Genetic Determination of Cardiac Mass in Normotensive Rats Results From an F3443WKY Cross Abdelkrim Sebkhi, Lan Zhao, Long Lu, Christopher S. Haley, Derek J.R. Nunez, Martin R. Wilkins
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Abstract—Genetic determinants affect adult cardiac mass and the predisposition to develop cardiac hypertrophy. The aim of this study was to identify quantitative trait loci (QTL) that control heart and left ventricular (LV) weight by use of normotensive inbred rat strains that differ in their adult cardiac mass phenotype. We studied 126 male F2 rats derived from a cross of normotensive Wistar-Kyoto and Fischer 344 rats. At 12 weeks of age, total heart weight and LV weight were measured. Genomic DNA from these animals was screened by use of polymorphic microsatellite markers across the whole genome (excluding the sex chromosomes). In this cross, the genetic contribution to total heart weight variation was 56%, and the genetic contribution for LV weight was 55%. Using the Mapmaker/QTL computer package, we identified a significant QTL on chromosome 3 with a log10 likelihood (LOD) score of 4.8, which accounted for 16.5% of the total variance of LV weight. This QTL was centered close to the marker D3Rat29. The QTL was also found to be significantly linked with total heart weight (LOD54.4). These data provide the first demonstration of a QTL on chromosome 3 that plays a role in determining the difference in LV mass between normotensive Fischer 344 and WistarKyoto inbred rat strains. The prostaglandin synthase 1 gene is located within the QTL. (Hypertension. 1999;33:949-953.) Key Words: genetics n rats, inbred strains n cardiac mass n heart hypertrophy n chromosome n genes
T
here is a paucity of information on the extent and importance of inherited variability in the response of the human heart to hypertrophic stimuli such as hypertension.1 However, animal studies have provided persuasive evidence for a genetic contribution to variation in cardiac mass. For instance, Tanase and colleagues2 conducted a detailed segregation study of 23 inbred normotensive and hypertensive rat strains, from which they estimated that 65% to 75% of the strain difference in heart weight was genetically determined. With the advent of highly polymorphic markers spread across the entire rat genome, it has become feasible to conduct combined segregation and linkage studies to define quantitative trait loci (QTL) that contribute to strain differences in cardiac mass. In most cases, these have involved the analysis of cosegregation in crosses between hypertensive strains or between a hypertensive and a normotensive strain.3–9 Although some QTL affecting cardiac mass seem to segregate independently of those controlling blood pressure (BP), it is not clear whether these loci also play a role in determining interstrain differences in cardiac mass when the BP of both parental strains is not elevated. To minimize the confounding effects of BP, we have therefore conducted a segregation and genetic linkage analysis using the F2 progeny
of a cross between 2 inbred normotensive rat strains, WistarKyoto (WKY) and Fischer 344 (F344), which exhibit a substantial difference in adult cardiac mass.
Methods Animals and Genetic Crosses Inbred WKY (Charles River, UK) and F344 rats (Olac, UK) were used to characterize the cardiac phenotype. Reciprocal mating of the parental strains, WKY(male)3F344(female) and WKY(female)3F344(male), produced the first-generation F1 hybrid animals, 6 pairs of which were chosen at random for brother-to-sister mating to produce an F2 segregating generation. Male F2 progeny (n5126) were selected for genotype and phenotype analysis. This number of animals is sufficient to detect 2 QTLs with 90% power by use of the traditional method of genotyping all progeny.10 All animals were given unlimited access to standard laboratory rat diet (Special Diet Services) and drinking water. All procedures were conducted in accordance with Imperial College of Science, Technology and Medicine guidelines.
