191

Differential Development of Vascular andCardiac Hypertrophy in Genetic Hypertension

Relation to Sympathetic Function

Michael A. Adams, Alexander Bobik, and Paul I. Korner

We compared blood pressure, hlndquarter vascular resistance properties, left ventricular weight,and norepinephrine kinetics, in spontaneously hypertensive rats (SHR) and weight-matchednormotensive Wistar-Kyoto (WKY) rats at 4, 9, 14, 20, 30, and 50 weeks of age- At 4 weeks,systolic and mean blood pressure measurements were the same in both strains, but the vascularresistance of the fully dilated hindquarter bed was significantly higher in SHR than in WKY rats,with a much larger difference during maximum constriction. Plots of resistance at maximumdilatation and at maximum constriction against body weight suggest that a component of theincrease hi vascular muscle mass in SHR occurred in the neonatal period preceding hypertensionfollowed by a later component related to the rise in blood pressure. By contrast, left ventricularhypertrophy was minimal at 4 weeks and most of its development paralleled the rise in bloodpressure. Sympathetic activity, assessed by norepinephrine fractional rate constant, was higher inSHR than in WKY rats in the left ventricle and kidney through most of the period between 4 and50 weeks, but was similar in both strains in the muscle bed. This pattern of sympathetic activitywill accentuate hypertension once cardiac and vascular hypertrophy are fully established. In allregions, norepinephrine tissue concentration was higher in young SHR and could potentiate thetrophic effects of growth factors in early vascular hypertrophy. We suggest that the initial(primary) component of vascular hypertrophy precedes the rise in blood pressure and may becritical in the pathogenesis of hypertension. Possible reasons for the short delay in the rise in bloodpressure in young SHR, once the vascular "amplifier" has been established, include highvascularity, immaturity of smooth muscle, and delay in the development of left ventricularhypertrophy. (Hypertension 1989; 14:191-202)

Folkow1-2 and colleagues were the first todemonstrate the importance of structuralchanges associated with hypertrophy of the

cardiovascular musculature in hypertension. In thespontaneously hypertensive rat (SHR), they showedthat, as a result of the increase in wall thickness andnarrowing of the lumen, changes in vascular resis-tance were "amplified" when compared with theresponses of Wistar-Kyoto (WKY) normotensiverats.3 The concentrically hypertrophied left ventri-cle also has amplifier properties, which help in themaintenance of cardiac output against a higherpressure load.4-5 In chronic secondary hypertension

From the Baker Medical Research Institute, Melbourne,Australia.

Supported by the National Heart Foundation of Australia, theNational Health and Medical Research Council, and through theaward of a postdoctoral fellowship to M.A.A. by the CanadianMedical Research Council.

Address for correspondence: Professor P.I. Korner, BakerMedical Research Institute, Commercial Rd., Prahran, Victoria,3181, Australia.

Received March 4, 1988; accepted March 6, 1989.

due to renal artery stenosis, the cardiovascularamplifiers contribute about 70% to the maintenanceof the elevated blood pressure (BP) and this isprobably similar in primary hypertension.6

In secondary hypertension, the hypertrophy isclearly a consequence of the elevated BP, and it hasgenerally been assumed that this is also the case inprimary hypertension. There has been speculationwhether, in primary hypertension, the early devel-opment of cardiovascular hypertrophy could ini-tiate the rise in BP.7-9 This hypothesis is not readilytestable in humans, because of the difficulty ofperforming an adequate longitudinal study. Such astudy is feasible in SHR, where the time course ofthe BP changes has been documented in greatdetail,10 but the range of ages in previous longitudi-nal studies on the development of cardiac andvascular hypertrophy has been too small for anadequate sequential analysis (see References 10-14). For example, we still do not know whether inSHR the vascular and cardiac hypertrophy developover the same age span.

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192 Hypertension Vol 14, No 2, August 1989

Present evidence suggests that sympathetic ner-vous system is important in the development ofhypertension in SHR.2-15-19 Folkow et al15 showedthat immunosympathectomy in very young ratsattenuated the development of hypertension.Recently Lee et al,20 using an improved ablationtechnique, found that hypertension could be pre-vented completely. One way in which increasedsympathetic neural activity in SHR could elevateBP is through a net increase in vasoconstrictor toneassociated with a low threshold of the "defense"reaction,1'2-16'17 so that the hypertrophy will occuras a direct consequence of the rise in BP. Analternative hypothesis is that the sympathetic ner-vous system exerts a local trophic role in thedevelopment of cardiovascular hypertrophy.7-18-19

Such a role presupposes elevation of regional nor-epinephrine tissue concentration [NE] and highturnover at the time of development of cardiovas-cular hypertrophy. However, there has been onlyone detailed longitudinal study by Patel et al,21 whodetermined the fractional rate constant of norepi-nephrine, but did not relate it to the development ofcardiovascular hypertrophy.

In the present experiments, we examined thetime course of development of cardiac and vascularhypertrophy and how this related 1) to the develop-ment of hypertension and 2) to the various mea-sures of regional sympathetic function. Accord-ingly, we compared the vascular properties of thehindquarters in SHR and WKY rats and measuredthe left ventricular and right ventricular weights atsix time points over an age span of 4-50 weeks ofage. Indexes of regional sympathetic function wereobtained in a parallel study in skeletal musclevessels, heart, and kidney.

