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Quantitative Trait Loci Mapping for Intracellular Calcium in Spontaneously Hypertensive Rats
AJH
2005; 18:666 – 671
Quantitative Trait Loci Mapping for Intracellular Calcium in Spontaneously Hypertensive Rats Yoichi Ohno, Hiromichi Suzuki, Hisao Tanase, Keiichi Otsuka, Takayuki Sasaki, Taichi Suzawa, Toshiyuki Morii, Yosuke Ando, Tatsuya Maruyama, and Takao Saruta Background: Increased intracellular calcium ([Ca2⫹]i) in platelets is also proposed as an intermediate phenotype for hypertension in spontaneously hypertensive rats (SHR). Increased [Ca2⫹]i in platelets is hypothesized to contribute to atherothrombotic events. Platelet hyperactivity is frequently associated with cardiovascular disease. Methods: In a genome scan, we performed the quantitative trait loci (QTL) mapping for [Ca2⫹]i in back-crossed rats derived from SHR and normotensive Fischer 344 rats, which demonstrated a single major QTL for hypertension on chromosome 1. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and in Ca2⫹-containing buffers was measured in platelets using the Fura-2 method. Results: Among the parental strains, systolic blood pressure and thrombin-stimulated [Ca2⫹]i were significantly greater in SHR than in Fischer 344 and F1 rats. The sarco(endo)plasmic reticulum Ca2⫹-dependent ATPase II gene locus (Serca2) between D12Mgh5 and D12Mgh6 showed the significant linkage for thrombin-stimulated
[Ca2⫹]i in Ca2⫹-free and Ca2⫹-containing buffers. The peak logarithm of the odds scores were 3.6 and 3.3, respectively. These QTL explained 19.8% and 17.4% of the total variances, respectively. D3Mit13 and DXMgh1 showed suggestive linkage for thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and in Ca2⫹-containing buffers, respectively. The peak logarithm of the odds scores were 2.6 and 2.1, respectively. Conclusions: A significant QTL for [Ca2⫹]i was mapped near Serca2 on chromosome 12, and suggestive QTL were identified near D3Mit13 and DXMgh1 in a genome scan. Genetic abnormalites in platelet [Ca2⫹]i may contribute to cardiovascular disease via platetet hyperactivity, independent of blood pressure elevation. Am J Hypertens 2005;18:666 – 671 © 2005 American Journal of Hypertension, Ltd. Key Words: Intracellular calcium, quantitative trait loci, spontaneously hypertensive rats, sarco(endo)plasmic reticulum calcium– dependent ATPase II gene, Sa gene.
latelet activation is essential in the development of preclinical atherosclerosis as well as in the clinical expression of atherothrombotic disease.1 Intracellular 2⫹ Ca ([Ca2⫹]i) is considered an essential second messenger in platelet activation. Moreover, abnormal [Ca2⫹]i homeostasis plays a key role in the pathogenesis of hypertension.2 [Ca2⫹]i in platelets is increased in patients with essential hypertension and in their offspring.3,4 Altered intracellular Ca2⫹ handling in platelets has also been reported in spontaneously hypertensive rats (SHR) and in young prehyperten-
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sive SHR.5,6 In the past decades, investigators have focused on the molecular genetics of arterial vascular disorders and have identified numerous polymorphisms in genes related to the cardiovascular system; however, the genetic contributions to augmented [Ca2⫹]i response in platelets remain highly controversial.7 Disturbed Ca2⫹ pump activity, abnormal function of Ca2⫹-binding proteins, and increased Na⫹–Ca2⫹ exchange activity reportedly increase [Ca2⫹]i.8 These studies suggest that increased [Ca2⫹]i in platelets could be a predictor of atherothrombotic events and a genetic hypertensive trait.
Received October 28, 2004. First decision December 2, 2004. Accepted December 22, 2004. From the Department of Internal Medicine (YO), Green Town Clinic Center, Saitama, Japan; Department of Internal Medicine (YO, KO, TS, TS, TM, TS), School of Medicine, Keio University, Tokyo, Japan; Department of Internal Medicine (HS), Saitama Medical College, Saitama, Japan; Medicinal Safety Research Laboratories (HT, YA), Sankyo Co. Ltd., Shizuoka, Japan; and Department of Internal Medicine
(TM), Sanno Hospital, Tokyo, Japan. This work was supported by a research grant from the National Dairy Promotion and Research Association, and by TEPCO Hospital Funds. Address correspondence and reprint requests to Dr. Yoichi Ohno, Department of Internal Medicine, Green Town Clinic Center, 2-23-3 Midoricho, Yashio City, Saitama, 340-0808, Japan; e-mail: [email protected]. ne.jp
0895-7061/05/$30.00 doi:10.1016/j.amjhyper.2004.12.001
© 2005 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
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QUANTITATIVE TRAIT LOCI MAPPING FOR INTRACELLULAR CALCIUM
We reported a unique back-cross analysis derived from SHR and normotensive Fischer 344 (F344) rats that approximates a polygenic model to a monogenetic form.9,10 Bimodality in blood pressure (BP) values of the backcrossed (BC) rats demonstrated an inferred single major gene locus or a major quantitative trait locus (QTL) for hypertension near the Sa gene locus (Sa).10 We measured [Ca2⫹]i in platelets to explore the “intermediate phenotype.” Increased [Ca2⫹]i in platelets was considered to be an inheritable hypertensive trait discriminated from the major QTL for BP on chromosome 1.9 We have already reported significant association between the sarco(endo)plasmic reticulum Ca2⫹-dependent ATPase (Serca) II gene locus (Serca2) and [Ca2⫹]i in platelets.11 In contrast, other studies have indicated that [Ca2⫹]i in platelets is also influenced by environmental factors such as shear stress and insulin resistance.4,12 It has not been clearly determined whether increased [Ca2⫹]i in platelets from hypertensive subjects or animals is a cellular marker of an underlying inherited abnormality or merely an epi-phenomenon secondary to endothelial dysfunction or an increase in BP. In this study, we attempted to map QTL for [Ca2⫹]i in platelets in BC rats derived from SHR and F344 rats by screening the total genome.
