Evidence for Primary Genetic Determination of Heart Rate Regulation
Chromosomal Mapping of a Genetic Locus in the Rat
Background We investigated whether an accelerated heart rate (HR), observed in the stroke-prone spontaneously hypertensive rat (SHRSPHD), is a primary, genetically determined trait and whether it contributes to blood pressure (BP) regulation in this model of polygenic hypertension.
Methods and Results We measured BP and HR in SHRSPHD and normotensive Wistar-Kyoto rats (WKY), as well as in F2 hybrids bred from crossing the two strains, at baseline and after 12 days of dietary NaCl loading. Random marker genome screening and cosegregation analysis were performed on F2 hybrids derived from SHRSPHD/WKY-0HD (n=115) and SHRSPHD/WKY-1HD (n=139) crosses (WKY-0HD and WKY-1HD are two congenic WKY strains). HR in SHRSPHD was significantly higher than in WKY-0HD both at baseline (404±30 versus 375±46 bpm; P=.0034) and after NaCl (437±23 versus 364±40 bpm; P=10−9). BP in F2 hybrids showed no significant correlation with HR either at baseline or after NaCl loading. HR after NaCl loading but not at baseline was significantly linked in a recessive fashion to a locus on chromosome 3: in animals homozygous for the SHRSPHD allele, HR was 414±49 compared with 383±44 bpm in heterozygotes and WKY homozygotes (F210,1=19.7, P=1.4×10−5, lod score=5.9). The putative BP-relevant gene at this locus, termed HR-SP1, showed no evidence of linkage to any of the BP parameters measured.
Conclusions Our results demonstrate that a genetic locus on rat chromosome 3, HR-SP1, contributes directly to the regulation of HR in SHRSPHD but exhibits no effect on BP. Thus, in addition to its modulation by reflex-mediated neurohumoral mechanisms, HR is also under the direct influence of primary genetic factors.
Cardiac output and total peripheral resistance are the major hemodynamic determinants of BP.1 In patients with established hypertension, an increase in total peripheral vascular resistance associated with normal or reduced cardiac index is a characteristic hemodynamic finding.1 Increased HR in these patients is commonly viewed as a compensatory response to a diminished stroke volume, which may be due to diastolic or systolic myocardial dysfunction.1 2 3 In contrast, in the early stages of hypertension in both humans and experimental animals, an increased HR is not uncommonly seen in the absence of compromised left ventricular function.1 In the presence of normal total peripheral resistance, this results in an increased cardiac output and consequently in a rise in peripheral BP. Thus, whereas an increased HR most likely represents a secondary phenomenon in established hypertension, it has been argued that an early increase in HR may contribute directly to the pathogenesis of hypertension.1 4 This hypothesis requires that one postulate a primary, presumably genetically determined alteration of baseline HR regulation. Thus far, no direct evidence has been presented to support such a possibility. The aim of the current study was to investigate this question by use of an experimental animal model that displays both hypertension and accelerated HR, namely, the SHRSPHD.
Animals and Genetic Crosses
Animals were obtained from our colonies of SHRSPHD, WKY-0HD, and WKY-1HD rats at the University of Heidelberg, Germany.5 WKY-0HD denotes the wild-type WKYHD strain, whereas WKY-1HD is a congenic line (two genetically almost identical strains that differ only in a defined area of their genome) that carries an SHRSP-homologous (ie, derived from the SHRSP genome), BP-relevant locus, BP/SP-1a, on chromosome 10.6 The experimental procedures for animal housing and breeding have been reported previously.5 The reciprocal (ie, either derived from a male SHRSP/female WKY mating or from the opposite constellation) F2(WKY-0HD×SHRSPHD) (n=115) and F2(WKY-1HD×SHRSPHD) (n=139) cohorts analyzed in the present study have been the subject of previous reports.5 6 7 8 9 10
The protocol for the hemodynamic characterization of the F2 rats and the WKY-0HD and SHRSPHD progenitor strains (n=11 and 12 males and females, respectively) was identical. It included femoral artery cannulation and intermittent on-line recordings as reported previously.10 Two consecutive femoral artery cannulations to measure BP and HR at baseline and after a period of 12 days of dietary NaCl exposure (1% NaCl in the drinking water) were performed.10 During measurements, the animals were shielded from the investigator, and three independent readings were taken while the animals were resting quietly.10
