Download Restriction Fragment Length Polymorphism of hsp70

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
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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
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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
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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.
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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
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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.
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