Materials and MethodsAnimals and Blood Pressure Measurements

Male SHR and WKY rats (Animal ResourcesCentre, Perth, Western Australia) were housed ingroups of three rats to a cage in a room with a12-hour light/dark cycle and an ambient tempera-ture of 22-24° C, with food and water provided adlibitum. We had intended to take special care toweight-match pairs of SHR and WKY rats at thedifferent ages (see below). However, no specialselection procedure turned out to be necessary, andin all experiments the difference in body weight wasless than 5%. The rats came originally from theNational Institutes of Health in the United Statesand the strain of WKY rats that we used was that inwhich the age-body weight relation was similar tothat of SHR.22

Systolic BP and heart rate were measured inconscious rats with an automated multichannel sys-tem (IITC Life Science Instruments, WoodlandHills, California, 24 channel) that used tail cuffs andphotoelectric sensors to detect the tail pulses. Atest chamber maintained at 27-28° C was used to

place rats in holders appropriate for their bodyweight. Over the last 2 weeks of each time period,there was a minimum of four BP recording sessions:the first two were regarded as training sessions andthe data was discarded; the systolic BP was takenas the mean of the last two sessions (4-6 sets ofmeasurements) recorded over the last 2-3 days. Inaddition, mean arterial pressure was measureddirectly in a separate series of 4-week-old SHR andWKY rats which were anesthetized with methohex-itone anesthesia (Brietal, Eli Lilly, Indianapolis,Indiana, 30 mg/kg i.p.) and were cannulated withaortic catheters 4-7 days before recording, asdescribed by Head and McCarty.23 In these ratspressure was recorded for a period of 3-4 hours onthe day of the study.

Hindquarter PreparationHindquarter perfusions were carried out in pairs

of age- and weight-matched SHR and WKY rats byusing a slight modification of the method of Folkowet al.24 The system included a common reservoir forboth rats, an injection port and bubble trap/mixingchamber in line with a peristaltic pump (Minipuls 2,Gilson Medical Elec, Inc., Middleton, Wisconsin).The perfusate consisted of 1.5% dextran T-40 (Phar-macia, Upsala, Sweden) in Tyrode's solution, whichwas aerated with 95% O2 and 5% CO2. The compo-sition (mM) of the Tyrode's solution was: KC1 20,CaCl2 • 2H2O 32.3, MgCl2 • 6H2O 5.1, NaH2PO4 • 2H2O6.2, NaHCO3 100, glucose 100, and NaCl 800 mg/100 ml fluid. The perfusate in the reservoir waswarmed, and we used a heating pad to maintainrectal temperature at 36-38° C.

The rats were anesthetized with sodium pento-barbital (60 mg/kg i.p.) and the lower abdominalaorta was exposed at the iliac bifurcation through amidline abdominal incision. After heparinization(1,000 IU/kg), the aorta was cannulated with a 19-or 23-gauge needle, with the tip immediately prox-imal to the iliac bifurcation. The middle caudal andthe caudal mesenteric arteries, which are close tothe bifurcation, were ligated and not perfused. Flowof perfusate was started immediately after transec-tion of the spinal cord and vena cava adjacent to thecannula entrance. The heart and other tissues wererapidly removed for catecholamine assays (seebelow). About 5 minutes after the start of perfusion,when the vasculature had been flushed clear ofblood, papaverine HC1 (12 mg/ml) was given as abolus (1.5 mg/100 g body wt) and the perfusionpressure at maximum vasodilatation (PPmudii)remained constant over a 20-minute washout period,indicating that edema was minimal. Subsequently,we obtained cumulative concentration-vascularresistance (=PP) response curves to the aradrenergic receptor agonist methoxamine (0.4-300/tg/ml, Burroughs Wellcome, Research TrianglePark, North Carolina) by stepwise increases in theinfusion rate (0.66-66 fiVmin i.v.) from a syringepump (Perfusor IV, Braun, Munich, FRG) contain-

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Adams et al Vascular and Cardiac Hypertrophy 193

TABLE 1. Linear Regression Equations in Spontaneously Hypertensive Rats and Wlstar-Kyolo Rats

GroupBW-HQW relationSHR+WKYPP-flow relationSHRWKY

SHR

WKY

SHR

WKY

PP-BW relationSHR

WKY

SHR

WKY

Age(wk)

4-40

4

4

14

14

27

27

40-5040-5040-5040-50

n

23

8*

1*

6 '

8*

4 '

4 '

31

32

31

32

Regression equation

HQW=24.97+0.031 BW

P P ^ do=10.07+0.72 HQQPPm». <ffl=6.83+0.72 HQQPPmrc.do'-13.45 + 1.48 HQQPPm«dii=9.84+1.48HQQPPmtdi=12.89+1.43HQQPPo-x ,8=8.70+1.43 HQQ

PPn»*<ffl=13.34+0.046 BWPPIOT<ffl«=11.33+0.034BWPPmDcc«=225+0.319BWPPm»cc»=159+0.274BW

SEb

0.0023

0.0880.0470.1810.1440.3870.543

0.00390.00390.320.27

r

0.95

0.850.940.910.900.950.91

0.910.850.890.85

n, number of rats; SEt,, standard error of regression coefficient; r, correlation coefficient; BW, body weight (g);HQW, hindquarter weight (% of BW); SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; PP,perfusion pressure; max dil, maximum dilatation; HQQ, hindquarter flow (ml/min/100 g HQW); max con, maximumconstriction.

'For PP-flow curves, number of data points were, in order, spontaneously hypertensive rats, Wistar-Kyoto rats:4 weeks, 32 and 28; 14 weeks, 17 and 24; 27 weeks, 12 and 12.

ing methoxamine. The concentration of methox-amine was related to the body weight of the rats, sothat in each experiment we used similar drug con-centrations and increments. For each dose a perfu-sion pressure plateau was reached before giving thenext dose, up to a dose of methoxamine where therewas no further elevation in perfusion pressure. Afterthis we tested whether this corresponded to perfu-sion pressure at maximum constriction (PP^cd)*which was obtained by means of a bolus of angio-tensin II (Ang II, 50/x.g) or of BaCl2 (100-200 ^g)-