A total genome screen was performed using polymorphic SSLPs (microsatellites: stretches of repetitive DNA that tend to be unstable, thus often polymorphic, ie, different from strain to strain, and thus useful for genetic mapping) obtained from previously published panels of SSLPs,7 8 11 12 from new library screenings,11 and from GenBank data searches. SSLP markers were amplified by PCR from 50 ng of genomic DNA in a final reaction volume of 10 μL, containing 100 nmol/L of each primer, 200 μmol/L dNTPs, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0 at 25°C), 0.1% Triton X-100, and 0.25 U of Taq DNA polymerase. The forward primer was labeled with [γ-32P]ATP (specific activity of 3000 Ci · mmol−1, DuPont/NEN) using T4 polynucleotide kinase (NEB). PCR reactions were processed on an MJ Research thermal cycler (PTC 100) using the following protocol: initial denaturation at 92°C for 3 minutes followed by 30 cycles of denaturation at 92°C for 15 seconds, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute, followed by a final extension step at 72°C for 7 minutes. Subsequent to the PCR, the samples were analyzed by polyacrylamide gel electrophoresis and autoradiography as described previously.7
Statistical Analysis and Linkage Analysis
Interstrain comparisons were performed by two-way ANOVA to test the effects of strain and sex. Statistical evaluation of the effect of genotypes on HR phenotype in the F2 intercrosses was performed by ANCOVA) to account for sex, cross, and parental constellation of the reciprocal crosses and to adjust for BP, respectively. For combined linkage analysis in both F2 intercrosses, means in each group were adjusted for cross, sex, and reciprocal cross status where appropriate.7 Linear regression analysis was performed by calculating the coefficient of determination, R2. Chromosomal mapping and lod score calculations were performed using the MAPMAKER/EXP and MAPMAKER/QTL programs.13 All phenotype parameters are expressed as mean±SD.
We previously reported10 that at 16 weeks of age, basal systolic BP differs by 70 and 90 mm Hg in female and male SHRSPHD compared with female and male WKY-0HD rats, respectively. Oral NaCl loading led to an additional BP increase of 30 mm Hg in both female and male SHRSPHD, whereas BP in WKY-0HD remained unchanged.10 HR at baseline conditions was significantly higher in male SHRSPHD than in male WKY-0HD (P=.0003), with no statistically significant difference among females (P=.55; Table 1⇓). In contrast, after oral NaCl loading, HR was significantly elevated among both female (+60 bpm) and male (+80 bpm) SHRSPHD rats (Table 1⇓). BP, as determined by radiotelemetry measurements, was modestly elevated in WKY-1HD compared with WKY-0HD, as previously reported,6 whereas HR demonstrated no difference among the two WKY strains (data not shown).
In both F2 cohorts, no significant correlation between systolic or diastolic BP and HR was observed either at baseline (R2=.09, P=.15 and R2=.02, P=.64, respectively) or after NaCl loading (R2=−.06, P=.35 and R2=−.10, P=.14, respectively). The genome screen identified suggestive linkage between a marker on chromosome 3 (Scn2a1) and HR after NaCl loading in both F2 crosses. ANCOVA revealed no significant differences between WKY homozygotes and heterozygotes. Accordingly, under assumptions of a recessive mode of inheritance (both alleles must carry the “mutant” genotype), zygosity (ie, constellation of alleles) at Scn2a1 demonstrated significant linkage to HR after NaCl loading in the F2(WKY-0HD×SHRSPHD) (P=.0062) and the F2(WKY-1HD×SHRSPHD) (P=.0012) populations. Basal HR showed evidence for linkage to the same locus in the F2(WKY-1HD×SHRSPHD) (P=.0008) cohort but not in the other cross (P=.33). Analysis for HR and BP phenotypes in both crosses combined demonstrated highly significant linkage between Scn2a1 and HR after NaCl loading (Table 2⇓). In animals homozygous (ie, carrying two identical alleles) for the SHRSP allele, HR was ≈30 bpm higher than in heterozygotes (ie, carrying one allele each from the progenitor strains) or WKY homozygotes. A maximum lod score of 5.9 was observed by QTL mapping, yielding a 100:1 CI for placement of the implicated gene, HR-SP1, that spans 32 cM (Figure⇓). HR-SP1 accounted for 14% of the overall variance of HR after NaCl loading. As expected, no linkage was found between BP phenotypes and HR-SP1 (Table 2⇓). Adjustment for BP by ANCOVA had no effect on the linkage data for HR-SP1 and HR either at baseline or after NaCl loading (data not shown).
Under physiological conditions, HR is predominantly determined via regulatory feedback loops integrated within the autonomous nervous system to ensure maintenance of an appropriate cardiac output. The increased HR observed in spontaneously hypertensive rats and SHRSP may be viewed as the result of increased sympathetic tone that has been implicated in the pathogenesis of hypertension in this model, particularly after oral NaCl loading.14 17 However, all these reports are based on the observation of mere associations, and no definite causal relationship between increased sympathetic activity and hypertension has ever been shown.