BP Measurement Twelve-week-old rats were anesthetized with an intraperitoneal injection of sodium pentobarbitone, 60 mg/kg body wt, and systemic arterial pressure was measured by means of a cannula inserted into the left carotid artery. The cannula was exteriorized through the back of the neck and attached to a pressure transducer (Cobe Laboratories
Received April 30, 1998; first decision May 19, 1998; revision accepted December 9, 1998. From the Section on Clinical Pharmacology, Division of Medicine, Imperial College of Science, Technology, and Medicine, Hammersmith Hospital, London, UK, and the Department of Genetics and Biometry (C.S.H.), Roslin Institute (Edinburgh), Roslin, Scotland, UK. Reprint requests and correspondence to D.J.R. Nunez, Section on Clinical Pharmacology, Division of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail
[email protected] © 1999 American Heart Association, Inc. Hypertension is available at http://www.hypertensionaha.org
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Inc) connected to a MacLab 4 integrated data acquisition system (AD Instruments). Once an animal had regained consciousness and its BP had stabilized, continuous pressure recordings were made for 20 minutes and the average of these readings was used to estimate mean arterial pressure.
Total Heart Weight, Left Ventricle Weight, and Right Ventricle Weight Determination At 12 weeks of age, each animal was weighed and then given a lethal overdose of pentobarbitone. The thorax was opened and the heart was removed from the great vessels, blotted with tissue paper to remove as much blood as possible, and weighed to give total heart weight (THW). Both atria were then removed, and the right ventricle was dissected free and weighed. The left ventricle and septum were weighed together (LVW).
Genomic DNA Preparation
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The liver and kidneys were removed from F2 animals, snap-frozen in liquid nitrogen, and stored at 280°C. Genomic DNA was extracted from these tissues with the use of a Nucleon genomic DNA extraction kit (Scotlab).
Genotyping Polymerase chain reaction (PCR) amplification was used to genotype the F2 animals at microsatellite loci that are polymorphic in the WKY3F344 cross. PCR primer pairs for the microsatellite markers were obtained from Research Genetics or Genosys Biotechnologies. The reagents for PCR were 50 ng of genomic DNA, 300 nmol/L of each primer, 200 mmol/L deoxyribonucleotide triphosphates, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0), 0.1% Triton X-100, and 0.25 U of Taq DNA polymerase (Promega), in a final reaction volume of 10 mL. PCR was performed for 35 cycles with an Omnigene thermocycler (Hybaid) and the program 1 minute at 94°C, 1 minute at 55°C, and 30 seconds at 72°C, followed by a final elongation step of 9 minutes at 72°C. The amplification products were resolved by electrophoresis through a 4% agarose gel (NuSieve 3:1 agarose, FMC Bioproducts) and visualized by staining with ethidium bromide. Only microsatellite markers that amplified reliably and distinguished the 2 parental alleles were used for further analysis. For some markers, it was necessary to alter the cycling parameters and MgCl2 concentration to minimize the presence of nonspecific bands.
Linkage and Statistical Analysis The Mapmaker/EXP 3.0 and Mapmaker/QTL 1.1 computer packages were used to construct genetic linkage maps and to localize QTL relative to the position of the microsatellite markers.10,11 At each 1-cM interval along the genetic map, assuming either a free, additive, dominant, or recessive mode of inheritance, the program computed the maximum log10 likelihood (LOD) to support the presence of a QTL at that position. A graphical representation of LOD versus chromosome position was then generated for each of the genetic models. In addition, it was possible to obtain an estimate of the fraction of the total variance explained by a particular QTL. The effect on THW and LVW of alleles at marker loci identified within a 1-LOD support interval of a QTL was evaluated by 1-way ANOVA. Depending on the genetic model used during the Mapmaker/QTL analysis, the LOD score thresholds for significant linkage were set as follows: free genetics model, 4.3; dominant and recessive models, 3.4; codominant model, 3.3. These stringent criteria, originally proposed by Lander and Kruglyak,12 for genome-wide QTL mapping involving experimental intercrosses are designed to reduce type 1 errors. The statistical significance of an interstrain difference in the phenotypic mean or variance was determined with the Student’s t test or the F test, with correction for multiple testing. In these cases, the significance level a was set at 0.05.
