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611 Restriction Fragment Length Polymorphism of hsp70 Gene, Localized in the RT1 Complex, Is Associated With Hypertension in Spontaneously Hypertensive Rats Pavel Hamet, Dewen Kong, Michal Pravenec, Jaroslav Kunes, Vladimir Kren, Pavel Klir, Yu-Lin Sun, and Johanne Tremblay Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017 Previous studies from our laboratory have demonstrated that the intermediate phenotype of thermosensitivity is present in hypertensive mice and rats. Increased expression of hsp70 caused by increased transcription rate was demonstrated in vivo, in organs, and in cultured cells from spontaneously hypertensive rats and hypertensive mice. In this study, a polymorphism of this gene was revealed with BamHl enzyme by using a human hsp70 probe. A 4.4-kb fragment was visualized in normotensive rats (Brown-Norway BN.Ix and Sprague-Dawley), and a 3.0-kb fragment was found in spontaneously hypertensive rats (SHR) of three different origins and in Wistar and Buffalo rats. Both fragments were present in the Wistar-Kyoto rat strain. The present study mapped the polymorphism of hsp70 into the RT1 complex in BN.1K and SHR.1N congenic strains. The hsp70 restriction fragment length polymorphism is associated with a blood pressure difference of IS mm Hg in recombinant inbred strains. These results justify the search for a mechanism by which hsp70 could influence blood pressure. (Hypertension 1992;19:611-614) • restriction fragment length polymorphisms spontaneously hypertensive rats KEYWORDS H eightened thermosensitivity potentially constitutes a distinct intermediate phenotype of hypertension. Increased environmental heat susceptibility has been demonstrated in hypertensive rats and mice.1-6 Our prior studies have suggested from observation of F2 and backcross mice populations that this abnormal thermosensitivity is determined by a specific locus cosegregating with blood pressure (Tms).1 Environmental temperature has an impact on the expression of hypertension in animals and humans.89 Data from our laboratory suggest that increased thermosensitivity persists even in vitro, i.e., in cultured cells from vascular smooth muscle and cardiomyocytes, and may be genetically determined. The best candidate genes for this thermosensitivity are heat stress genes. This group of genes, classified by the molecular weight of their From the Centre de Recherche Hotel-Dieu de Montreal (P.H., D.K., M.P., J.K., Y.-L.S., J.T.), Universite de Montreal, Montreal, Canada, the Faculty of Medicine (V.K.), Charles University of Prague, and the Institute of Physiology (P.K.), Czechoslovak Academy of Sciences, Prague, Czechoslovakia. Supported by grants from the Medical Research Council of Canada and the Dairy Bureau of Canada. J.T. is a senior scholar from Fonds de la Recherche en Sante du Quebec, and D.K. is a recipient of a Canadian International Development Agency fellowship. J.K. and M.P. are visiting professors from the Czechoslovak Academy of Sciences and are supported by Servier Canada. Address for reprints: Dr. Pavel Hamet, Laboratory of Molecular Pathophysiology, Centre de Recherche Hotel-Dieu de Montreal, Pavilion Marie-de-Ia-Ferre, 3850 rue Saint-Urbain, Montreal, Quebec, Canada H2W 1T8. heat-shock proteins • DNA, recombinant protein products, is expressed in a variety of cells of all organisms after application of stimuli, including heat, mechanical stress, a-adrenergic agonists, and calcium ionophore. A major representative of this group of genes is hsp70 (for review, see Reference 10). Using mouse hsp68 and human hsp70 probes from the hsp70 family of genes, we have established that there is an increased transcription rate as well as messenger RNA (mRNA) accumulation of a major representative of hsp70.u'12 However, preliminary studies indicate that the mRNA accumulation is only transient and that the protein product becomes actually even less abundant with time in hypertensive animals after stress exposure of whole animals, isolated organs, and cultured cells. n.13-15 At least two copies of hsp70 were localized in the major histocompatibility complex of humans,16 mice,17 and rats.18 The area in which hsp70 is localized is of interest since it contains other genes potentially relevant to environmental interactions, such as 21-hydroxylase (21-OH) and