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1 Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0636
2 Institute of Biology and Medical Genetics, Charles University, 12800 Prague, Czech Republic
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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hypertension; genetic; salt; SHR; telemetry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The present study was initiated to dissect out the locus in or around P9Ka and to begin fine mapping of specific variants responsible for increased arterial pressure. Four congenic strains were produced by introgression of various segments of chromosome 2 between WKY/lj and SHR/lj, both from our La Jolla colony. After confirming the presence of arterial pressure loci within the segments transferred using telemetry, the segments were further narrowed by constructing congenic substrains from the original reciprocal congenic strains. Since studies of Dahl R/S strains (11) indicated that chromosome 2 also contains salt-sensitive arterial pressure loci, we tested the interaction between high dietary salt intake (8%) and the introgressed chromosome 2 segments on arterial pressure. The results of these studies point to a cluster of arterial pressure alleles on chromosome 2 with opposing influences on arterial pressure and extensive sensitivity to dietary and environmental stressors.
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METHODS |
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Designation of congenic strains and substrains.
Four initial chromosome 2 congenic strains were produced in the present study and are designated as WKY/lj-SHR/lj-2a, WKY/lj-SHR/lj-2b, SHR/lj-WKY/lj-2c, and SHR/lj-WKY/lj-2d. In each of these strains, the first strain denotes the recipient, while the second refers to the donor; "2" refers to the chromosome targeted, and the lowercase letter identifies each congenic strain. These congenic strains will be referred to by the chromosome and lower case identifier, i.e., 2a, 2b, 2c, and 2d. A total of nine congenic substrains were then constructed from the original congenics, 2a and 2c. All congenic substrains derived from WKY/lj-SHR/ lj-2a are identified by an uppercase letter designation, 2a-A, 2a-B, 2a-C, 2a-D, 2a-E, and 2a-F, while substrains derived from SHR/lj-WKY/lj-2c are 2c-G, 2c-I, and 2c-J.
Congenic crosses.
The original congenics, 2a through 2d, were derived by the intercrossing of WKY/lj and SHR/lj to yield F1 hybrids which were then backcrossed to the desired recipient strain, WKY/lj or SHR/lj. Starting with the second backcross generation, 10–15 polymorphic markers spanning the segment being transferred were genotyped in the offspring and used to identify optimum male or female breeders. After nine successive generations of selective backcrossing, the donor’s genetic background was eliminated by over 99.9%. The differential chromosome 2 segment was then fixed and made homozygous by crossing appropriate male and female animals. Following marker-based selection, the homozygous congenic strains were maintained through brother sister mating. Animals phenotyped and reported in the present study were in the 5th to 7th generation of inbreeding
Congenic substrain crosses.
To obtain the congenic substrains following initial phenotyping of the original congenic strains, the WKY/lj-SHR/lj-2a (2a) congenic was backcrossed to WKY/lj, while the SHR/lj-WKY/lj-2c (2c) was backcrossed to SHR/lj, to yield rats heterozygous within the original introgressed chromosomal segments. These heterozygous (F1) rats were intercrossed, and progeny were marker-selected for a second round of breeding to generate animals with segmental homozygosity. The fractionated chromosomal segments were then fixed in the new congenic substrains through brother-sister mating. The congenic substrains phenotyped in the present study were in the 5th to 6th generation of inbreeding.
Genotyping.
Post-weaning pups (4–6 wk of age) were briefly anesthetized with halothane-oxygen, a 10-mm tip from the tail was surgically removed, the wound was cauterized, and the tissue was snap-frozen and stored at -70°C. Genomic DNA was isolated from tail snips by phenol-chloroform extraction and ethanol precipitation. Genotyping used the polymerase chain reaction (PCR) amplification of DNA microsatellites. Primers (Research Genetics, Huntsville, Al) were selected based on their map locations (http://rgd.mcw.edu/) and on their being polymorphic between parental strains. The PCR reaction volume of 25 µl contained 1.5 mM MgCl2, 50 µM of each dNTP (Boehringer Mannheim, Indianapolis, IN), 0.264 µM of each primer, 1 U Taq polymerase (Promega, Madison, WI), and 0.5–1.5 µg rat genomic DNA. PCR cycling consisted of an initial 94°C denaturation for 3 min followed by 34 cycles of 93°C (40 s), 55°C (40 s), and 72°C (90 s) using an Ericomp TwinBlock Cycler System (Ericomp, San Diego, CA). PCR products were analyzed on a 7% polyacrylamide gel at 25 mA constant current, and amplified DNA products were visualized with ethidium bromide. Gels were photographed, and each gel and photograph was scored independently by two readers.
