Increased Rigidity of Red Blood Cell Membranein Young Spontaneously Hypertensive Rats

ANNE CHABANEL, DAVID SCHACHTER, AND SHU CHIEN

SUMMARY The micropipette test was used to study the effects of age on the elasticity of red bloodcell (RBC) membrane in spontaneously hypertensive rats (SHR) and age-matched normotensiveWistar-Kyoto rats (WKY), ranging from 3 to 23 weeks of age. The development of hypertension in theSHR started at 3 weeks and was fully established at 7 to 8 weeks. In the developmental phase ofhypertension (3-5 weeks), the SHR showed a significant increase in RBC membrane elastic modulus(i.e., a decrease in RBC membrane deformability) when compared with the age-matched normoten-sive control rats (WKY). After the establishment of hypertension (7-8 weeks), however, the deforma-bility of the RBC membrane of SHR improved and became comparable to that of the WKY. Theseresults indicate that abnormal erythrocyte membrane elasticity is an early event in SHR and thatadaptive recovery occurs when hypertension is fully developed. (Hypertension 10: 603-607, 1987)

KEY WORDS • erythrocyte deformability • erythrocyte membrane elasticityhypertension • spontaneously hypertensive rats

SPONTANEOUSLY hypertensive rats (SHR) areregarded as the best animal model of humanessential hypertension.1 In patients with essen-

tial hypertension, blood viscosity can be correlatedwith both systolic and diastolic pressure.2 Moreover,in essential hypertensive patients with high plasmarenin activity, blood viscosity is significantly elevated,and the increase has been attributed in part to reducedred blood cell (RBC) deformability.3 Erythrocyte fil-terability is reduced in subjects with untreated essentialhypertension.4 The diameter of RBCs is generally larg-er than that of precapillaries or capillaries. Under hy-pertensive conditions with an increase in vessel tone,RBC deformability could be a limiting factor in RBCtransit through the microcirculation. Therefore, thepresent experiments were undertaken to investigate ifRBC deformability is altered in the animal model ofhypertension (SHR), as determined by measuring theRBC membrane elasticity with the micropipette tech-nique.

From the Department of Physiology and Cellular Biophysics,College of Physicians and Surgeons, Columbia University, NewYork, New York.

Supported by National Institutes of Health Grants HL 16851 andAM 21238.

Address for reprints: Dr. S. Chien, Department of Physiologyand Cellular Biophysics, College of Physicians and Surgeons, Co-lumbia University, 630 West 168th Street, New York, NY 10032.

Received January 12, 1987; accepted July 21, 1987.

Materials and MethodsPreparation of Red Blood Cell Suspensions

Male SHR (Tac: N[SHR]fBR) and Wistar-Kyotocontrol rats (Tac:N[WKY]fBR) were obtained fromTaconic Farms (Germantown, NY, USA). Three se-ries of experiments were performed. In the first series,two age groups were studied: 4 and 8 weeks old. Tenanimals of each group were killed, and the exsanguin-ated blood was heparinized; the organs were used for astudy on calcium-binding protein.5 In the second seriesof experiments, to further document the age-relatedvariation of RBC mechanical properties, blood wasdrawn through venous puncture from 10 awake SHRand 10 awake WKY and pooled into heparinized tubes.The animals were kept alive and fed a nutritionallycomplete pellet diet (Camm Maintenance Rodent Diet,Camm Research Institute, Wayne, NJ, USA; 0.9%Ca, 0.8% P), with water ad libitum. Blood was drawnfrom these animals at intervals beginning at the age of3 weeks and continuing to 23 weeks of age. In the thirdseries of experiments, blood was taken from anesthe-tized animals by heart puncture from 3 SHR and 3WKY at 3, 5, and 7 weeks of age and organs wereisolated for studies on calcium-binding protein.5

Systolic blood pressure (SBP) was measured by tailsphygmomanometry, after the awake animals hadbeen warmed for 15 minutes (29 °C). On each occa-sion, two determinations of SBP were performed and

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604 HYPERTENSION VOL 10, No 6, DECEMBER 1987

the values were averaged. For the micropipette test,the pooled blood was centrifuged at 1500 g for 5 min-utes and the plasma and buffy coat were removed. Theerythrocytes were washed three times in a standardwash buffer composed of 8 mM sodium phosphate,145 mM NaCl, and 5 mM KC1 (pH 7.4). The washederythrocytes were resuspended in the wash buffer con-taining 0.0025% bovine serum albumin to yield a he-matocrit of 0.01%.