TNFa. To evaluate whether hsp70, which is increasingly expressed in hypertensive cells, segregates with blood pressure, we have searched for polymorphisms in various hypertensive and normotensive rat strains and studied segregation with hypertension in recombinant inbred (RI) and congenic strains. Methods Animals Spontaneously hypertensive rats (SHR) of three different origins were used in the present study. Two of 612 Hypertension Vol 19, No 6, Part 2 June 1992 RE: Bam HI PROBE: Human 11ST 70 SHHp sum SURc WKY .4 Kb 3.0 Kb Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017 them were purchased from commercial North American sources (Taconic Farms, Inc., Germantown, N.Y., and Charles River, Wilmington, Mass.), and the third was obtained from the Czechoslovak Academy of Sciences, Prague (but originated from OLAC, England). The normotensive animals were Brown-Norway BN.lx progenitors (hereafter referred to as BN) of RI strains and Wistar-Kyoto (WKY) rats from Taconic Farms. The RI strains were partly from the Czechoslovak Academy of Sciences and partly from the Biology Department, Faculty of Medicine, Charles University, as previously reported," representing a set of 30 fully inbred homozygous strains. The congenic strains were BN.lx.lK with the RTl k haplotype on a normotensive background and SHR.1N with the RT1" haplotype on a hypertensive background.19 Normotensive strains of Wistar, Buffalo, and Sprague-Dawley rats were also used. DNA Preparation, Electrophoresis, and Southern Hybridization High molecular weight genomic DNA was processed from adult livers of different rat strains as described by Sambrook et al.20 After restriction enzyme digestion, DNA was electrophoresed on 0.8% agarose gel for 42 hours at 25 V and then transferred onto a Nylon N+ membrane (Amersham Canada Ltd., Oakville, Canada) by using Vacugen (Pharmacia-LKB, Baie D'Urfe, Canada). The hybridization process has been described previously.21 Autoradiography was performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). DNA Probes To prepare human hsp70 probes, Hindlll-Sph I insert (2.78 kb) and 5'-Pst I fragment (1.2 kb) in PHHSP70 plasmid, kindly donated by Dr. R.I. Morimoto, were processed by elution from agarose gel after restriction enzyme digestion and electrophoresis.21 These DNA fragments were labeled with [a-32P]dCTP and DNA polymerase (Klenow fragment) by random-primer extension.22 Mouse /wp6S-containing plasmid was provided by Dr. L.A. Moran.23 The probe was prepared as described elsewhere.11 Results Digestion by several restriction enzymes, such as Pst I, Xba I, Xho I, and Sac I, did not reveal any polymorphism of hsp70. Only BamHl digestion allowed the visualization of a clear polymorphism between nor- FIGURE 1. Autoradiogram showing restriction fragment length polymorphism between normotensive Wistar-Kyoto (WKY), Brown-Norway (BN), and spontaneously hypertensive rat (SHR) strains. Strains of several origins were used in this experiment, SHRp from Prague, SHRt from Taconic Farms, and SHRcfrom Charles River Laboratories, as described in "Methods." Genomic DNA was digested with Bam/// restriction enzyme (RE) and hybridized with human hsp70 probe. motensive and hypertensive animals. This enzyme also demonstrated 15-kb and 8-kb fragments identical in all animals, but a distinct 4.4-kb fragment was present only in BN and WKY strains. A shorter fragment of 3 kb, although also evident in WKY rats, was seen in all SHR strains of Prague, Taconic, and Charles River origin. Noticeably, in WKY rats, both bands were visible but the intensity of hybridization was less as compared with that of BN and SHR for both fragments. An example of this polymorphism is illustrated in Figure 1. Both human hsp70 probes (2.78 kb and 1.2 kb) and the mouse hsp68 probe gave the same results. These data demonstrate the homogeneity of this gene polymorphism in hypertensive strains of various origins and the presence of larger fragments in both normotensive strains. Additional experiments with other normotensive strains have revealed the presence of the 3.0-kb fragment in Buffalo and Wistar rats, and the 4.4-kb fragment was found in BN and Sprague-Dawley rats. The different hsp70 alleles characterized by hsp70 polymorphism are summarized in Table 1. Use of the RT1 congenic strains SHR.1N and BN.1K enabled us to map this polymorphism to