Methods Animals The SHR, Wistar-Kyoto (WKY) rats, and F344 rats were maintained at the Medicinal Safety Research Laboratories, Sankyo Co. Ltd. (Shizuoka, Japan). The F1 females from male SHR and female F344 parents were back-crossed with male SHR as previously described.10 The same BC population was used for a genome scan of hypertension by targeting on a potential epistasis for BP between a chromosome-1 QTL and the Serca2 locus on chromosome 12. Systolic BP was measured by the tail-cuff method at 10, 13, and 15 weeks of age; the mean values were used for statistical analysis. Data from 78 male BC rats underwent complete analysis. This experimental protocol was approved by the institutional animal care committee. Measurement of [Ca2ⴙ]i The [Ca2⫹]i in platelets was measured by the Fura-2 method as previously described.9,11,13 Thrombin-stimulated (0.3 U/mL) [Ca2⫹]i was measured in Ca2⫹-free buffer and in 1 mmol/L Ca2⫹-containing buffer. Genotyping of Microsatellite Markers and [Ca2ⴙ]i-Regulating Genes A total of 80 highly polymorphic microsatellite markers were selected to be spaced at 20-cM intervals, which spanned the entire genome as previously described.10 The BC rats were genotyped as homozygotes (ie, SHR) or heterozygotes (ie, F1) in autosomal chromosomes or as the
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identical genotype with SHR or F344 rats in X chromosome. Serca II gene was genotyped by restriction fragment length polymorphisms (RFLP) we previously discovered.11 The phospholipase C (PLC) ␦-1 gene was genotyped by RFLP using XhoI.14 Quantitative Linkage Analysis The QTL mapping was performed by using MAPMAKER/ Exp 3.0 and MAPMAKER/QTL 1.1b (Whitehead Institute, Cambridge, MA). Logarithms of the odds (LOD) scores of 1.9 and 3.3 were used as thresholds for suggestive and significant linkage, respectively.15 Statistical Analysis Data are reported as mean ⫾ SD. Comparisons were performed by analysis of variance (ANOVA) followed by the Scheffé F test to evaluate the differences in BP and [Ca2⫹]i between the genotyped groups. These statistical computations were performed with the SPSS statistical program (SPSS Inc., Chicago, IL). A level of P ⬍ .05 was considered to be statistically significant.
Results Among the parental strains (SHR, F1, and F344), the most genetically similar to SHR had a significantly higher systolic BP and greater thrombin-stimulated [Ca2⫹]i in platelets (Fig. 1). Selection of F344 rats as a contrasting strain was advantageous to map QTL for [Ca2⫹]i because thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer in platelets in SHR is ⬃5 SD higher than in F344 rats whereas it was ⬃3 SD higher than in WKY rats. Basal [Ca2⫹]i in platelets was not different among the parental strains. Serca2 was mapped between D12Mgh5 and D12Mgh6 on chromosome 12 and exhibited significant linkage to thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and in Ca2⫹containing buffers (Fig. 2). The peak LOD scores were 3.6 and 3.3, respectively. These QTL explained 19.8% and 17.4% of the total variances, respectively. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and Ca2⫹-containing buffers was significantly higher in Serca II homozygotes than in Serca II heterozygotes (Table 1). However, their systolic BP levels were similar. D3Mit13 on chromosome 3 showed suggestive linkage for thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer (Fig. 3). The peak LOD score was 2.6, and this QTL explained 14.6% of the total variances. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer was significantly higher in D3Mit13 homozygotes than in D3Mit13 heterozygotes (Table 1), although their systolic BP levels were similar. DXMgh1 on chromosome X demonstrated suggestive linkage for thrombin-stimulated [Ca2⫹]i in Ca2⫹-containing buffer (Fig. 4). The peak LOD score was 2.1, and this QTL explained 11.3% of the total variances. Thrombinstimulated [Ca2⫹]i in Ca2⫹-containing buffer was significantly higher in BC rats with the SHR than those with the
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and suggestive QTL on rat chromosome 3 and x in BC rats derived from SHR and normotensive F344 rats. To our knowledge, this is the first reported observation pertaining to QTL mapping for [Ca2⫹]i in the platelets in hypertension. Serca II and Chromosome 12 Serca is thought to be a determinant factor that regulates [Ca2⫹]i stores in vascular smooth muscle cells.16 Serca II mRNA is reported to be overexpressed and the function of sarcoplasmic reticulum increased in purified microsomal fractions in vascular smooth muscle cells of SHR.17,18 Because we did not identify any amino acid substitution in the Serca II coding region of SHR compared with WKY and Sprague-Dawley rats,13 an increase in Serca II mRNA may contribute to the increased thrombin-stimulated [Ca2⫹]i. However, in platelets of SHR, compared with WKY, measurements of the level of expression of Serca IIb mRNA have yielded inconsistent results. Bobe et al measured an increase in Serca IIb mRNA up to 130%,19 whereas Mountian et al reported that it was significantly decreased to 60%.20 Platelet isolation and extraction of mRNA from platelets requires delicate techniques, which may explain the divergent results of their mRNA analysis. Recent genetic engineering experiments suggest that the level of Serca II mRNA expression may determine the
FIG. 1. Relationship between systolic blood pressure (BP) and thrombin-stimulated intracellular Ca2⫹ concentration ([Ca2⫹]i) in platelets of spontaneously hypertensive rats (SHR) (n ⫽ 9) (), normotensive Fischer 344 (F344) rats (n ⫽ 10) (●), and F1 rats (n ⫽ 5) (‘) derived from SHR, and F344 rats, and Wistar-Kyoto (WKY) rats (n ⫽ 6) (夝). Values are means ⫾ SD. (a) [Ca2⫹]i in Ca2⫹-free buffer. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer were significantly higher in SHR than in F344, F1, and WKY rats (ANOVA followed by Scheffé F test). SHR v F344, P ⬍ .0001, SHR v F1, P ⬍ .001, and SHR v WKY, P ⬍ .001. (b) [Ca2⫹]i in Ca2⫹-containing buffer. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-containing buffer was significantly higher in SHR than in F344 and WKY rats (ANOVA followed by Scheffé F test). SHR v F344 and SHR v WKY, P ⬍ .0001.