The rats were perfused at a constant flow of 4ml/min/100 g body wt, which in adult rats corre-sponds to a flow of 10 ml/mih/100 g hindquarterwt.24 The relation between hindquarter weight andbody weight was closely similar in SHR and WKYrats, but differed at different stages of growth (Table1). Thus, in low body weight (young) rats of eachstrain, the hindquarter weight was a lower fractionof the total body weight than in adult rats. Forexample, in a 50-g rat, hindquarter weight was26.5±0.4% body wt, whereas in a 450-g rat, hind-quarter weight averaged 38.8±0.6% body wt, whichwas close to the value of 40% that Folkow et al24

reported in adult rats. Thus, at constant perfusionper unit body weight, young rats will receive ahigher flow per gram hindquarter weight than adultrats, so that the vascular resistance at P P ^ m willbe overestimated. The body weight-hindquarterweight relation accounted for about 90% of thevariance, and we used it to correct P P ^ ^ to thevalue corresponding to a standard perfusion rate of10 ml/min/100 g hindquarter wt. This was done fromperfusion pressure-flow lines obtained in a separate

series of 4-, 14-, and 27-week-old SHR and WKYrats (see Table 1 and Results). The correction wasapplied as shown in Figure 1, where P P ^ ^ isplotted against both flow/unit body weight andflow/unit hindquarter weight. In rats of this bodyweight, perfusion at 4 ml/min/100 g body wt corre-sponds to perfusion of 14.6 ml/min/100 g hindquar-ter wt; PPmaxdj at this flow was 24.1 mm Hg while,at a flow of 10 ml/min/100 g hindquarter wt, it was20.4 mm Hg, so that the first value requires acorrection of -3.7 mm Hg. We have assumed thatthe perfusion pressure-flow relation at 9 weeks washalfway between that at 4 and 14 weeks (Table 1).The average corrections applied to normalize thePPm»x dii values to perfusion at 10 ml/100 g hindquar-ter wt at 4, 9, 14, 20, 30, and 50 weeks were asfollows: in SHR -3.7, -3 .1 , -2 .3 , -1.5, -0.8, and-0.2 mm Hg; in WKY rats -3.0, -2 .3 , -1.6, -1.2,-0.8, and -0.2 mm Hg. For the PP^a,,,, a similarcorrection is not required, since the muscle "yields"instead of developing more tension.3

Tissue NorepinephrineThe rats were anesthetized with sodium pento-

barbital (60 mg/kg i.p.) and killed by exsangui-nation.25 The left ventricle plus septum, the kid-neys, and the forelimb muscle (caput longum oftriceps brachii) were quickly dissected from theother tissues, weighed, rapidly frozen in liquidnitrogen, and then stored at —80° C until analysis.

Norepinephrine was extracted from the tissue by aslight modification of a procedure described previ-ously.26 Briefly, the tissues were homogenized (Pory-tron PT 20) in ice-cold 0.4 M perchloric acid (4.0 ml,

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194 Hypertension Vol 14, No 2, August 1989

0.4 M) containing 3,4-dihydroxybenzylamine(DHBA). After the homogenate was centrifuged topellet-precipitated proteins, the pH of the superna-tant was adjusted to pH 8.6 with 1 M Tris buffer 8.6containing ethylenediaminetetraacetate (EDTA, 10mg%). Norepinephrine was then extracted from thissolution onto 300 mg alumina (active/neutral form,Merck Sharp & Dohme, West Point, Pennsylvania),which was washed twice with water. Norepinephrinewas eluted with 0.4 M perchloric acid.

Quantitation of norepinephrine was carried outby high-performance liquid chromatography(HPLC), using either fluorometric (Schoeffel, FS-970) or electrochemical (LC-4A controller, Bioana-lytical Sys., Inc., West Lafayette, Indiana)detection.26-27 Fluorescence of catecholamines wasmeasured through a Corning (Corning Glass Works,Corning, New York) 7-60 glass filter (band pass250-400 nm) on excitation at 200 nm. For electro-chemical estimation of the catecholamines, an elec-trochemical cell with a glassy carbon electrode(TL-8A, Bioanalytical Sys., Inc.) was used, withthe potential set at 0.8 V against a Ag/AgCl refer-ence electrode. The use of 0.8 V was validated bycomparison of redox curves (detector response vs.oxidation potential) for norepinephrine and for alu-mina eluates containing norepinephrine extractedfrom heart, kidney, and muscle tissue. Signals fromthese detection systems were recorded on a Shi-madzu data system (C-R3A Chromatopac, Shi-madzu Sci. Instrs., Columbia, Maryland), and nor-epinephrine was quantified by using peak area ratiosrelative to the internal standard (DHBA). The liquidchromatographic system consisted of a Varian 5000Pump equipped with a refrigerated autosampler(AS-48, Bio-Rad Labs., Richmond, California) andan Altex Ultrasphere-ODS column (3 /Am; 46x7.5cm). When fluorescence detection was used, themobile phase consisted of 10 mM perchloric acid(pH 2). Since with electrochemical detection sensi-tivity is relatively poor at this pH, the mobile phasefor this system consisted of dihydrogen phosphate(0.05 M), sodium citrate (0.05 M), EDTA (2 mM),and octyl sodium sulfate (100 mg%), dissolved inmethanol/water (3%/10% vol/vol). The pH of theprepared solution was adjusted to 4.5. Quantita-tively the two systems provided identical estimatesof norepinephrine in the three tissues.

Norepinephrine TurnoverThe rate of norepinephrine synthesis (turnover)

can be expressed as .KxfNE], where K (the frac-tional rate constant) is the fraction of norepineph-rine lost in unit time after suppression of norepi-nephrine biosynthesis by inhibition of the enzymetyrosine hydroxylase by a-methyl-DL-/?-tyrosinemethyl ester HC1 (AMPT) and [NE] is the steady-state tissue norepinephrine concentration. K isclosely related to sympathetic nerve impulsetraffic21 •29-30 and was calculated from the rate ofdecline of the tissue [NE] according to the equation

K=(ln [NEJ-ln [NEt.0])/t

where [NE,_o] and [NE,] are the norepinephrineconcentrations at zero time and after 4 or 8 hours ofinhibition of synthesis with 300 mg/kg AMPT i.p.every 4 hours, in a solution of 60 mg/ml in 0.9%NaCl, which is known to inhibit tyrosine hydrox-ylase.29-30 For determination of K, we used all datapoints in three subgroups of rats killed at 0, 4, or 8hours after administration of AMPT, using the slopeof the In [NEJ-time regression equation. Norepi-nephrine turnover is the amount of norepinephrinereleased per gram of tissue per unit time, and iscalculated from the product of K times the endog-enous steady-state tissue [NE]. An individual Kwas estimated in each rat, based on the rate ofdecline from the animals' own zero time concentra-tion to the average [NE] of the group, after 4 or 8hours inhibition of synthesis. This method providesa reasonable estimate of the standard error of themean of the group's norepinephrine turnover rate,and makes allowance for the errors associated withthe determination of both K and [NE].