TABLE 1. Average Body Weight, HW, LVW, RVW, Relative LVW, and Blood Pressure in WKY and F344 Strains WKY
F344
P
Body weight, g
273.4620.8 (22)
262.8621.5 (23)
131021
HW, mg
945.3659.8 (22)
721.2653.8 (23)
9310217
LVW, mg
689.0644.4 (22)
546.2640.2 (23)
1310214
RVW, mg
208.1621.9 (22)
136.6615.4 (23)
3310216
Relative LVW, mg/g
2.5360.14 (22)
2.160.10 (23)
1310216
Blood pressure, mm Hg
80.463.6 (9)
Phenotype
86.066.0 (9)
431022
Values are mean6SD (number of animals).
Results Characterization of Cardiac Phenotype of the F344 and WKY Strains THW and LVW of WKY rats (n522) were significantly greater than those of F344 rats (n523). The strains differed by 23% (224.1 mg) for THW and 20.7% (142.8 mg) for LVW (Table 1). Scatter diagrams of the relationship between body weight and THW or LVW, together with the corresponding coefficients of correlation (r), are shown in Figure 1. In both strains, THW and LVW showed a positive correlation with body weight. However, in the WKY strain, r for THW versus body weight was only 0.61 compared with 0.85 in the F344 strain. The weaker correlation of THW with body weight in the WKY strain seemed to be because of the lack of a correlation between RVW and body weight in this strain (r50.19, P.0.05). In contrast, there was a strong correlation between RVW and body weight in the F344 strain (r50.70, P,0.05). Because LVW was the only phenotypic parameter that showed a strong linear correlation with body weight in both strains, the values of LVW were made proportional to body weight by dividing LVW by body weight. There was a small but statistically significant (P,0.05) difference between mean systemic BP of the 2 parental strains (WKY BP580.463.6 mm Hg [n59]; F344 BP586.066.0 mm Hg [n59]) (Table 1). Despite their lower systemic pressures, the WKY rats had considerably larger hearts than F344 rats. No significant correlation was found between BP and THW, LVW, or RVW phenotypes in either strain (data not shown).
Segregation Analysis The average phenotypic variances of the 3 nonsegregating generations (WKY, F344, and F1) were used to estimate the environmental variance of THW and LVW. The proportion of the total F2 variance that was due to genetic variation, expressed as heritability, was calculated according to the formula ([total F2 variance2environmental variance]3100/ total F2 variance) and was found to be 56% for THW and 55% for LVW.
Linkage Analysis A genetic linkage map for each of the 20 autosomes was constructed with genotypic data from polymorphic microsatellite markers (the X and Y chromosomes were not screened) (Table 2). The best map order for the markers was determined by multipoint linkage analysis using Mapmaker/EXP 3.0.
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phenotype were typed across all the autosomes. The remaining 61 F2 animals were then genotyped with the use of markers for regions showing evidence of linkage (LOD.1.5) in the primary screen. A genetic linkage map for chromosome 3 was generated with data from 11 polymorphic microsatellite markers for all 126 F2 progeny (Figure 2). Using the Mapmaker/QTL program, we found a significant QTL on chromosome 3 affecting both THW and LVW, supported by peak LOD scores of 4.4 for THW and 4.8 for LVW, that accounted for 15.1% and 16.5% of the total variance in THW and LVW, respectively. This region containing the QTL was centered around the 3 markers D3Rat29, D3Rat34, and D3Rat38, which span a genetic distance of 9.5 cM. The peak LOD scores for both THW and LVW were associated with the marker D3Rat29. The 1-LOD support interval for the position of the QTL was 16 cM. Under the dominant genetic model, linkage with TWH and LVW was supported by LOD scores of 3.0 and 2.3, respectively, compared with LOD scores of 4.8 and 4.4 in the free genetic model. Thus, the likelihood ratio against the dominant model was 125:1 for THW and 63:1 for LVW. When tested as a likelihood ratio test against x2 distribution with 1 degree of freedom, the dominant model is significantly less likely (P50.01 to 0.001) for both traits. The results of 1-way ANOVA of the cosegregation of THW, LVW, or LVW/body weight, with the QTL characterized by these 3 markers, are shown in Table 3. The locus marked by D3Rat29 seemed to affect both THW and LVW in a codominant fashion, inasmuch as the F2 animals homozygous for the WKY (WW) or F344 (FF) allele had the largest and smallest mean THW and LVW, respectively, and the mean values for the heterozygotes (WF) fell between the phenotypic means of the 2 homozygous groups. Rats homozygous for the F344 allele at the marker D3Rat29 have LVW and THW '62 mg (P,0.05) and 80 mg (P,0.05) lower than those homozygous for the WKY allele.