F344 genotype at DXMgh1 (Table 1). Furthermore, systolic BP tended to be slightly higher in BC rats with the SHR genotype (P ⫽ .06). DXMgh1 has a potential to raise BP slightly through abnormal intracellular Ca2⫹ homeostasis. The PLC ␦-1 gene locus was mapped 37.1 cM apart from D8Mgh1 on chromosome 8. Neither the QTL for [Ca2⫹]i nor the QTL for BP was detected around the PLC ␦-1 gene locus.
Discussion The current study identified a significant QTL for thrombin-stimulated [Ca2⫹]i in platelets on rat chromosomes 12
FIG. 2. A quantitative trait locus (QTL) for thrombin-stimulated intracellular calcium (Ca2⫹) (a) in Ca2⫹-free buffer and (b) in Ca2⫹containing buffer on chromosome 12. Peak logarithms of odds (LOD) scores were 3.6 and 3.3, respectively. Dashed lines indicate significant linkage; dotted lines indicate suggestive linkage.
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Table 1. Thrombin-stimulated intracellular calcium (Ca2⫹) concentration ([Ca2⫹]i) according to the genotypes at D3Mit13, Serca2, and DXMghl Genotype Serca2 Systolic blood pressure (mm Hg) Thrombin-stimulated [Ca2⫹]i Ca2⫹-free buffer (nmol/L) Ca2⫹-containing buffer (nmol/L) D3Mit13 Systolic blood pressure (mm Hg) Thrombin-stimulated [Ca2⫹]i Ca2⫹-free buffer (nmol/L) Ca2⫹-containing buffer (nmol/L) Genotype DXMgh1 Systolic blood pressure (mm Hg) Thrombin-stimulated [Ca2⫹]i Ca2⫹-free buffer (nmol/L) Ca2⫹-containing buffer (nmol/L)
Homozygotes (SHR)
Heterozygotes (F1)
P Value
n ⫽ 43 162 ⫾ 8
n ⫽ 35 160 ⫾ 12
.42
263 ⫾ 34 570 ⫾ 74 n ⫽ 41 161 ⫾ 9
234 ⫾ 22 515 ⫾ 53 n ⫽ 37 161 ⫾ 11
⬍.0001 ⬍.0001
260 ⫾ 34 557 ⫾ 71
236 ⫾ 24 526 ⫾ 62
⬍.001 .04
SHR Type
.42
F344 Type
n ⫽ 37 163 ⫾ 9
n ⫽ 41 159 ⫾ 10
.06
257 ⫾ 36 566 ⫾ 75
244 ⫾ 28 526 ⫾ 60
.08 ⬍.01
The results of Serca2 have been published in Ref. 11.
intensity of Ca2⫹ signaling and Ca2⫹ contraction coupling in cardiovascular cells.21–23 However, it would be an oversimplification to consider Serca II itself as the only causal gene in the chromosomal segment, because we have not found any functionally significant variants between the Serca II alleles in SHR versus F344. Several genes related to [Ca2⫹]i homeostasis could consist of a [Ca2⫹]i-regulating gene complex near Serca2 and could synergistically contribute to the increased Ca2⫹ response to thrombin.
(322 ⫺ 202)/322, or approximately 0.6. Similarly, the ratio for BP is calculated by (102 ⫺ 52)/102, or approximately 0.75. The proportion of the heritable component of [Ca2⫹]i was smaller than that of BP. Williams et al reported that in a human twin study, additive genetic effects were smaller in [Ca2⫹]i in platelets than in systolic BP.29 Therefore, [Ca2⫹]i in platelets could be an inheritable cellular marker, although heritability of [Ca2⫹]i seems lower than that of BP.
Candidate Genes on Chromosome 3 and X
Vincent et al30 reported that a QTL for BP responses to administration of a dihydropyridine calcium antagonist was mapped between D2Wox8 and D2Mit15 on chromosome 2 in BC rats derived from a cross of the Lyon hypertensive and normotensive rats. In that chromosomal
The rat– human comparative map24 and the human genome project have provided information about several [Ca2⫹]i-regulating genes located in the vicinities of QTL for [Ca2⫹]i. The PLC ␥-1 gene in the vicinity of D3Mit13 on chromosome 3 is an important candidate, as it is directly related to the process of Ca2⫹ mobilization.25 The vitamin D– dependent Ca2⫹-binding protein (calbindinD9k) gene, which plays a key role in intestinal Ca2⫹ transport, is mapped near DXMgh1. Intestinal calbindinD9k expression was found to be decreased in SHR compared with WKY rats.26,27
QTL for BP and [Ca2ⴙ]i
Inheritance or Epi-Phenomenon? Whether increased [Ca2⫹]i in platelets is a cellular marker for underlying inherited abnormality is controversial. Genetic variance is calculated by subtracting environmental variance from phenotypic variance.28 The SD of thrombinstimulated [Ca2⫹]i in Ca2⫹-free buffer in each of the parental strains and BC rats were approximately 20 and 32 nmol/L, respectively. The ratio of the heritable variance to the total variance in BC rats for [Ca2⫹]i is calculated by
FIG. 3. Quantitative trait locus (QTL) for thrombin-stimulated intracellular calcium (Ca2⫹) in Ca2⫹-free buffer on chromosome 3. Peak logarithm of odds (LOD) score was 2.6. Dashed lines indicate significant linkage; dotted lines indicate suggestive linkage.