Data AnalysisWe found that a logistic function curve described

the relation between methoxamine concentrationand change in perfusion pressure; a computer pro-gram was used to fit the data points using thealgorithm of Marquardt.31 Each curve was charac-terized by the following parameters: 1) PPnumdai 2)ECJOJ

t ne concentration of methoxamine to reach50% of the pressure range of the curve betweenminimum and maximum pressures with methox-amine, 3) the maximum slope of the concentration-response curve with methoxamine, and 4) the rangebetween P P ^ & and P P ^ „„ with methoxamine orAng II. The slope of the logistic curve depends inpart on the range between the two plateaus (i.e.,PPmMcoo-PPmix<ni)» and we therefore also calculatedthe range-independent (normalized) slope of thelogistic function, where the range is regarded as100% under all conditions.32

The various age and strain differences were com-pared by one-way analysis of variance andcovariance.33 Individual comparisons betweenstrains at particular ages were done using an unpairedStudent's t test and, where appropriate, the Mann-Whitney test. A /?<0.05 was taken to indicatestatistical significance.

ResultsAge Relation to Body Weight, Blood Pressure,and Heart Rate

The relation between age and body weight wasclosely similar in SHR and WKY rats (Figure 2).The rate of body weight gain was about 25 g/wkover the period 4-14 weeks, compared with theaverage increase of about 4 g/wk over the subse-quent 36 weeks. The similar body weights in thepaired perfusion experiments ensured that, at any

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Adams et al Vascular and Cardiac Hypertrophy 195

40

g 30EI

_j

Q 20X

g-10

Q/100g BVV,

ml/mln/100g BW2 4 6 8

wk

10 15 20 0 5 10FLOW- ml/min/100a HQW

15 20

FIGURE 1. Left panel, relation ofperfusion pressure atmaximum dilatation (PPmaxdii) to flow (Q) in isolatedhindquarters of 4-week-old spontaneously hypertensiverats (SHR) (n=8), plotted as ml/min/100 g body wt (BW)(left line) and ml/min/100g hindquarter wt (HQW). In ratsof 75.5 g, PPmcx m corresponds to flows of 4 ml/min/100 gBWandto 14.6 ml/min/100 g HQW. PP^ m, at a flow of10 ml/min/100 g HQW lies at the tip of the arrowhead.Right panel, relation of PPn^du to flow (ml/min/100 gHQW) in 4-week-old SHR (lower solid line and symbols;n=8) and Wistar-Kyoto (WKY) rats (lower interruptedline and symbols; n=7) and in corresponding pooled datain 14- and 27-week-old SHR (n=10) and WKY rats(n=12). Points are expected means from the regressionlines ±SEM.

given age, strain differences in vascular resistanceproperties did not depend on differences in bodysize.

The difference in systolic BP between the twostrains increased progressively during the period of

rapid growth between 4 and 14 weeks (Figure 2).The difference was minimal and not significant at 4weeks (n=5 in each strain). We also performeddirect measurements of mean arterial pressure in4-week-old SHR and WKY rats, which was 113±5and 110 ±6 mm Hg, respectively (n=7 and 6; AN.S.), in agreement with the systolic BP findingswhen using tail-cuff measurements. At all timesafter and including 9 weeks, the systolic BP washigher in SHR than in WKY rats (/XO.OOOI). InWKY rats the "adult" BP value was reached byabout 9 weeks of age, whereas the correspondingvalue in SHR was reached by about 14 weeks(Figure 2). There was a progressive increase in thedifference in systolic BP throughout the period ofrapid growth, to a stable difference in the adult ofabout 75 mm Hg.

In both SHR and WKY rats, heart rate declinedwith increasing age at a rate of 1-2 beats/min/wk.The rate of decline was similar in both strains, butat all ages the heart rate was higher in SHR thanin WKY rats. The average heart rate over alltime intervals was 371 ±38.9 beats/min in SHRand 349±40.3 beats/min in WKY rats (A=22;F ( 1 , 2 6 4 ) = 2 3 . 2 , / J < 0 . 0 0 1 ) .

Heart WeightThe left ventricle plus septum weight/body weight

ratio and the ratio of right ventricular weight/bodyweight declined in both SHR and WKY rats from4 weeks to reach stable values at about 14-20weeks (Figure 2). At 4 weeks the left ventricle plusseptum weight/body weight ratio was only 8%higher in SHR than in WKY rats (p=0.05), risingto =*25% at 14 weeks and to a final value of =*40%

400

i

£3003H* 200ao" IOO

/f1

11•1 1 • 1 •

?240EE'.200oc

Q.

B 1 6 0

m

J1200)

80

•

/ ^

/r-*-n T^ , ^ .' ^ WKY

• • • • • • •

4 9 14 20 30 40 60AGE - weeks

FIGURE 2. Relation between age (weeks)and 1) (top left) body weight (g) in spon-taneously hypertensive rats (SHR) (n=126)and Wistar-Kyoto (WKY) rats (n=131); 2)(lower left) tail-cuff systolic blood pressurein the same SHR and WKY rats; 3) (upperright) left ventricle plus septum (LVS)weight/body weight ratio (g/kg), with aster-isks representing significant strain differ-ences; 4) (lower right) average values ofLVS and right ventricle (RV) weight/bodyweight ratio, expressed as percentages ofcorresponding values in WKY rats at eachage. In 3 and 4, number of SHR and WKYrats were: 4 weeks (5,5); 9 weeks (6,5); 14weeks (6,6); 20 weeks (5,5); 30 weeks (4,6);and 50 weeks (4,6).

4 0 14 20 30 40 60AGE - weeks

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196 Hypertension Vol 14, No 2, August 1989

400

xEI 300 •

ir3toUJ

tr 200

OCO3

100 •

WKY

^ SO _—— 20i

g

-4

SHR/

I

J

^ 60

r/T

30_ , . 9 n

" ' — 14

9

100 1METHOXAMINE - jjfl/ml

above the corresponding values in WKY rats after20 weeks (/?<0.001; Figure 2). At no time periodwas there a significant difference in right ventric-ular weight/body weight ratio between SHR andWKY rats (Figure 2).