Figure 1. Relationship between THW, LVW, or RVW and body weight in WKY and F344 parental strains. The correlation coefficient, r, was calculated for the degree of association of each pair of variables.
Discussion Genetic distances between markers were calculated with the Haldane mapping function. One hundred sixty-one microsatellite markers with a difference in allele size $5% between WKY and F344 parental strains were used in a whole genome search for QTL affecting THW, LVW, or the ratio of LVW to body weight. In a primary screen, 65 F2 animals comprising the top and bottom quartiles of the cumulative frequency distribution of LVW TABLE 2.
Here we report the results of the first segregation and genetic linkage analysis that has used the F2 progeny of a cross between inbred normotensive WKY and F344 strains to identify QTL that affect the substantial difference in adult cardiac mass between the 2 strains. We and others2,13 have found that the cardiac mass of adult F344 rats is considerably lower than that of WKY animals and that this trait breeds true.
Distribution of the Microsatellite Markers Used in the Whole Genome Screen Chromosome
Marker Distribution
1
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
No. of markers
10
10
11
4 9
7
9
10
6
10
8
7
7
8
8
8
7
8
7
7
6
Maximal distance (cM) between adjacent markers
30
28
29
31
30
19
26
28
20
31
15
31
29
27
17
30
23
27
17
19
% Chromosome within 10 cM of markers
69
83
80
76
50
69
67
50
84
58
46
55
47
64
57
57
61
65
49
43
% Chromosome within 20 cM of markers
100
90
99
100
90
98
99
92
100
85
76
90
82
100
86
86
100
81
80
79
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Genetic Determination of Cardiac Mass
Figure 2. LOD plots of LVW across the entire chromosome 3. The 4 plots were computed assuming either a free, dominant, codominant, or recessive genetic model. The plots represent a genetic linkage map of chromosome 3 that was produced with the use of Mapmaker/EXP 3.0 and data obtained from the analysis of 11 polymorphic microsatellite markers in 126 F2 progeny of the WKY3F344 cross. Distances between markers are given in cM. The filled area denotes the 1-LOD support interval of the QTL. The positions of 5 previously assigned genes are shown below the map. The prostaglandin synthase 1 gene (Ptgs1) lies within this QTL.
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We performed an initial segregation analysis of the cardiac mass phenotypes that suggested that 1 to 2 genes were involved in determining the interstrain difference. From the graphs published by Lander and Botstein,10 our study has 90% power to detect 2 QTL by the use of the traditional method of genotyping all progeny. We then used polymorphic markers for the WKY3F344 cross that permitted us to perform a comprehensive screen of .99% of the rat genome for QTL (based on a recent reevaluation of the size of the rat genome).14 This search identified a significant QTL for LVW and THW on chromosome 3 that accounts for '16% of the total variance of these phenotypes. Even though the maximal distance between markers was never greater than 31 cM, it is possible that we may have missed genetic factors with an autosomal recessive or sex-linked pattern of inheritance or autosomal dominant loci of small effect. We analyzed only male rats because at the time of the breeding program, there was no evidence of sex-dependent effects on cardiac mass. Since then, there have been 2 studies describing sex-dependent effects in crosses of hypertensive with nonhypertensive rat strains (not F344 or WKY).6,8 Our segregation data provide evidence for a significant genetic contribution to the total variance of cardiac mass and confirm the work of Tanase and colleagues2 who studied the segregation of cardiac mass in 23 inbred rat strains. They