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4.
5.
6.
7. 8. FIG. 4. Quantitative trait locus (QTL) for thrombin-stimulated intracellular calcium (Ca2⫹) in Ca2⫹-free buffer on chromosome X. Peak logarithm of odds (LOD) score was 2.1. Dashed lines indicate significant linkage; dotted lines indicate suggestive linkage.
region, we could not detect any QTL for either BP or [Ca2⫹]i. It is possible that a QTL for BP with modest effects became undetectable because the contribution of the single major QTL for BP was augmented by epistasis in our study.10 Brzustowicz et al31 have identified QTL for plateletactivating factor-evoked [Ca2⫹]i responses in Epstein-Barr virus–transformed lymphoblasts. A significant linkage was discovered in one family at D16S151 and a suggestive linkage at two distinct loci on chromosome 11 in another family. Because the rat– human comparative map indicates the QTL for [Ca2⫹]i in the rat platelets of our study, D3Mit13, Serca2, and DXMgh1, correspond to 20q, 12q, and Xp in human chromosomes, respectively,24 the QTL for [Ca2⫹]i in the rat platelets are different from those in their human lymphoblasts. In conclusion, QTL for [Ca2⫹]i are mapped in the vicinity of D3Mit13, Serca2, and DXMgh1 on chromosomes 3, 12, and X, respectively, in SHR. The Serca II gene is a prime candidate for increased [Ca2⫹]i in SHR. DXMgh1 has a potential to raise BP slightly through abnormal intracellular Ca2⫹ homeostasis. Genetically increased [Ca2⫹]i in platelets may contribute to the onset of cardiovascular disease independently from the major gene for hypertension. Establishing a congenic strain of this chromosomal region is required to confirm that the region is responsible for the abnormal intracellular Ca2⫹ regulation. Further studies are necessary to determine whether or not genetic variation in the Serca II gene, especially in the upstream regulatory region, is responsible for genetic difference in [Ca2⫹]i in platelets between SHR and F344.
References 1. 2. 3.
Ross R: Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115–126. McCarron DA: Calcium metabolism and hypertension. Kidney Int 1989;35:717–736. Erne P, Bolli P, Burgisser E, Buhler FR: Correlation of platelet calcium with blood pressure. Effect of antihypertensive therapy. N Engl J Med 1984;310:1084 –1088.
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Ohno Y, Suzuki H, Yamakawa H, Nakamura M, Otsuka K, Saruta T: Impaired insulin sensitivity in young, lean normotensive offspring of essential hypertensives: possible role of disturbed calcium metabolism. J Hypertens 1993;11:421– 426. Oshima T, Young EW, Bukoski RD, McCarron DA: Abnormal calcium handling by platelets of spontaneously hypertensive rats. Hypertension 1990;15:606 – 611. Oshima T, Young EW, McCarron DA: Abnormal platelet and lymphocyte calcium handling in prehypertensive rats. Hypertension 1991;18:111–115. Voetsch B, Loscalzo J: Genetic determinants of arterial thrombosis. Arterioscler Thromb Vasc Biol 2004;24:216 –229. Orlov SN, Li JM, Tremblay J, Hamet P: Genes of intracellular calcium metabolism and blood pressure control in primary hypertension. Semin Nephrol 1995;15:569 –592. Ohno Y, Matsuo K, Suzuki H, Tanase H, Takano T, Saruta T: Increased intracellular Ca2⫹ is not coinherited with an inferred major gene locus for hypertension (HT) in the spontaneously hypertensive rat. Am J Hypertens 1997;10:282–288. Ohno Y, Tanase H, Nabika T, Otsuka K, Sasaki T, Suzawa T, Morii T, Yamori Y, Saruta T: Selective genotyping with epistasis can be utilized for a major quantitative trait locus mapping in hypertension in rats. Genetics 2000;155:785–792. Ohno Y, Matsuo K, Suzuki H, Tanase H, Serikawa T, Takano T, Saruta T: Genetic linkage of the sarco(endo)plasmic reticulum Ca2⫹-dependent ATPase II gene to intracellular Ca2⫹ concentration in the spontaneously hypertensive rat. Biochem Biophys Res Commun 1996;227:789 –793. Levenson J, Devynck MA, Pithois MI, Le Quan Sang KH, Filitti V, Simon A: Dynamic association between artery shear flow condition and platelet cytosolic free Ca2⫹ concentration in human hypertension. Clin Sci Colch 1990;79:613– 618. Ohno Y, Matsuo K, Suzuki H, Tanase H, Ikeshima H, Takano T, Saruta T: Genotypes of sarco(endo)plasmic reticulum Ca2⫹-dependent ATPase II gene in substrains of spontaneously hypertensive rats. J Hypertens 1996;14:287–291. Yagisawa H, Tanase H, Nojima H: Phospholipase C-delta gene of the spontaneously hypertensive rat harbors point mutations causing amino acid substitutions in a catalytic domain. J Hypertens 1991; 9:997–1004. Lander E, Kruglyak L: Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 1995; 11:241–247. Burk SE, Lytton J, MacLennan DH, Shull GE: cDNA cloning, functional expression, and mRNA tissue distribution of a third organellar Ca2⫹ pump. J Biol Chem 1989;264:18561–18568. Levitsky DO, Clergue M, Lambert F, Souponitskaya MV, Jemtel TH, Lecarpentier Y, Lompre A-M: Sarcoplasmic reticulum calcium transport and Ca2⫹-ATPase gene expression in thoracic and abdominal aortas of normotensive and spontaneously hypertensive rats. J Biol Chem 1993;268:8325– 8331. Monteith GR, Kable EP, Kuo TH, Roufogalis BD: Elevated plasma membrane and sarcoplasmic reticulum Ca2⫹ pump mRNA levels in cultured aortic smooth muscle cells from spontaneously hypertensive rats. Biochem Biophys Res Commun 1997;230:344 –346. Bobe R, Bredoux R, Wuytack F, Quarck R, Kovacs T, Papp B, Corvazier E, Magnier C, Enouf J: The rat platelet 97-kDa Ca2⫹ATPase isoform is the sarcoendoplasmic reticulum Ca2⫹ATPase 3 protein. J Biol Chem 1994;269:1417–1424. Mountian I, Baba-Aissa F, Jonas JC, Humbert S, Wuytack F, Parys JB: Expression of Ca2⫹ transport genes in platelets and endothelial cells in hypertension. Hypertension 2001;37:135–141. Periasamy M, Reed TD, Liu LH, Ji Y, Loukianov E, Paul RJ, Nieman ML, Riddle T, Duffy JJ, Doetschman T, Lorenz JN, Shull GE: Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2⫹-ATPase isoform 2 (SERCA2) gene. J Biol Chem 1999;274:2556 –2562.