Hindquarter Vascular Resistance PropertiesPerfusion pressure-flow relation. We obtained

perfusion pressure-flow curves in the fully dilatedvascular bed of 4-, 14-, and 27-week-old SHR andWKY rats (see Table 1 for numbers). In 4-week-oldrats of both strains, the relation between PPmaxdiiand flow was linear up to flow rates of about 25ml/min/100 g hindquarter wt (Figure 1 and Table 1).From analysis of covariance of the regression linesin SHR (n=8) and in WKY rats (n=7), there was nosignificant strain difference in regression coeffi-cients, but there was a significant difference inintercepts (Table l,p<0.001); P P ^ ^ was on aver-age 3.2 mm Hg higher in SHR than in WKY rats.The pressure-flow curves in 14- and 27-week-oldrats were linear up to flow rates of about 15 ml/mm/100 g hindquarter wt. Over this range, therelation was virtually the same at the two timeperiods, so that we pooled the results from bothgroups (Figure 1). Again, there were no significantdifferences in regression coefficients but the inter-cepts differed, with PP,™* ̂ on average 3.7 mm Hghigher in SHR than in WKY rats (Table l,p<0.001).The slopes of the 14- and 27-week-old SHR andWKY rats were about twice as steep as those of the4-week-old rats, indicating a greater resistance toflow in the older animals (Table 1 and Figure 1). Ata flow of 10 ml/min/100 g hindquarter wt, P P ^ da inthe young and older SHR averaged 17.3 and 27.8mm Hg, respectively, with the corresponding fig-ures in WKY rats 13.6 and 24.1 mm Hg (bothp<0.01). Thus, there was a similar age-relatedincrease in vascular resistance in both strains.

10 100

FIGURE 3. Methoxamine con-centration perfusion pressureresponse curves of hindquarters,with values adjusted to constantflow per unit hindquarter weight(see Materials and Methods).Each curve was reconstructedfrom average values of the curveparameters obtained in spontane-ously hypertensive rats (SHR)(right panel) and Wistar-Kyoto(WKY) rats (left panel) at 4, 9,14, 20, 30, and 50 weeks, asmarked on the right of eachcurve, n at each age were, inorder, SHR and WKY: 4 weeks(5,5); 9 weeks (6,5); 14 weeks(6,6); 20 weeks (5,5); 30 weeks(5,6); and 50 weeks (4,6).

In 4-week-old SHR, increasing flow beyond thelinear part of the pressure-flow curve resulted in aplateau value of P P ^ ^ of =37 mm Hg at highflows greater than 35-40 ml/min/100 g hindquarterwt. In WKY rats, the slope of the perfusion pressure-flow curve at these high flows was significantly lessthan in the more physiological range, but there wasno definite plateau at flows as high as 50 ml/min/100g hindquarter wt. The difference between SHR andWKY rats suggests that the hindquarter bed wasless distensible in young SHR. In the older SHR,we did not study flows greater than 20-25 ml/min/100 g hindquarter wt and the perfusion pressure-flow curves were of similar appearance in bothstrains.

Responses to methoxamine. In other SHR andWKY rats, we obtained dose-response curves tomethoxamine at different ages adjusted to constantflow perfusion of 10 ml/100 g hindquarter wt (seeMaterials and Methods) (Figure 3). When the hind-quarters were maximally dilated with papaverine,PPm«dii was higher at all ages in SHR than in WKYrats (p<0.01 except at 4 weeks, Figure 4). Therelation between age and PP,,** da was curvilinear ineach strain (Figure 4). The (PP^du in SHR)/(PPmo dii in WKY rats) ratio was similar at all agesand averaged 1.26.

Similarly, at every age PP^c*, was higher inSHR than in WKY rats (Figures 3 and 4). Onefinding common to both strains was that in 4- and9-week-old rats, the maximum methoxamine-induced constrictor response was below the maxi-mum constriction induced by Ang II or BaCl2 (Table2). But in rats at or older than 14 weeks, a radrenergic receptor stimulation elicited the samemaximum constrictor responses as supramaximaldoses of Ang II or BaCl2. The (PP^co,, in SHR)/(PPmocon in WKY rats) ratio (produced by eithermethoxamine or Ang II) was similar at all timeperiods and averaged 1.35.

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Adams et al Vascular and Cardiac Hypertrophy 197

FIGURE 4. Mean±SEM of parameters ofmethoxamine-perfusion pressure (PP)response curves of spontaneously hyper-tensive rats (SHR) and Wistar-Kyoto(WKY) rats at different ages. "Significantdifference between strains p<0.05. Param-eters were 1) PP^M at maximum dilata-tion; 2) PPmaxcan at maximum constriction(see Materials and Methods); and 3) ECX

dose of methoxamine at PP halfwaybetween PPmiu M and PP^ c^

1004 9 14 20 30

AQE - »••»€4 9 14 20 30

AQE - *••»•

In both SHR and WKY rats, we determined therelation between body weight on the one hand andPPm x̂dj and P P ^ e c on the other (Figure 5 andTable 1). Analysis of covariance showed that, withPPm»x dii, the slopes of the regression lines divergedsignificantly (p<0.05), and the slope in SHR wasabout 35% steeper than in WKY rats. Thus, in a60-g rat the P P ^ ^ averaged 16.1 ±0.9 in SHR and13.4±0.9 mm Hg in WKY rats (A=2.7 mm Hg;p=0.05); at the end of the rapid growth phase (400-grat), PPn^da in SHR was 31.8±0.7 mm Hg com-pared with 25.1±0.6 mm Hg in WKY rats (A=6.7mm Hg; p<0.001). By contrast, the slopes of thelines relating body weight to P P ^ ^ were only 16%steeper in SHR than in WKY rats (N.S., Table 1and Figure 5), with the perfusion pressure in 60-grats 69 mm Hg higher in SHR than in WKY rats, ascompared with an only slightly greater difference in400-g rats of 85 mm Hg (p<0.001 for both sets).