the rat major histocompatibility complex (RT1) on chromosome 20 where the RT1 complex is now assigned to linkage group IX.24 The mean blood pressure of the Prague SHR strain was 181 ±4 mm Hg, while that of the SHR.1N congenic strain was significantly (p<0.001) lower (156 ±4 mm Hg). It is evident from Figure 2 that the congenic strains with an RT1" haplotype, whether on a hypertensive (SHR.1N) or normotensive (BN.lx) TABLE 1. Alleles Characterized by hsp70 Polymorphism hsp70 alleles Rat strain 3.0 kb 4.4 kb Wistar SHR (Prague) SHR (Charles River) SHR (Taconic) BUF BN Sprague-Dawley WKY SHR, spontaneously hypertensive rats; BUF, Buffalo normotensive rats; BN, Brown-Norway normotensive rats; WKY, WistarKyoto rats. Harriet et al Restriction Fragment Length Polymorphism of hsp70 RE: Baa HI PROBE: Human HSP 70 SHRp SHR A. 4Kb 3.0 Kb Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017 background, possess the 4.4-kb fragment, while those with the RTl k haplotype on SHR or normotensive backgrounds (BN.1K) have the 3.0-kb fragment. This was confirmed in RI strains. Figure 3 illustrates a complete association between the RTl k haplotype and the 3.0-kb fragment, and the 4.4-kb fragment was observed in all RI strains with the RT1" haplotype, demonstrating the absence of intraRT1 recombination in these strains. Mean blood pressure in the RI strains with the 4.4-kb fragment was 128±3 mm Hg, and in the RI strains with the 3.0-kb fragment it was 143±6 mm Hg. This difference of 15 mm Hg was statistically significant (p<0.02) and corresponded with data reported previously for RTl k and RTl n haplotypes.19 Discussion A polymorphism of hsp70 was demonstrated between normotensive and hypertensive rats. Although hsp70 constitutes a family of genes, the probe used in the present studies revealed that a major representative of this family maps to the RT1 complex. This is unequivocally demonstrated by our results in RI and congenic strains of BN and SHR progenitors and is concordant with the findings in other strains by Wurst and others. 182526 These data underline the relevance of the rat RT1 complex as one of the components determining the expression of hypertension and are compatible with the 613 FIGURE 2. Autoradiogram showing restriction fragment length polymorphism in spontaneously hypertensive rat (SHR) and Brown-Norway (BN) strains as progenitors and in congenic strains with the RT1" haplotype on an SHR background (SHR. IN) and the RTlk haplotype on a BN background (BN.1K). Genomic DNA was digested with Bam/// restriction enzyme (RE) and hybridized with human hsp70 probe, as described in "Methods." possibility that the genomic abnormality is in the area where hsp70 is located. It is clear that although the 3.0-kb allele, localized in the RTl k haplotype, is present in all SHR tested, it also occurs in the Wistar and Buffalo strains. On the other hand, it should be borne in mind that the RT1 complex contributes by only 15 mm Hg to the expression of hypertension. Our current hypothesis is that the locus for environmental susceptibility lies within the RT1 complex potentially associated with hsp70. Heat susceptibility and hsp70 expression will have to be tested in other strains possessing the 3.0-kb fragment. Interestingly, it has recently been demonstrated that a strain with susceptibility to stress may be selected from the Wistar background.27 In conclusion, the polymorphism of the hsp70 gene, a member of the hsp70 family that has previously been found to be excessively expressed in hypertension, maps to the RT1 complex and is thus associated with a 15 mm Hg increment of blood pressure. Acknowledgments We thank Dr. Laurence A. Moran of the University of Toronto and Dr. Richard I. Morimoto of Northwestern University in Evanston, 111., for providing the Mmshsp68 (pMHS 243) and human hsp70 probes, Regis Tremblay and Carole Long for their technical assistance throughout these investigaSHR FIGURE 3. Systolic RT1 0 n 1 k 200 180 160 BN n.I Oiastolic 140 120 100 80 RFLP hsp70 (Bam HI) 4.4 Kb 3.0 Kb M.B.P. ± SEM 128 ± 3 143 ± 6 Blood pressure and restriction fragment length polymorphism (RFLP) of hsp70 in recombinant inbred strains. The upper part of the figure shows a graph of systolic and diastolic blood pressure in all recombinant inbred strains. Shaded columns indicate strains with the RTF haplotype, and dark