Chromosome 2 mapping.
To determine the length of the differential chromosome 2 segments transferred, we typed the congenic strains using 40 microsatellite markers polymorphic between the SHR/lj and WKY/lj parental strains (Fig. 1). The map positions of the markers were determined utilizing the rat genome data available at http://rgd.mcw.edu. The congenic status of the background was confirmed using an additional 64 polymorphic microsatellite markers scattered throughout the genome and confirmed to be polymorphic between our SHR/lj and WKY/lj (Table 1).
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Data analysis.
Over a 24-h period, all traits (i.e., SP, DP, MAP, HR, and locomotor activity) exhibited circadian variation with peak values during the dark (active) period and minimal values during the light (resting) period. Since the onset and offset transitions would confound averaging of arterial pressure and HR values, data were selected for analysis from a 6-h window in the middle of the 12-h rhythms between the hours of 9:00 AM to 3:00 PM (resting) and 9:00 PM to 3:00 AM (active). From the 24 h data collected every day (288 data points/day), the 72 data points from each window were extracted and averaged for each rat for each day. The averaged light or dark period values were then taken as repeated measurements. Likewise, since changes between low- and high-NaCl diets caused a transient lag in cardiovascular responses, values obtained between the 4th and the 10th day of each diet was averaged. A repeated measure ANOVA and one-way ANOVA with a Duncan multiple comparison test was performed to compare the values obtained between and within groups and P < 0.05 was set in advance as the level of significance.
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RESULTS |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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When comparing 2a and 2b WKY/lj congenic strains harboring SHR/lj segments, the length of the transferred segment is larger but the magnitude of arterial pressure increase is smaller in the former than the latter. If one assumes that there is a single arterial pressure QTL in the introgressed part of the segment, then the congenic with the larger segment should have exhibited a similar arterial pressure increase to that with the smaller; however, this is not our finding. Deng et al. (3) reported a similar finding in two congenic strains with introgressed chromosome 2 segments from the WKY or MNS onto the Dahl-S background. They found that arterial pressures were decreased (44 and 29 mmHg, respectively), compared with the Dahl-S, although the region introgressed from the MNS congenic was a larger chromosomal segment than that of the WKY. They hypothesized that this difference could have arisen 1) if the QTL allele of the WKY rat was different from that of the MNS rat, 2) if the WKY and MNS rats have the same QTL alleles in the D2Mgh12 region but the larger substitution in the MNS congenic strain also contained the D2Mit6 locus, which modified the arterial pressure effects of the former QTL, or 3) if there are additional arterial pressure QTL located in this region of chromosome. In a related study by Jeffs et al. (13) a similar finding and explanation was advanced.
These disparate findings lead to the second major conclusion of our study, namely, the presence of multiple and contrasting arterial pressure loci within these segments of rat chromosome 2. Based on the results of this study, we would propose that arterial pressure effects observed in any chromosome 2 congenic strain derive from the composite effects of multiple and separate QTL. Our results also argue for the presence of arterial pressure lowering QTL whose contributions may be evident or masked, depending on the segments transferred and the genetic background. For example, the smaller arterial pressure increase observed in the 2a congenic, relative to 2b, could be explained by the presence of a depressor QTL in the region not shared by 2b, and a pressor QTL in the region common to both 2a and 2b. Therefore, the resultant effect of in the 2a congenic would be a smaller blood pressure (BP) increase compared with the 2b where the pressor QTL action is unopposed. The findings from the substrains derived from congenic 2a, discussed next, also support this hypothesis.