In both SHR and WKY rats, EQo decreasedprogressively after the age of 9 weeks, with thevalues in the two strains virtually identical afterabout 14 weeks (Figure 4). Between 4 and 14 weeks,EQo was significantly less in SHR than in WKYrats, so that only about 50% of the concentration ofmethoxamine was required to elicit the same con-strictor response (p=0.02).

The maximum slope of the sigmoid curve wassignificantly greater in SHR than in WKY rats, andin both strains there was a linear increase with age,with the two lines approximately parallel (Figure 4).The slope parameter is related to the perfusionpressure range between PPn^da and P P ^ a * (seeData Analysis under Materials and Methods), sothat overall the greater maximum slope in SHR

compared with WKY rats at a given age was partlydue to the greater perfusion pressure range in SHRat a given age. However, there was also a "true"increase in responsiveness in SHR between 4 and20 weeks, since their range-independent slope param-eter was significantly greater than in WKY rats(Table 3). At 30 and 50 weeks, however, the range-independent slope was similar in both strains (Table3), so that during this period the absolute value of

40

30

QX

20

0.Q.

10

400

SHR

z 300OOx

2 200a.a.

100L0100 200 300

BODY WEIGHT - g4 0 0

FIGURE 5. Relation between body weight and perfusionpressures at maximum dilatation (PPmm &J and maximumconstriction (PP^a*) in 31 spontaneously hypertensiverats (SHR) (•) and 32 Wistar-Kyoto (WKY) rats (o) aged4-50 weeks.

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198 Hypertension Vol 14, No 2, August 1989

TABLE 2. Differences Between Perfuskm Pressure at Maximum Constriction Produced by Methoxamine and byAngiotensin II

Age (wk)

49

14-50t

Methoxamine

176.5±4.19287.3±5.53347.0±5.00

SHR

Angiotensin II

231.5±4.82*314.8±7.15*347.0±5.00

Methoxamine

130.2±15.09210.0+9.51264.3+3.90

WKY

Angiotensin II

161.8+19.39*231.2±10.36*264.3+3.90

Values for differences in perfusion pressure (mm Hg) are mean±SEM. SHR, spontaneously hypertensive rats;WKY, Wistar-Kyoto rats.

*p for difference <0.05.tValues for methoxamine and angiotensin II responses between 14 and 50 weeks were similar at all times.

the slope of the SHR was higher only because ofelevation of the perfusion pressure range.

Regional Sympathetic FunctionIn the left ventricle plus septum there is a sub-

stantial sympathetic innervation of the myocar-dium. However, in skeletal muscle and kidney mostof the innervation goes to blood vessels.34-35

In the left ventricle plus septum, [NE] was nearlytwice as great in SHR as in WKY rats from 4 to 14weeks of age (Figure 6, /?<0.01). However, at 20weeks and older the [NE] was closely similar inboth strains. The left ventricle plus septum frac-tional rate constant K tended to be higher in SHRthan in WKY rats at most times between 4 and 50weeks, but the variability was large, so that, at theindividual time intervals, only the strain differencesat 9 and 50 weeks were statistically significant. Theaverage norepinephrine turnover (i.e., A"x[NE])was also higher at most ages in SHR than in WKYrats, but again because of the variability the differ-ence was statistically significant only at 4 and 9weeks.

Forelimb muscle [NE] was about 40% higher inSHR than in WKY rats during the rapid growthperiod from 4 to 14 weeks (/><0.01) (Figure 6), butafter that period the difference in tissue concentra-tion was much smaller and not significant. In con-trast to the findings in heart and kidney, K wasvirtually identical in SHR and WKY rats between 4and 30 weeks, but at 50 weeks K in SHR was about40% below the value observed in WKY rats. Nor-epinephrine turnover tended to be higher at 4 and 9

weeks in SHR than in WKY rats, but the differencewas significant only at 4 weeks (/?<0.01).

In the kidney, [NE], K, and norepinephrine turn-over were all higher in SHR than in WKY rats at mosttime periods (Figure 6). The only exception was at 20weeks when K and norepinephrine turnover were thesame as in WKY rats. On average, K and norepineph-rine turnover in SHR were, respectively, 1.66 and1.92 times the corresponding values in WKY rats.

DiscussionThe present study provides the most detailed

analysis to date of the time course of cardiovascularhypertrophy and sympathetic function in SHR dur-ing the first year of life. We found that: 1) a highervascular resistance in SHR compared with WKYrats was already established at 4 weeks, suggestingthat vascular hypertrophy was present at a timewhen there was no difference in BP between thetwo strains; 2) left ventricular hypertrophy laggedbehind the development of vascular hypertrophyand occurred in parallel with the rise in BP; 3) inboth strains a^-adrenergic receptor stimulation withmethoxamine did not elicit maximum hindquarterbed vasoconstriction until after 14 weeks of age;between 4-14 weeks the resistance vessels of SHRwere relatively hyperresponsive compared withthose of WKY rats, as assessed by the differencesin EQo and in range-independent slope; and 4) [NE]and turnover was increased in the heart, kidney,and skeletal muscle vessels of young SHR; in theskeletal muscle bed these changes were indepen-

TABLE 3. Comparison of Range-Independent and Maximum Slopes in Spontaneously Hypertensive Rats andWIstar-Kyoto Rats of Methoxamine/ConcentratJon-Response Curves

Age (wk)

49

14203050

Range-independent slope(normalized units)

SHR

119±16.990±4.6

122±11.8127+11.8130±3.6181±12.8

WKY

97±14.571±7.3*95 ±4.7*97±6.2

123±4.8186±25.8

Maximum slopeSHR

190±26.4234±15.3366±36.3397±40.8421±11.3616±43.0

(pressure/unit dose)WKY

123±28.5133±15.2*214±14.2'242±19.9*284±19.5*452±47.7*

*p for difference <0.05.Values are mean±SEM. SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats.