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22. Shull GE: Gene knockout studies of Ca2⫹-transporting ATPases. Eur J Biochem 2000;267:5284 –5290. 23. Baker DL, Hashimoto K, Grupp IL, Ji Y, Reed T, Loukianov E, Grupp G, Bhagwhat A, Hoit B, Walsh R, Marban E, Periasamy M: Targeted overexpression of the sarcoplasmic reticulum Ca2⫹-ATPase increases cardiac contractility in transgenic mouse hearts. Circ Res 1998;83:1205–1214. 24. Watanabe TK, Bihoreau MT, McCarthy LC, Kiguwa SL, Hishigaki H, Tsuji A, Browne J, Yamasaki Y, Mizoguchi-Miyakita A, Oga K, Ono T, Okuno S, Kanemoto N, Takahashi E, Tomita K, Hayashi H, Adachi M, Webber C, Davis M, Kiel S, Knights C, Smith A, Critcher R, Miller J, Thangarajah T, Day P, Hudson JR, Irie Y, Takagi T, Nakamura Y, Goodfellow PN, Lathrop GM, Tanigami A, James MR: A radiation hybrid map of the rat genome containing 5,255 markers. Nat Genet 1999;22:27–36. 25. Berk BC, Corson MA: Angiotensin II signal transduction in vascular smooth muscle: role of tyrosine kinases. Circ Res 1997;80:607– 616. 26. Drueke TB, Chabanis S, Roullet C, Duchambon P, Lacour B, McCarron DA: Abnormal regulation of intestinal calbindin
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(CaBP9k) and calmodulin in the spontaneously hypertensive rat. Am J Med Sci 1994;307(Suppl 1):S96 –S101. Roullet CM, Roullet JB, Duchambon P, Thomasset M, Lacour B, McCarron DA, Drueke T: Abnormal intestinal regulation of calbindin-D9K and calmodulin by dietary calcium in genetic hypertension. Am J Physiol 1991;261:F474 –F480. Falconer DS: Variance, in Falconer DS (eds): Introduction to Quantitative Genetics. Longman Scientific & Technical, Harlow, England 1989, pp 125–147. Williams PD, Puddey IB, Martin NG, Beilin LJ: Platelet cytosolic free calcium concentration, total plasma calcium concentration and blood pressure in human twins: a genetic analysis. Clin Sci Colch 1992;82:493–504. Vincent M, Samani NJ, Gauguier D, Thompson JR, Lathrop GM, Sassard J: A pharmacogenetic approach to blood pressure in Lyon hypertensive rats. A chromosome 2 locus influences the response to a calcium antagonist. J Clin Invest 1997;100:2000 –2006. Brzustowicz LM, Gardner JP, Hopp L, Jeanclos E, Ott J, Yang XY, Fekete Z, Aviv A: Linkage analysis using platelet-activating factor Ca2⫹ response in transformed lymphoblasts. Hypertension 1997;29: 158 –164.
2005; 18:666 – 671
Quantitative Trait Loci Mapping for Intracellular Calcium in Spontaneously Hypertensive Rats Yoichi Ohno, Hiromichi Suzuki, Hisao Tanase, Keiichi Otsuka, Takayuki Sasaki, Taichi Suzawa, Toshiyuki Morii, Yosuke Ando, Tatsuya Maruyama, and Takao Saruta Background: Increased intracellular calcium ([Ca2⫹]i) in platelets is also proposed as an intermediate phenotype for hypertension in spontaneously hypertensive rats (SHR). Increased [Ca2⫹]i in platelets is hypothesized to contribute to atherothrombotic events. Platelet hyperactivity is frequently associated with cardiovascular disease. Methods: In a genome scan, we performed the quantitative trait loci (QTL) mapping for [Ca2⫹]i in back-crossed rats derived from SHR and normotensive Fischer 344 rats, which demonstrated a single major QTL for hypertension on chromosome 1. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and in Ca2⫹-containing buffers was measured in platelets using the Fura-2 method. Results: Among the parental strains, systolic blood pressure and thrombin-stimulated [Ca2⫹]i were significantly greater in SHR than in Fischer 344 and F1 rats. The sarco(endo)plasmic reticulum Ca2⫹-dependent ATPase II gene locus (Serca2) between D12Mgh5 and D12Mgh6 showed the significant linkage for thrombin-stimulated
[Ca2⫹]i in Ca2⫹-free and Ca2⫹-containing buffers. The peak logarithm of the odds scores were 3.6 and 3.3, respectively. These QTL explained 19.8% and 17.4% of the total variances, respectively. D3Mit13 and DXMgh1 showed suggestive linkage for thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and in Ca2⫹-containing buffers, respectively. The peak logarithm of the odds scores were 2.6 and 2.1, respectively. Conclusions: A significant QTL for [Ca2⫹]i was mapped near Serca2 on chromosome 12, and suggestive QTL were identified near D3Mit13 and DXMgh1 in a genome scan. Genetic abnormalites in platelet [Ca2⫹]i may contribute to cardiovascular disease via platetet hyperactivity, independent of blood pressure elevation. Am J Hypertens 2005;18:666 – 671 © 2005 American Journal of Hypertension, Ltd. Key Words: Intracellular calcium, quantitative trait loci, spontaneously hypertensive rats, sarco(endo)plasmic reticulum calcium– dependent ATPase II gene, Sa gene.