concluded that the effect of genetic factors on cardiac mass was greater than that of BP. Several studies have reported QTL contributing to the regulation of adult cardiac mass independent of the influence of BP on chromosomes 2,9 8,7 10,4 12,5,6 and 17.3 In crosses with hypertensive strains however, it is unclear whether differences in cardiac mass are truly independent of arterial pressure or whether they reflect technical artifact caused by the use of mechanical pressure transducers that may be less sensitive to cumulative pressure increments than myocardial sensors. In a cross between Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats, Cicila and colleagues15 found that the endothelin-3 locus (Edn3) on chromosome 3, a region at least 70 cM from our QTL, cosegregated with BP and relative heart weight in an SS/Jr3F1 (SS/Jr3SR/Jr) population. In a subsequent study, reported only in abstract form,16 this group has introgressed the Dahl salt-resistant Edn3 allele into the salt-sensitive strain. Their data indicate the presence of 2 QTL on chromosome 3 controlling BP and relative heart weight: one is associated with marker Pck1 (closely linked to Edn3), and the other is associated with marker D3Wox3, '100 cM away. Our QTL lies at least 70 cM away from the Edn3 locus, and it is possible that the markers D3Wox3 and D3Rat29 are defining the same locus in these crosses. The improved linkage map for chromosome 3 reported recently
TABLE 3. Effects of Genotypes at Markers D3Rat29, D3Rat34, and D3Rat38 on THW, LVW, and Relative LVW/Body Weight (rLVW) of F2 Animals D3Rat29
D3Rat34
D3Rat38
Genotype
THW
LVW
rLVW
n
THW
LVW
rLVW
n
THW
LVW
rLVW
n
WW
858.0
641.4
2.24
39
861.1
643.1
2.24
37
858.3
640.4
2.21
32
WF
836.3
623.4
2.17
51
832.6
621.7
2.18
54
839.1
625.7
2.19
59
FF
776.5
579.1
2.13
36
778.6
579.8
2.11
35
777.0
578.9
2.11
35
10.6
11.3
5.8
9.9
10.9
6.3
9.3
9.9
531025
331025
431023
431025
231023
F score
P
Values are mean.
131024
131024
631025
4.9 931023
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by Dene and colleagues17 does not clarify this issue. In any case, it may be necessary to produce congenic strains to establish conclusively whether 1 or 2 QTLs for heart weight are present in chromosome 3. We have searched the rat genome databases and syntenic regions in mouse and human databases to identify potential candidate genes on chromosome 3. Four genes have been assigned to a region within the 1-LOD support interval of the QTL: the glucagon gene (Gsg), the gene for the a-polypeptide of the type II voltage-gated sodium channel locus (Scn2a1), the prostaglandin synthase 1 gene (Ptgs1), and the adenylate kinase gene (Ak1) (Figure 2). The relevance of these genes to the phenotypic variance in cardiac mass is not clear, but the prostaglandin synthetase gene is an interesting candidate because it has prostaglandin endoperoxidase/ cyclooxygenase activity and catalyzes the conversion of arachidonic acid to prostaglandin H2, from which various vasoactive prostaglandins (PGE2, PGF2a, prostacyclin, thromboxane) are synthesized.18 Although there are no previous reports of cosegregation of the Ptgs1 locus with cardiac mass phenotype, a role for PGF2a in cardiac myocyte hypertrophy has been suggested by the ability of various prostaglandins to induce hypertrophy of ventricular myocytes in culture to an extent comparable to that induced by several well-known hypertrophic factors, such as phenylephrine, and angiotensin II.19 In conclusion, we report the results of a genome scanning study to search for chromosomal regions that control the difference in cardiac mass between the normotensive WKY and F344 inbred rat strains. We have detected a locus on chromosome 3 that includes the prostaglandin synthase 1 gene.
Acknowledgments This work was funded by the British Heart Foundation. C.S.H. is supported by the Biotechnology and Biological Sciences Research Council.
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Genetic Determination of Cardiac Mass in Normotensive Rats: Results From an F344×WKY Cross Abdelkrim Sebkhi, Lan Zhao, Long Lu, Christopher S. Haley, Derek J. R. Nunez and Martin R. Wilkins Downloaded from http://hyper.ahajournals.org/ by guest on January 21, 2018
Hypertension. 1999;33:949-953 doi: 10.1161/01.HYP.33.4.949 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1999 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
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