Congenic substrain 2a-C, with a fractionated part of the original congenic 2a, exhibited a very small change in arterial pressure compared with 2a. Since 2a-C differs from 2a largely in lacking the transferred SHR/lj segment between markers D2Rat21 and D2Rat161, this result would support a hypothesized depressor "dBP QTL" between markers D2Rat161 and D2Mgh10, a region shared by 2a and 2a-C and excluded from 2b. The hypotensive effect of dBP QTL appears to completely counterbalance a pressor QTL, here designated as "pBP2 QTL," and represented in 2b, since substrain 2a-C did not exhibit arterial pressure change. This raises the possibility of a third pressor QTL, designated as "pBP1 QTL," located between markers D2Rat21 and D2Rat161 and responsible for the increased arterial pressure exhibited by 2a. In support of this thesis, congenic substrain 2a-B, which has the introgressed segment between D2Rat21 and D2Rat27, exhibited a relatively similar increase in SP to that observed in 2a, particularly during nighttime, thereby further narrowing the location of pBP1 QTL to this 8 cM region. Thus our results from the WKY/lj congenic strains and substrains study, would suggest at least three arterial pressure-related QTL in the transferred SHR/lj chromosome 2 segment; two pressor QTL, pBP1 QTL, positioned between 
30 to 40 cM on the rat map, and pBP2 QTL, positioned 80 to 100 cM [D2Mgh10 and D2Rat62]; and one depressor QTL, dBP QTL, tentatively positioned at 45 to 80 cM; however, this segment length will be narrowed when the SHR/lj congenics are discussed below.
Our findings are in agreement with studies which have reported arterial pressure-related QTL in the vicinity of pBP1 QTL. For example, in an F2 intercross between Lyon hypertensive and normotensive strains, a significant linkage was established between PP and the carboxypeptidase B gene (CPB) positioned between 30 to 40 cM on the rat map (6). In addition, from an F2 cross between SHRSP and WKY, a basal BP QTL was reported with its peak close to D2Mit6, 9.8 cM from the CPB gene (2). We would conclude that the physiological effect of the SHR/lj pBP1 QTL, when transferred onto the WKY/lj genome, is to increase basal and high-salt SP, DP, and PP by an average of 8, 4, and 4 mmHg, respectively.
Evidence supporting the presence of arterial pressure-related QTL in the vicinity of pBP2 QTL come from linkage studies in F2 progeny from WKY x S, MNS x S, and SHRSP x WKY in an area bracketed between 80 and 100 cM on the rat map (2, 4). Additional evidence comes from the backcross population derived from a Lyon hypertensive and Lyon normotensive cross, where a QTL was proposed (positioned between 72 and 101 cM, Fig. 1) that influences the systolic and diastolic arterial pressure responses to administration of a dihydropyridine calcium antagonist, PY108-068 (22). Thus we would conclude that the physiological effect of the SHR/lj pBP2 QTL, when transferred onto the WKY/lj, is to increase baseline and salt loaded SP, DP, and PP by an average of 18, 12, and 6 mmHg, respectively.
The finding of putative hypotensive loci in hypertensive strains is not unreasonable given the random segregation of genes in their derivation. Further, Garrett et al. (8) analyzed F2 progeny from a cross between Dahl-S and Lewis rats and reported an arterial pressure QTL on chromosome 2 with the pressor allele for arterial pressure coming from the Lewis normotensive strain. Our results from the WKY/lj congenics is that the physiological effect of the SHR/lj dBP QTL, when transferred onto the WKY genome, is to lower SP and DP by -14 and - 11 mmHg, respectively, as calculated from the differences between 2b congenic strain and 2a-C substrain. It would be interesting if this allele were lacking in the SHRSP, thereby potentially contributing to the greater arterial pressure in this strain.