latelet activation is essential in the development of preclinical atherosclerosis as well as in the clinical expression of atherothrombotic disease.1 Intracellular 2⫹ Ca ([Ca2⫹]i) is considered an essential second messenger in platelet activation. Moreover, abnormal [Ca2⫹]i homeostasis plays a key role in the pathogenesis of hypertension.2 [Ca2⫹]i in platelets is increased in patients with essential hypertension and in their offspring.3,4 Altered intracellular Ca2⫹ handling in platelets has also been reported in spontaneously hypertensive rats (SHR) and in young prehyperten-
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sive SHR.5,6 In the past decades, investigators have focused on the molecular genetics of arterial vascular disorders and have identified numerous polymorphisms in genes related to the cardiovascular system; however, the genetic contributions to augmented [Ca2⫹]i response in platelets remain highly controversial.7 Disturbed Ca2⫹ pump activity, abnormal function of Ca2⫹-binding proteins, and increased Na⫹–Ca2⫹ exchange activity reportedly increase [Ca2⫹]i.8 These studies suggest that increased [Ca2⫹]i in platelets could be a predictor of atherothrombotic events and a genetic hypertensive trait.
Received October 28, 2004. First decision December 2, 2004. Accepted December 22, 2004. From the Department of Internal Medicine (YO), Green Town Clinic Center, Saitama, Japan; Department of Internal Medicine (YO, KO, TS, TS, TM, TS), School of Medicine, Keio University, Tokyo, Japan; Department of Internal Medicine (HS), Saitama Medical College, Saitama, Japan; Medicinal Safety Research Laboratories (HT, YA), Sankyo Co. Ltd., Shizuoka, Japan; and Department of Internal Medicine
(TM), Sanno Hospital, Tokyo, Japan. This work was supported by a research grant from the National Dairy Promotion and Research Association, and by TEPCO Hospital Funds. Address correspondence and reprint requests to Dr. Yoichi Ohno, Department of Internal Medicine, Green Town Clinic Center, 2-23-3 Midoricho, Yashio City, Saitama, 340-0808, Japan; e-mail: [email protected]. ne.jp
0895-7061/05/$30.00 doi:10.1016/j.amjhyper.2004.12.001
© 2005 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
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We reported a unique back-cross analysis derived from SHR and normotensive Fischer 344 (F344) rats that approximates a polygenic model to a monogenetic form.9,10 Bimodality in blood pressure (BP) values of the backcrossed (BC) rats demonstrated an inferred single major gene locus or a major quantitative trait locus (QTL) for hypertension near the Sa gene locus (Sa).10 We measured [Ca2⫹]i in platelets to explore the “intermediate phenotype.” Increased [Ca2⫹]i in platelets was considered to be an inheritable hypertensive trait discriminated from the major QTL for BP on chromosome 1.9 We have already reported significant association between the sarco(endo)plasmic reticulum Ca2⫹-dependent ATPase (Serca) II gene locus (Serca2) and [Ca2⫹]i in platelets.11 In contrast, other studies have indicated that [Ca2⫹]i in platelets is also influenced by environmental factors such as shear stress and insulin resistance.4,12 It has not been clearly determined whether increased [Ca2⫹]i in platelets from hypertensive subjects or animals is a cellular marker of an underlying inherited abnormality or merely an epi-phenomenon secondary to endothelial dysfunction or an increase in BP. In this study, we attempted to map QTL for [Ca2⫹]i in platelets in BC rats derived from SHR and F344 rats by screening the total genome.
Methods Animals The SHR, Wistar-Kyoto (WKY) rats, and F344 rats were maintained at the Medicinal Safety Research Laboratories, Sankyo Co. Ltd. (Shizuoka, Japan). The F1 females from male SHR and female F344 parents were back-crossed with male SHR as previously described.10 The same BC population was used for a genome scan of hypertension by targeting on a potential epistasis for BP between a chromosome-1 QTL and the Serca2 locus on chromosome 12. Systolic BP was measured by the tail-cuff method at 10, 13, and 15 weeks of age; the mean values were used for statistical analysis. Data from 78 male BC rats underwent complete analysis. This experimental protocol was approved by the institutional animal care committee. Measurement of [Ca2ⴙ]i The [Ca2⫹]i in platelets was measured by the Fura-2 method as previously described.9,11,13 Thrombin-stimulated (0.3 U/mL) [Ca2⫹]i was measured in Ca2⫹-free buffer and in 1 mmol/L Ca2⫹-containing buffer. Genotyping of Microsatellite Markers and [Ca2ⴙ]i-Regulating Genes A total of 80 highly polymorphic microsatellite markers were selected to be spaced at 20-cM intervals, which spanned the entire genome as previously described.10 The BC rats were genotyped as homozygotes (ie, SHR) or heterozygotes (ie, F1) in autosomal chromosomes or as the
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identical genotype with SHR or F344 rats in X chromosome. Serca II gene was genotyped by restriction fragment length polymorphisms (RFLP) we previously discovered.11 The phospholipase C (PLC) ␦-1 gene was genotyped by RFLP using XhoI.14 Quantitative Linkage Analysis The QTL mapping was performed by using MAPMAKER/ Exp 3.0 and MAPMAKER/QTL 1.1b (Whitehead Institute, Cambridge, MA). Logarithms of the odds (LOD) scores of 1.9 and 3.3 were used as thresholds for suggestive and significant linkage, respectively.15 Statistical Analysis Data are reported as mean ⫾ SD. Comparisons were performed by analysis of variance (ANOVA) followed by the Scheffé F test to evaluate the differences in BP and [Ca2⫹]i between the genotyped groups. These statistical computations were performed with the SPSS statistical program (SPSS Inc., Chicago, IL). A level of P ⬍ .05 was considered to be statistically significant.