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Adams et al Vascular and Cardiac Hypertrophy 199

FORELIMB MUSCLES KIDNEY LEFT VENTRICLE & SEPTUM

tNE]

300

200

ng/g 100

00.2

Rata 0.1Constant

NE 20

Turnover10

(ng/g)/h

— 17S

50

25

04 9 14 20 30 60 4 9 14 20 30 50

AQE - weeks

SHR

WKY

4 9 14 20 30 SO

FIGURE 6. Relation betweenage and tissue norepinephrineconcentration [NE], fractionalNE rate constant and NE turn-over in forelimb muscles, kid-ney, and left ventricle and sep-tum. 'Significant differencesbetween strains, n of spontane-ously hypertensive rats (SHR)and Wistar-Kyoto (WKY) rats,respectively: 4 weeks (5,5); 9weeks (6,6); 14 weeks (6,6); 20weeks (5,5); 30 weeks (5,5); and50 weeks (7,5).

dent of increased neural activity, as assessed fromthe fractional rate constant.

Vascular and Cardiac ChangesIn our colony there were no significant differ-

ences at 4 weeks between SHR and WKY rats, ineither systolic or mean arterial BP. However, by 6weeks in the same colony, there was a definite risein mean arterial pressure, which increased in sub-sequent weeks in a manner similar to our systolicpressure findings.36 Gray's review37 of blood pres-sure changes in young SHR suggests that BP mayalready be raised in SHR during the neonatal period,but, in our experiments, there was no evidence ofthis at 4 weeks. Thus, our findings indicate that inSHR there already was considerable vascular hyper-trophy by 4 weeks, preceding the rise in BP. Later,there was a further increase in vascular hypertro-phy, suggesting that there also was a componentassociated with the rise in BP. But at 4 weeks, leftventricle plus septum weight in SHR was only 8%above that in WKY rats. Thus, before the rise inBP, left ventricular hypertrophy was slight, andover 90% of the rise in left ventricular weightfollowed the rise in BP.

We have assumed that the greater vascular resis-tance at full dilatation and during maximum con-striction in SHR is due to the greater muscle mass inthe media of their resistance vessels. Medial hyper-trophy in SHR is the main determinant of theincreased wall/lumen ratio of individual resistancevessels, which, by causing narrowing of the vesselcompared with the lumen of WKY rats, is respon-sible for the greater values of the vascular resis-tances at full dilatation and at maximum constrictionand for the greater slope of the stimulus-responsecurve. 1Aii^8 There is controversy concerning towhat extent anatomic "rarefaction" (i.e., reductionin vascular density of resistance vessels per unittissue volume) contributes to the strain differences inresistance properties.39-40 Experimentally,microsphere-induced anatomic rarefaction in the hind-

quarter bed causes elevation of P P ^ &, but leavesunaltered P P ^ ^ and slope.41 By contrast, anincreased wall/lumen ratio and associated vascularnarrowing increases all three parameters of the dose-response curve, both in individual small arteries42

and in the whole vascular bed.41

From the perfusion pressure-flow curves, thedifferences in PP,,^ ̂ between SHR and WKY ratswere similar at 4, 14, and 27 weeks (Figure 1).However, the main study, which encompasses awider age and body weight range, suggests that thedifference between the strains widened progres-sively and that at 4 weeks, the difference was abouthah0 that observed in adult rats (Figure 5). The twoseries included a substantial number of rats, thusproviding strong evidence that, by 4 weeks, a con-siderable amount of vascular hypertrophy had devel-oped. However, the strain difference in PPnuxcco a t 4weeks was only =20% below the value in adults(Figure 5). One reason why PP,^,™ in SHR wascloser to the final adult value than P P ^ ^ is that theamplification is accentuated during constriction,since resistance increases in proportion to the fourthpower of the radius. In addition, the stiffer vesselsin the young SHR compared with the WKY ratsmay accentuate the degree of luminal encroachmentduring maximum constriction.

We did not determine vascular resistance beforethe age of 4 weeks and extrapolation from the bodyweight-PPn^ da and body weight-PP,^ „„ relation inFigure 5 is somewhat hazardous. Had we extrapo-lated the body weight-PP^ ^ relation to a lowerbody weight value, we would have concluded thatthe amount of vascular hypertrophy soon after birthwas small or absent. However, the body weight-PPm»x con relation is consistent with some degree ofhypertrophy at, or soon after, birth. The anatomicliterature on this point is conflicting.43-44 To date,only the structure of large arteries has been exam-ined in these very young animals, with evidence ofhypertrophy in one study,43 but not in another.44

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200 Hypertension Vol 14, No 2, August 1989

Although the rise in P P ^ dn for a given increasein body weight was somewhat steeper in SHR thanin WKY rats, in each strain there was a substantialincrease in this parameter with increasing bodyweight and age (Figures 4 and 5). With increasingbody weight, most of the narrowing in averagecross-sectional area per unit tissue of the hindquar-ter bed was similar in SHR and WKY rats (e.g.,Figure 1). We do not know the mechanisms involvedin these body weight-related and age-related vascu-lar resistance rises, but each of the following couldplay a role: 1) reduction in the rats' metabolic rateduring the early postnatal period,45 which may beassociated with a reduced density of resistancevessels and a rise in resistance as the rat gets older;2) growth-related increases in the length of resis-tance vessels; 3) changes in the proportion of skel-etal muscle to fat in the hindquarters at differentstages of development; 4) changes in collagen andelastin composition of the fibrous matrix of thevessel, which affect wall distensibility2-3; and 5)changes in proportion of different isoforms of vas-cular smooth muscle actin, reflecting alterations inthe proportion of the various phenotypes of smoothmuscle in the vasculature.46

In the absence of autonomic support, the devel-opment of vascular hypertrophy ahead of left ven-tricular hypertrophy in SHR will result in somedegree of mismatching of the intrinsic properties ofthe vascular amplifier and the cardiac pumpingcapacity.5 This occurs with normal left ventricularmyocardial function per gram of myocardium, asindicated in studies performed under highly con-trolled conditions in renal hypertensive dogs5 and inSHR.4 The development of additional myocardialelements in left ventricular hypertrophy allows theheart to maintain a given stroke volume and cardiacoutput against a higher BP than is possible withouthypertrophy.5 In the event, there is enhanced leftventricular neural activity at an early age (Figure 6,rate constant data), which could provide sufficientinotropic support to raise BP. Possibly, the reasonwhy the pressure does not rise to higher values thanin WKY rats is that renal sympathetic activity isalso elevated at the time, so that there is still somedegree of mismatch.