The third major finding of the present study comes from the reciprocal congenics where WKY/lj segments were transferred onto the SHR/lj genome. The two congenic substrains, 2c-I and 2c-J, which contain only part of the original introgressed segment of the 2c congenic, exhibit a similar magnitude of arterial pressure decrease as the 2c implying a major QTL is located within the overlap region of 2a, 2c-I, and 2c-J (Fig. 1). The third congenic substrain, 2c-G, which did not exhibit a decrease in arterial pressure but shares segments in common with 2c-I and 2c-J, narrows the location of the QTL to a segment of 16 cM. between markers D2Rat161 and D2Mit8 and positioned 46.6 to 62.5 cM (Fig. 1). We would tentatively designate this site "pBP3 QTL" and propose it lies within markers D2Rat161 and D2Mit8 (Fig. 1). Deng et al. (3) reported the existence of an arterial pressure QTL on chromosome 2 in the region corresponding to that of pBP3 QTL region based on two congenic strains introgressing WKY or MNS segments onto the Dahl-S. This QTL region was further narrowed by constructing congenic substrains from the original MNS congenic and localized to a region bracketed by gene markers NEP (neutral endopeptidase) and GCA (guanylyl cyclase A/atrial natriuretic peptide receptor), which corresponds to 58.3 to 70 cM on the map (7). This is also in proximity to our originally estimated marker, P9Ka (14). Recently, Pravenec et al. (15) reported on a congenic derived by transferring a segment of BN chromosome 2 onto the SHR between D2Rat171 and D2Arb24 (50 to 85 cM position). They found the BN alleles significantly lowered systolic and diastolic pressures and ameliorated cardiac hypertrophy. Jeffs et al. (13) transferred a segment of WKY chromosome 2, containing a region corresponding to the pBP3 QTL, onto the SHRSP genetic background and reported lowered baseline and salt-loaded systolic arterial pressure in male congenics, compared with SHRSP. Our results would suggest that the physiological effect of the WKY/lj pBP3 QTL, when placed onto the SHR/lj genome, lowers baseline and salt loaded SP, DP, and PP by an average of -12, -7, and -8 mmHg, respectively.
The SHR/lj congenic strains and substrains permit a further localization of the proposed "dBP QTL." Specifically, inspection of Fig. 1 shows that the arterial pressure lowering effects of transferring the WKY/lj segments in 2c, 2c-I, and 2c-J are equivalent and maximal. However, these segments also overlap that within which we have attributed an arterial pressure lowering effect to an SHR allele. Although one explanation could be that expression of the dBP QTL is affected by genetic background interactions, a second explanation, consistent with our data, is that dBP QTL lies within the short segment transferred into 2d. Transfer of this segment has, however, selective effects on arterial pressure; namely, it lowers diastolic but not SP, since its replacement with a WKY/lj allele in the 2d congenic strain increased DP. If dBP QTL were located within or adjacent to this segment of 2d, then it would also explain the large diastolic pressure elevation observed in 2b, since the effect of the proposed SHR/lj pBP2 QTL would be unopposed. It is hypothesized that the SHR arterial pressure lowering QTL lies within this short segment on chromosome 2.
The fourth finding of our study is that an 8% high-salt diet induced greater increases in arterial pressure in SHR/lj parental, congenic strains and substrains than in WKY/lj parental, congenic strains and substrains. Furthermore, most strains exhibited salt-induced bradycardia, the magnitude of which was generally greater in WKY/lj congenic and parental strains than in SHR/lj congenic and parental strains. Congenic strain 2d, with the smallest introgressed WKY/lj chromosome 2 segment lacked the ability to decrease HR during high salt, whereas substrain 2c-I, which has a region between markers D2Rat40 and D2Mgh10 in common with the 2d congenic, roughly 6.9 cM, also exhibited a significantly less salt-induced HR decrease, particularly during the active (night) period. This finding may identify a heart rate locus, tentatively designated as "HR1 QTL," which exerts a complex effect potentially limited through interaction with other genes, as its effect appears to depend, in part, on the simultaneous presence of an SHR/lj allele between markers D2Mgh10 and D2Rat63, since the reciprocal congenic strain 2b also lacked the ability to decrease HR during high salt. We would conclude that a WKY/lj allele (within D2Rat40-D2Rat50) along with an SHR/lj allele (within D2Mgh10-D2Rat63) is required for the phenotypic expression of the HR1 QTL effect. Recently, Jaworski et al. (12) reported finding a chromosome 2 QTL for the air-puff stimulus-induced orienting response bradycardia centered around marker D2Rat61/62 (positioned at 96.7 cM, Fig. 1) which may provide support for the presence of heart rate loci in this region.