Results Among the parental strains (SHR, F1, and F344), the most genetically similar to SHR had a significantly higher systolic BP and greater thrombin-stimulated [Ca2⫹]i in platelets (Fig. 1). Selection of F344 rats as a contrasting strain was advantageous to map QTL for [Ca2⫹]i because thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer in platelets in SHR is ⬃5 SD higher than in F344 rats whereas it was ⬃3 SD higher than in WKY rats. Basal [Ca2⫹]i in platelets was not different among the parental strains. Serca2 was mapped between D12Mgh5 and D12Mgh6 on chromosome 12 and exhibited significant linkage to thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and in Ca2⫹containing buffers (Fig. 2). The peak LOD scores were 3.6 and 3.3, respectively. These QTL explained 19.8% and 17.4% of the total variances, respectively. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free and Ca2⫹-containing buffers was significantly higher in Serca II homozygotes than in Serca II heterozygotes (Table 1). However, their systolic BP levels were similar. D3Mit13 on chromosome 3 showed suggestive linkage for thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer (Fig. 3). The peak LOD score was 2.6, and this QTL explained 14.6% of the total variances. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer was significantly higher in D3Mit13 homozygotes than in D3Mit13 heterozygotes (Table 1), although their systolic BP levels were similar. DXMgh1 on chromosome X demonstrated suggestive linkage for thrombin-stimulated [Ca2⫹]i in Ca2⫹-containing buffer (Fig. 4). The peak LOD score was 2.1, and this QTL explained 11.3% of the total variances. Thrombinstimulated [Ca2⫹]i in Ca2⫹-containing buffer was significantly higher in BC rats with the SHR than those with the
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and suggestive QTL on rat chromosome 3 and x in BC rats derived from SHR and normotensive F344 rats. To our knowledge, this is the first reported observation pertaining to QTL mapping for [Ca2⫹]i in the platelets in hypertension. Serca II and Chromosome 12 Serca is thought to be a determinant factor that regulates [Ca2⫹]i stores in vascular smooth muscle cells.16 Serca II mRNA is reported to be overexpressed and the function of sarcoplasmic reticulum increased in purified microsomal fractions in vascular smooth muscle cells of SHR.17,18 Because we did not identify any amino acid substitution in the Serca II coding region of SHR compared with WKY and Sprague-Dawley rats,13 an increase in Serca II mRNA may contribute to the increased thrombin-stimulated [Ca2⫹]i. However, in platelets of SHR, compared with WKY, measurements of the level of expression of Serca IIb mRNA have yielded inconsistent results. Bobe et al measured an increase in Serca IIb mRNA up to 130%,19 whereas Mountian et al reported that it was significantly decreased to 60%.20 Platelet isolation and extraction of mRNA from platelets requires delicate techniques, which may explain the divergent results of their mRNA analysis. Recent genetic engineering experiments suggest that the level of Serca II mRNA expression may determine the
FIG. 1. Relationship between systolic blood pressure (BP) and thrombin-stimulated intracellular Ca2⫹ concentration ([Ca2⫹]i) in platelets of spontaneously hypertensive rats (SHR) (n ⫽ 9) (), normotensive Fischer 344 (F344) rats (n ⫽ 10) (●), and F1 rats (n ⫽ 5) (‘) derived from SHR, and F344 rats, and Wistar-Kyoto (WKY) rats (n ⫽ 6) (夝). Values are means ⫾ SD. (a) [Ca2⫹]i in Ca2⫹-free buffer. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-free buffer were significantly higher in SHR than in F344, F1, and WKY rats (ANOVA followed by Scheffé F test). SHR v F344, P ⬍ .0001, SHR v F1, P ⬍ .001, and SHR v WKY, P ⬍ .001. (b) [Ca2⫹]i in Ca2⫹-containing buffer. Thrombin-stimulated [Ca2⫹]i in Ca2⫹-containing buffer was significantly higher in SHR than in F344 and WKY rats (ANOVA followed by Scheffé F test). SHR v F344 and SHR v WKY, P ⬍ .0001.
F344 genotype at DXMgh1 (Table 1). Furthermore, systolic BP tended to be slightly higher in BC rats with the SHR genotype (P ⫽ .06). DXMgh1 has a potential to raise BP slightly through abnormal intracellular Ca2⫹ homeostasis. The PLC ␦-1 gene locus was mapped 37.1 cM apart from D8Mgh1 on chromosome 8. Neither the QTL for [Ca2⫹]i nor the QTL for BP was detected around the PLC ␦-1 gene locus.
Discussion The current study identified a significant QTL for thrombin-stimulated [Ca2⫹]i in platelets on rat chromosomes 12
FIG. 2. A quantitative trait locus (QTL) for thrombin-stimulated intracellular calcium (Ca2⫹) (a) in Ca2⫹-free buffer and (b) in Ca2⫹containing buffer on chromosome 12. Peak logarithms of odds (LOD) scores were 3.6 and 3.3, respectively. Dashed lines indicate significant linkage; dotted lines indicate suggestive linkage.