The lack of correlation between HR and BP phenotypes in our analysis argues against a causal contribution of increased HR to hypertension in SHRSP at the developmental stage examined. It also rules out the possibility that the observed alteration of HR in SHRSP is a secondary, hypertension-induced event. The genome screening analysis, on the other hand, demonstrated that HR variance is, at least in part, directly determined by the influence of a genetic locus, HR-SP1, on rat chromosome 3. Allelic status at HR-SP1 was not associated with differences in BP, and adjustment for BP before linkage analysis had no effect on the results. These data indicate that the influence of HR-SP1 was maintained across the entire range of blood pressures (136 to 210 mm Hg systolic) observed in the F2 crosses.
The finding that significant linkage to HR-SP1 in the overall population was only detected after oral NaCl loading may point to an ecogenetic (gene-environment) interaction between HR-SP1 and dietary NaCl content. Alternatively, the failure to obtain overall significant linkage to HR-SP1 at baseline may be attributed to a higher environmental noise present during normal salt intake that may act as a confounder, preventing the detection of the HR-SP1 effect under normal dietary conditions.7
The presence of the gene that encodes the brain isoform of the α-1 polypeptide of the type 2 voltage-gated sodium channel (Scn2a1) at the center of the HR-SP1 locus allowed the identification of a homologous region on human chromosome 2q23-2q24.3.18 This segment carries the human SCN2A gene and exhibits conservation with a corresponding region on mouse chromosome 2.19 Interestingly, the gene encoding a human G-protein–coupled, inwardly rectifying K+ channel, GIRK1, was recently mapped to human chromosome 2q24.1.20 GIRK1 represents one of the two inwardly rectifying K+ channel subunits that make up KACh, which is involved in HR regulation in response to vagal stimulation via activation of muscarinic receptors.21 Therefore, we may speculate that the rat homologue of GIRK1, Kir3.4, may represent a candidate gene for HR-SP1, even though one obviously needs to be mindful of the fact that the 100:1 CI for mapping HR-SP1 spans a genetic distance of 32 cM. This is a segment large enough to be expected to contain some 1500 genes, among which multiple other candidate genes may be present. Analysis of the currently available homology data for human, mouse, and rat revealed no other plausible candidate genes.
At present, without knowledge of the nature of the relevant gene at HR-SP1 and in the absence of information concerning a large number of potentially interesting intermediate phenotypes that relate to HR, we can only speculate whether the effect of HR-SP1 is a “direct” one (eg, a mutation affecting the cardiac pacemaker channel) or a more “remote” phenomenon (eg, mediated via modulation/resetting of one of the complex regulatory circuits that modulate cardiovascular function). We can say with certainty, however, that the portion of HR variance determined by HR-SP1 is not correlated nor collinear with BP and therefore is not a function of resetting the threshold or gain of classic pathways.
As is always the case in experiments of this kind, any conclusions and generalizations from these results regarding the effects of HR-SP1 must be made with great caution. Strictly speaking, our data and interpretations apply only to the strains and crosses as well as the particular experimental settings examined and, equally important, only to the specific developmental period under investigation in this study. Therefore, we cannot exclude the possibility that at a different developmental stage HR, and thus genes affecting HR regulation, may contribute to BP variance in SHRSPHD.
In summary, our study provides the first evidence that allelic variation at an HR-relevant genetic locus has the potential to affect HR in a primary, direct fashion that is independent of BP. Moreover, it provides the first example of identifying such an HR-relevant genetic locus by means of linkage analysis. Our data highlight the power of dissecting complex cardiovascular traits by molecular genetic means. Continued efforts along this line of investigation will lead to a better understanding of the complex interplay of heritable and environmental factors and of primary genetic and secondary, reflex-mediated mechanisms that govern cardiovascular function.
Selected Abbreviations and Acronyms
|PCR||=||polymerase chain reaction|
|QTL||=||quantitative trait mapping, in contrast to “classic” linkage analysis for categorical traits|
|SHRSPHD||=||stroke-prone spontaneously hypertensive rats|
|SSLP||=||simple sequence-length polymorphism|
|WKY-0HD||=||wild-type hypertensive Wistar-Kyoto strain of rats|
|WKY-1HD||=||a congenic line to WKY-0HD rats|
This work was supported by NIH SCOR (P50HL5500) from the National Heart, Lung, and Blood Institute, the EurHypGen concerted action of the European Economic Community, and the Deutsche Forschungsgemeinschaft (Dr Kreutz, Kr1152/1-2 and GA175/8-1). Dr Kreutz is the recipient of a Howard Hughes Medical Institute Research Fellowship for Physicians, and Dr Lindpaintner is the recipient of NIH Research Career Development Award K04HL03039.
- Received May 16, 1997.
- Revision received June 4, 1997.
- Accepted June 6, 1997.
- Copyright © 1997 by American Heart Association
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