Sympathetic InnervationAt most periods there was elevation in heart rate

and in regional sympathetic activity (assessed fromthe fractional norepinephrine rate constants) in heartand kidney, but sympathetic regional activity in themuscle was normal (Figure 6). Such a patternresembles the defense reaction.2'16-17 The elevationof cardiac sympathetic activity in young SHR willhelp to compensate for any inadequacy in intrinsicpumping capacity (see above) and later on, willprovide enhanced cardiac pumping once left ven-tricular hypertrophy has fully developed. In thehypertrophied vessels of the hindlimb bed, theamplifier properties will ensure that resting vascular

resistance is elevated, even at normal levels ofsympathetic activity. Assuming the early develop-ment of the renal vasculature is similar to that of thehindlimb, the rise of resting renal vascular resis-tance in SHR will tend to be relatively greater thanin the hindlimb. We cannot say from the availabledata how the defense pattern of sympathetic activ-ity will affect the mismatch between vascular ampli-fier and cardiac pump before left ventricular hyper-trophy has fully developed. However, at the lattertime, the elevated neural activity (fractional rateconstant) will accentuate the elevation of BP.

Tissue [NE] was raised in all three regions up to14 weeks of age, and in the kidney for most of the 50weeks. Chronic increases in sympathetic activityhave been shown to increase steady-state concen-trations of the enzymes tyrosine hydroxylase anddopamine ^hydroxylase, thereby increasing tissue[NE].47 In SHR, higher levels of both enzymes havebeen reported in the mesenteric vessels, comparedwith those in WKY rats.48 The effect of modulationof transmitter release on [NE] by, for example,increased reuptake42 are difficult to predict. How-ever, another mechanism capable of increasing tis-sue [NE] independently of neural activity (as in thecase in the muscle bed in our experiments) may bean increased innervation density in the vasculatureof young SHR, as recently demonstrated by Headand colleagues.49-50 Thus, both a chronic increase inneural activity and an increased innervation couldalter postjunctional properties and contribute to thelower ECJO (and threshold) and to the higher range-independent slope observed in 4-14-week-old SHRduring methoxamine stimulation (Figure 4, Table3). Neuromuscular coupling between vascular aradrenergic receptors and the contractile machineryof the smooth muscle cell develops slowly in bothSHR and WKY rats, as judged by the inability todevelop maximum constriction with methoxamineuntil they are above 14 weeks of age, as has alsobeen observed in rabbits.51 Below this age bothstrains have a lower response for a given level ofaradrenergic receptor stimulation than the corre-sponding group of older rats (Figures 3 and 4), butthe lower threshold and increased slope of youngSHR will tend to enhance the resistance vesselresponses, compared with those of young WKYrats.

Recent studies by Lee et al20 have shown thathypertension and hypertrophy are completely pre-vented by sympathetic ablation (see above), but wedo not know how this comes about. The observedpattern of sympathetic activity will elevate BPthrough enhanced constrictor and inotropicresponses once cardiovascular hypertrophy hasdeveloped, but it may have an additional trophicgrowth-promoting role at an earlier stage, which isindependent of the level of neural activity.52 Innormal rabbits in vivo, trophic effects of the sym-pathetic innervation have been found to contributeto the development of the smooth muscle of the ear

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Adams et at Vascular and Cardiac Hypertrophy 201

artery18 and to the development of medial hypertro-phy of cerebral vessels in SHR.53 In tissue culture,catecholamines stimulate growth through adrener-gic receptor-mediated mechanisms,19-54 but the pro-cess is complex and appears to involve interactionswith numerous growth factors.7'55

In the hindlimb bed, in contrast to the otherregions, elevation of [NE] and turnover occurs atnormal levels of sympathetic activity.47 From thetime course of P P ^ da, hypertrophy develops in theimmediate postnatal period and any trophic role ofthe sympathetic activity should be apparent by theage of 4-6 weeks. In the light of tissue culturestudies,19-53 our findings of high [NE] at 4 weeks inthe skeletal muscle bed provide a pointer of apossible trophic role of the sympathetic in earlyvascular hypertrophy. We may hypothesize that thetransiently increased density of sympathetic vascu-lar innervation49-50 elevate the amount of norepi-nephrine released in the tissue, where it can interactwith local growth factors released by immaturevascular smooth muscle.54-55 A more definitiveanswer must await further experiments.

Is There Primary Cardiovascular Hypertrophy?Our findings suggest that a significant component

of vascular hypertrophy precedes the elevation ofhypertension, while the rest occurs pari passu withthe rise in BP. The first can be regarded as theprimary component that is critical to the develop-ment of hypertension, while the additional laterhypertrophy occurs as the conventional adaptiveresponse associated with the pressure rise. Bycontrast, there is little, if any, primary left ventric-ular hypertrophy and this process appears to bepredominantly adaptive.

A priori we would expect that the simultaneousestablishment of cardiovascular hypertrophy willproduce an almost immediate elevation of BP, dueto the properties of the vascular amplifier. How-ever, though the latter was well established by 4weeks, we observed no rise in BP at this time,though hypertension developed soon afterwards(e.g., Reference 37). We do not know either thereason for the small delay, despite the defensepattern of sympathetic activity, or why it took14-20 weeks before the adult BP level was reachedin SHR. Factors discussed earlier, such as themetabolically related high vascularity in the youngrat, slow development of adrenergic receptor cou-pling to the contractile apparatus of the smoothmuscle cell, and mismatch between left ventricularpumping capacity and vascular impedance may allplay a role in delaying the manifestation of thevascular amplifier on the BP of SHR.

AcknowledgmentsWe are grateful to Susan Timmins for her able

technical assistance and to Judith Segal, FrancesCribbin, and Tessa Morton for their help in prepar-ing the manuscript.

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KEY WORDS • primary hypertension • hypertrophy •vascular resistance • norepinephrine • spontaneouslyhypertensive rats

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M A Adams, A Bobik and P I KornerRelation to sympathetic function.

Differential development of vascular and cardiac hypertrophy in genetic hypertension.

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