columns represent the RTlk haplotype. The lower part of the figure illustrates the RFLP with Bam///, revealing the 4.4-kb or 3.0-kb fragments in available individual strains. M.B.P., mean of the mean blood pressure; BN, Brown-Norway normotensive rats; SHR, spontaneously hypertensive rats. 614 Hypertension Vol 19, No 6, Part 2 June 1992 tions, and Louise Chevrefils for preparing and Ovid Da Silva for editing this manuscript. References Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017 1. Schlager G: Selection for blood pressure levels in mice. Genetics 1974;76:537-549 2. McMurthy JP, Wexler BC: Hypersensitivity of spontaneously hypertensive rats (SHR) to heat, ether, and immobilization. Endocrinology 1981;108:1730-1736 3. McMurthy JP, Wexler BC: Hypersensitivity of spontaneously hypertensive rats to heat and ether before the onset of high blood pressure. Endocrinology 1983;112:166-171 4. Wright G, lams S, Knecht E: Resistance to heat stress in the spontaneously hypertensive rat. Can J Physiol Pharmacol 1977;55:975-982 5. Wright G, Knecht E, Toraason M: Cardiovascular effects of whole-body heating in spontaneously hypertensive rats. 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Hashimoto T, Mosser RD, Tremblay J, Hamet P: Increased accumulation of hsp70 messenger RNA due to enhanced activation of heat-shock transcription factor in spontaneously hypertensive rats. J Hypertens 1991;9(suppl 6):S170-S171 13. Hamet P, Malo D, Hashimoto T, Tremblay J: Heat stress genes in hypertension. J Hypertens 1990;8(suppl 7):S47-S52 14. Hamet P, Tremblay J, Malo D, Kunes J, Hashimoto T: Genetic hypertension is characterized by the abnormal expression of a gene localized in MHC, HSP70. Transplant Proc 1990;22:2566-2567 15. Tremblay J, Hadrava V, Kruppa U, Hamet P: Enhanced growthdependent expression of TGF/31 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats. Can J Physiol Pharmacol (in press) 16. Sargent CA, Dunham I, Campbell RD: Identification of multiple HTF-island associated genes in the human major histocompatibility complex class III region. EMBO J 1989;8:2305-2312 17. Gaskin HR, Prochazka M, Nadeau JH, Henson VW, Leiter EH: Localization of a mouse heat shock hsp70 gene within the H-2 complex. Immunogenetics 1990;32:286-289 18. Wurst W, Benesch C, Drabent B, Rothermel E, Benecke BB, Gunther E: Localization of heat shock protein 70 genes inside the rat major histocompatibility complex close to class III genes. Immunogenetics 1989;30:46-49 19. Pravenec M, Klir P, Kren V, Zicha J, Kunes J: An analysis of spontaneous hypertension in spontaneously hypertensive rats by means of new recombinant inbred strains. J Hypertens 1989;7: 217-222 20. Sambrook J, Fritsch EF, Maniatis T: Isolation of high-molecular. weight DNA from mammalian cells, in Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989, pp 9-14 21. Sun YL, Xu YZ, Bellard M, Chambon P: Digestion of the chicken /S-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal assay characterized by an atypical ladder of DNA fragments. EMBO J 1986;5:293-300 22. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13 23. Perry MD, Moran LA: Isolation of a mouse heat-shock gene (hsp68) by recombinational screening. Gene 1987;51:227-236 24. Hedrich HJ: Linkage map of the laboratory rat (Rattus norvegicus). Rat Newsletter July 1991;No. 25:19-22 25. Lambracht D, Hanisch L, Wurst W, Gunther E, Wonigeit K: Restriction fragment-length polymorphism analysis of a second laboratory-derived mutant RT1 haplotype. Transplant Proc 1990;22:2512-2513 26. Rothermel E, Wurst W, Heine L, Gunther E: Analysis of a nonclassical class I gene of the rat major histocompatibility complex, (abstract) Transplant Proc 1990;22:2509 27. Markel AL, Amstylavski SY, Naumenko EV: The central adrenergic mechanisms of blood-pressure regulation in rats with inherited arterial hypertension. Biogenic Amines 1987;4:329-338 Restriction fragment length polymorphism of hsp70 gene, localized in the RT1 complex, is associated with hypertension in spontaneously hypertensive rats. P Hamet, D Kong, M Pravenec, J Kunes, V Kren, P Klir, Y L Sun and J Tremblay Downloaded from http://hyper.ahajournals.org/ by guest on May 7, 2017 Hypertension. 1992;19:611-614 doi: 10.1161/01.HYP.19.6.611.a Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. 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