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Table 1. Thrombin-stimulated intracellular calcium (Ca2⫹) concentration ([Ca2⫹]i) according to the genotypes at D3Mit13, Serca2, and DXMghl Genotype Serca2 Systolic blood pressure (mm Hg) Thrombin-stimulated [Ca2⫹]i Ca2⫹-free buffer (nmol/L) Ca2⫹-containing buffer (nmol/L) D3Mit13 Systolic blood pressure (mm Hg) Thrombin-stimulated [Ca2⫹]i Ca2⫹-free buffer (nmol/L) Ca2⫹-containing buffer (nmol/L) Genotype DXMgh1 Systolic blood pressure (mm Hg) Thrombin-stimulated [Ca2⫹]i Ca2⫹-free buffer (nmol/L) Ca2⫹-containing buffer (nmol/L)
Homozygotes (SHR)
Heterozygotes (F1)
P Value
n ⫽ 43 162 ⫾ 8
n ⫽ 35 160 ⫾ 12
.42
263 ⫾ 34 570 ⫾ 74 n ⫽ 41 161 ⫾ 9
234 ⫾ 22 515 ⫾ 53 n ⫽ 37 161 ⫾ 11
⬍.0001 ⬍.0001
260 ⫾ 34 557 ⫾ 71
236 ⫾ 24 526 ⫾ 62
⬍.001 .04
SHR Type
.42
F344 Type
n ⫽ 37 163 ⫾ 9
n ⫽ 41 159 ⫾ 10
.06
257 ⫾ 36 566 ⫾ 75
244 ⫾ 28 526 ⫾ 60
.08 ⬍.01
The results of Serca2 have been published in Ref. 11.
intensity of Ca2⫹ signaling and Ca2⫹ contraction coupling in cardiovascular cells.21–23 However, it would be an oversimplification to consider Serca II itself as the only causal gene in the chromosomal segment, because we have not found any functionally significant variants between the Serca II alleles in SHR versus F344. Several genes related to [Ca2⫹]i homeostasis could consist of a [Ca2⫹]i-regulating gene complex near Serca2 and could synergistically contribute to the increased Ca2⫹ response to thrombin.
(322 ⫺ 202)/322, or approximately 0.6. Similarly, the ratio for BP is calculated by (102 ⫺ 52)/102, or approximately 0.75. The proportion of the heritable component of [Ca2⫹]i was smaller than that of BP. Williams et al reported that in a human twin study, additive genetic effects were smaller in [Ca2⫹]i in platelets than in systolic BP.29 Therefore, [Ca2⫹]i in platelets could be an inheritable cellular marker, although heritability of [Ca2⫹]i seems lower than that of BP.
Candidate Genes on Chromosome 3 and X
Vincent et al30 reported that a QTL for BP responses to administration of a dihydropyridine calcium antagonist was mapped between D2Wox8 and D2Mit15 on chromosome 2 in BC rats derived from a cross of the Lyon hypertensive and normotensive rats. In that chromosomal
The rat– human comparative map24 and the human genome project have provided information about several [Ca2⫹]i-regulating genes located in the vicinities of QTL for [Ca2⫹]i. The PLC ␥-1 gene in the vicinity of D3Mit13 on chromosome 3 is an important candidate, as it is directly related to the process of Ca2⫹ mobilization.25 The vitamin D– dependent Ca2⫹-binding protein (calbindinD9k) gene, which plays a key role in intestinal Ca2⫹ transport, is mapped near DXMgh1. Intestinal calbindinD9k expression was found to be decreased in SHR compared with WKY rats.26,27
QTL for BP and [Ca2ⴙ]i
Inheritance or Epi-Phenomenon? Whether increased [Ca2⫹]i in platelets is a cellular marker for underlying inherited abnormality is controversial. Genetic variance is calculated by subtracting environmental variance from phenotypic variance.28 The SD of thrombinstimulated [Ca2⫹]i in Ca2⫹-free buffer in each of the parental strains and BC rats were approximately 20 and 32 nmol/L, respectively. The ratio of the heritable variance to the total variance in BC rats for [Ca2⫹]i is calculated by
FIG. 3. Quantitative trait locus (QTL) for thrombin-stimulated intracellular calcium (Ca2⫹) in Ca2⫹-free buffer on chromosome 3. Peak logarithm of odds (LOD) score was 2.6. Dashed lines indicate significant linkage; dotted lines indicate suggestive linkage.
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4.
5.
6.
7. 8. FIG. 4. Quantitative trait locus (QTL) for thrombin-stimulated intracellular calcium (Ca2⫹) in Ca2⫹-free buffer on chromosome X. Peak logarithm of odds (LOD) score was 2.1. Dashed lines indicate significant linkage; dotted lines indicate suggestive linkage.
region, we could not detect any QTL for either BP or [Ca2⫹]i. It is possible that a QTL for BP with modest effects became undetectable because the contribution of the single major QTL for BP was augmented by epistasis in our study.10 Brzustowicz et al31 have identified QTL for plateletactivating factor-evoked [Ca2⫹]i responses in Epstein-Barr virus–transformed lymphoblasts. A significant linkage was discovered in one family at D16S151 and a suggestive linkage at two distinct loci on chromosome 11 in another family. Because the rat– human comparative map indicates the QTL for [Ca2⫹]i in the rat platelets of our study, D3Mit13, Serca2, and DXMgh1, correspond to 20q, 12q, and Xp in human chromosomes, respectively,24 the QTL for [Ca2⫹]i in the rat platelets are different from those in their human lymphoblasts. In conclusion, QTL for [Ca2⫹]i are mapped in the vicinity of D3Mit13, Serca2, and DXMgh1 on chromosomes 3, 12, and X, respectively, in SHR. The Serca II gene is a prime candidate for increased [Ca2⫹]i in SHR. DXMgh1 has a potential to raise BP slightly through abnormal intracellular Ca2⫹ homeostasis. Genetically increased [Ca2⫹]i in platelets may contribute to the onset of cardiovascular disease independently from the major gene for hypertension. Establishing a congenic strain of this chromosomal region is required to confirm that the region is responsible for the abnormal intracellular Ca2⫹ regulation. Further studies are necessary to determine whether or not genetic variation in the Serca II gene, especially in the upstream regulatory region, is responsible for genetic difference in [Ca2⫹]i in platelets between SHR and F344.
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