Finally, our studies provide insight into relationships between reversibility of arterial pressure to transient increases in dietary NaCl. In most studies utilizing the SHR, 8% NaCl is placed in the chow with water ad libitum; however, with SHRSP this salt concentration is too high, as it leads to significant morbidity and mortality (1), and 1–2% NaCl is generally placed in the drinking water (13). In the current study, the use of 8% NaCl diet led to evidence of altered systemic hemodynamics as reflected in irreversibility of arterial pressures and/or altered time-dependent rates of change in WKY-substituted congenic strains and substrains. We have no explanation for this phenomenon. In the use of 8% NaCl diets, we have never observed mortality in SHR strains. Additionally, the duration of the 8% NaCl diets were relatively short (16 to 21 days). Griffin et al. (10) recently reported on a comparison between SHR and SHRSP and failed to document significant renal damage in the SHR resulting from the 8% NaCl diet; however, they did show significant effects on the SHRSP. We cannot attribute our observations as indicating alleles on chromosome 2 which determine renal damage, especially since no chromosome 2 QTL have been reported. However, the loss of reversibility in the WKY strains with introgressed SHR segments raises questions on studies seeking the genetic bases for hypertension related renal damage. Further salt studies are needed on all congenic substrains.
In conclusion, this study confirms the presence of multiple arterial pressure QTL on rat chromosome 2. Utilizing reciprocal congenic strains and substrains, we identified and narrowed the location and physiological effects of these loci. Our results are interpreted to indicate the likely identification of three pressor QTL and one putative heart rate locus in four reciprocal congenic strains and nine substrains. We also present evidence for a depressor QTL in SHR/lj. The finding of a depressor allele in the SHR rat might explain, in part, discrepancies in the literature about the magnitude of arterial pressure effects of various reported QTL. The physiological effects of the loci appear to reflect the influence of gene and gene-product interactions and, in particular, on effects of the genetic background. Based on our findings, we tentatively assigned identifications to these four arterial pressure and one heart rate QTL to facilitate comparisons between laboratories and crosses, and across different genetic maps. Chromosome 2 appears from our other studies to be a chromosome rich in genes that determine cardiovascular and behavioral traits, stress responses, and the coordinated coupling between stress and the cardiovascular system. The finding of multiple arterial pressure QTL on this chromosome implies that identifying specific genes will ultimately necessitate sequencing through the genomes of multiple congenic substrains, all of which have been accurately phenotyped for cardiovascular parameters, including dietary NaCl manipulations. These new chromosome 2 congenic strains and substrains can be used to define loci more precisely and to correlate genetics with other traits, including neurohumoral indices, and we offer them to other laboratories interested in pursuing these goals.
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ACKNOWLEDGMENTS |
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These studies were supported by National Heart, Lung, and Blood Institute Grants HL-35018 (to M. P. Printz) and HL-07444 (to A. Alemayehu).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. P. Printz, Univ. of California, San Diego Dept. of Pharmacology 0636, Basic Sciences Bldg., Rm. 3084, 9500 Gilman Drive, La Jolla, CA 92093-0636 (E-mail: mprintz{at}ucsd.edu).
10.1152/physiolgenomics.00065.2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1 Na,K-ATPase gene is a susceptibility gene in the Dahl salt-sensitive HSD rat. J Clin Invest 102: 1102–1111, 1998.[ISI][Medline]
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HR) (A and D), systolic pressure (
P < 0.05 vs. corresponding light period. Right: 
P < 0.05 vs. corresponding WKY/lj; 
P < 0.05 vs. corresponding light period.