactivate the myosin binding subunit of myosin phosphatase causing an accumulation of phosphorylated myosin.7,8 Consistent with a role for ROCK in vascular contraction, the addition of catalytically active ROCK to permeabilized vessels increases contraction,9 and a pharmacological inhibitor of the kinase, Y-27632, blocks agonist and guanine nucleotide–stimulated contraction.10
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Recent findings suggest that dysregulation of the ROCK signaling pathway plays a role in vascular diseases, such as hypertension (reviewed in References 14,15 and Table 5). A seminal study by Uehata et al10 showed that the ROCK inhibitor Y-27632 reduced BP in 3 different experimental models of hypertension, raising the possibility that Rho/ROCK upregulation contributes to the development or maintenance of hypertension. In support of this notion, we12 observed that RhoA expression, RhoA activation, and -dependent proliferation were greater in vessels and VSMCs from genetically inbred SHR and N-nitro-L-arginine methyl ester–treated (NO-deficient) hypertensive rats compared with that from WKY normotensive rats. It was subsequently shown that Rho activity is greater in vessels and VSMCs from animals in a variety of models of hypertension.37,38 In addition to alterations in Rho, Rho kinase activity has also been shown to be upregulated in SHR, because Rho kinase inhibitors have a greater effect on vessel dilation and relaxation in these animals.16–18,39,40
Rho/ROCK signaling not only mediates VSMC contraction that is dysregulated in hypertension, but this pathway is also involved in VSMC proliferation, migration, and gene expression.11,41,42 These responses contribute to the pathophysiological progression of vascular disorders, such as restenosis injury and atherosclerosis; therefore, it is not surprising that ROCK inhibitors have been shown to also limit the progression of these vascular disorders in animal models.43–45
The trait-associated nonsynonymous coding region single nucleotide polymorphism (Thr431Asn) in ROCK2 occurs in or near a coiled-coil region (available online at http://us.expasy.org/cgi-bin/niceprot.pl?O75116), a structural domain of likely functional importance (Figure 1). Within the coiled-coil region but downstream of Thr431Asn lies the RhoA binding (transactivation) domain (Figure 1). ROCKs are likely to exist as dimers by parallel association at the coiled-coil domain.46 Inference from Rac1 binding to PAK1 suggests that dimerization of the effector might induce an autoinhibitory form of the kinase and that GTPase binding can trigger conformational changes disrupting the dimer, resulting in kinase activation.47,48 Therefore, changes in the coiled-coil region could be hypothesized to effect dimerization, Rho binding, and thereby ROCK activation and phosphorylation of its substrates. Future studies will be aimed at comparing the relative abilities of Thr431 versus Asn431 to phosphorylate substrates, such as the myosin binding subunit of myosin phosphatase, and produce cellular responses, such as stress fiber formation and cellular contraction.
Unique Strengths and Limitations of This Study
Normotensive Subjects
This study concerned largely healthy, younger individuals (median age, 40.8 years), with basal BPs averaging 117/64 mm Hg (Table 1). Thus, this population does not allow us to directly test whether allelic variation at ROCK2 predisposes to later development of hypertension. However, longitudinal studies in human populations now clearly document that normotensive individuals in the higher BP strata are at increased risk to progress to hypertension.49,50 Indeed, even "prehypertension" (in the BP stratum 120 to 139/80 to 89 mm Hg) in a population-based cohort is a predictor of elevated cardiovascular morbidity.51 The Thr431 allele influenced stress BP responses, which may be a predictor of development of later cardiovascular events, such as hypertension52–54 (Table 5).
Twins
Multiple autonomic phenotypes, both biochemical and physiological, allowed for inference of an integrated picture of the effects of particular genetic variants. Furthermore, twin data offer the unique advantage of direct measurement of heritability, the fraction of phenotypic variance accounted for by genetic variance.55 Heritability results from our study are similar to previous studies for SBP and DBP.56–58 Genetic conclusions drawn from twin studies are likely to be generalizable to the population from which they were sampled.55
Ethnicity
We restricted our analyses to subjects of European ancestry, because allelic association studies can be susceptible to artifactual conclusions resulting from even inapparent population admixture.59 Studies of additional ethnic or population groups will be required to evaluate whether our ROCK2 results are of more general importance in the overall population.
Linkage Disequilibrium at the ROCK2 Locus
Although SNPs C125592A and C151504G (Table 3) lie 25 912 bp apart, they were in almost complete linkage disequilibrium (D' 0.98), and the genotypes at each SNP showed very similar trait associations. Such extended local linkage disequilibrium is typically found in subjects of relatively homogeneous European ancestry,60 such as our twin sample. Nonetheless, other variants within this ROCK2 haplotype block are also likely to be associated with the traits we have described here. Indeed, the extended size of the haplotype block (region of linkage disequilibrium) at this chromosomal position may define the limit of resolution of genetic mapping as a tool to position genotypes influencing phenotypes. Publicly available data in National Center for Biotechnology Information dbSNP also suggest an extended block of LD in the ROCK2 locus in the region of SNP rs9808232 (Asn431Thr). Hence, further experiments on the functional consequences of the Asn431Thr variant, both in vitro and in vivo, will be required to solidify any conclusions about the functional role of this polymorphism in humans.
Conclusions and Perspectives
Common genetic variation in the ROCK2 locus, especially the nonsynonymous coding region single nucleotide polymorphism Asn431Thr, predicts changes in both resting and stress-elevated BP, contributing up to 5% of the heritable variation in such traits in twin pairs. ROCK2 Asn431Thr variation affected SVR, but not CO, vascular compliance, or vasoactive hormone (catecholamine, renin, aldosterone, and cortisol) secretion, indicating a small arterial effect of this SNP on the BP phenotype, likely by changing the coupling of systemic resistance to the renin-angiotensin system in an example of genetic pleiotropy. The results suggest that common variation in ROCK2 exerts early vascular resistance–mediated changes on BP, documenting novel pathophysiological mechanisms for human circulatory control, and suggesting new possibilities for diagnostic profiling and treatment of subjects at risk of developing systemic hypertension.
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Acknowledgments |
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This work was supported by the National American Heart Association scientist development grant (to T.M.S.), Department of Veterans Affairs (to D.T.O.), and National Institutes of Health (to D.T.O., J.H.B., N.J.S.).
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Footnotes |
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The first 2 authors contributed equally to this work.
Received November 23, 2005; first decision December 13, 2005; accepted March 2, 2006.
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References |
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