The mean corpuscular volume (MCV) of RBCs wascalculated from the hematocrit value determined in amicrocentrifuge (15,000 g for 5 minutes) divided bythe RBC count measured in an electronic counter(Model ZB> Coulter Electronics, Hialeah, FL, USA).

Micropipette TechniqueThe micropipette technique has been described else-

where.6 Micropipettes with an internal radius of 0.45to 0.70 fim were prepared with a micropipette puller(Narishige Scientific Instrument Laboratory, Tokyo,Japan). The micropipette was filled with the buffersolution and mounted on a micromanipulator (Nari-shige). The wide end of the micropipette was connect-ed to a pressure-regulating system, which consisted oftwo reservoir bottles and a damping chamber. By ad-justing the relative heights of the reservoir bottles witha micrometer device, desired pressure levels were pre-set and then imposed on the micropipette by turning astopcock. The applied pressure was measured with atransducer (Model 23 BC, Statham Instruments, Ox-nard, CA, USA) and recorded with an amplifier-re-corder system (Gould, Cleveland, OH, USA). A sus-pension of erythrocytes at a hematocrit level ofapproximately 0.01% was placed in a small roundchamber located on the stage of a Nikon inverted mi-croscope (Ehrenreich Photo-Optical, Garden City,NY, USA). The erythrocytes were viewed with the useof a 100 X objective and a 20 x eyepiece. The imagewas recorded with a video camera and a tape recordersystem (Panasonic, division of Matsuchita ElectricCorp. of America, Franklin Park, IL, USA). The mi-cropipette tip was manipulated for positioning at thesurface of the erythrocyte membrane. A small portionof the erythrocyte was aspirated by a preset negativepressure for 20 seconds. The length of the aspiratedtongue and the radius of the pipette were measured on atelevision screen. The membrane elastic modulus,which reflects the steady state resistance to deforma-tion, was calculated from the relationship between thestress applied, (AP)RP, and the strain induced,DpJRp,6 where AP is the applied negative pressure, RPis the internal radius of the micropipette, and Dp,,, is themaximum length of the aspirated portion within themicropipette. When the aspiration pressure was re-moved, the deformed erythrocyte segment in the mi-cropipette decreased in length with time and the cellrecovered its original shape. All measurements weremade at room temperature (21-24°C). For each ex-periment the same micropipette was used to test RBCsfrom SHR and WKY. Statistical analysis of the datawas performed using a Wilcoxon rank sum test and apaired Student's t test.

ResultsThe increase in SBP with age is shown in Figure 1.

These data were obtained from the same sets of ani-mals studied over a 20-week period starting at the ageof 3 weeks. The only test performed on the rats wasdrawing 0.2 to 0.8 ml of blood from tail vein punctureat intervals. The SBP was always higher in SHR thanin WKY, and the difference was already significant at5 weeks of age (p< 0.005). After the sharp initial risein pressure over the first 10 weeks, the blood pressureincreased more slowly thereafter.

The variation in MCV is shown in Figure 2. MCVdeclined with age in both WKY and SHR. Both sets ofanimals exhibited a sharp initial decrease in MCV untilthe age of 9 weeks, followed by a plateau. The MCV ofthe erythrocytes from SHR was always smaller thanthat from the WKY (from 3 to 10 weeks of age;p<0.05).

The values for the elastic modulus as determined bythe micropipette test are summarized in Table 1. At 3,4, 5, 7, and 8 weeks of age, the values given in Table 1

Q 140Oo

3 5 7 13 15 17 19 21 23

AGE (weeks)

FIGURE 1. Systolic blood pressure of SHR and WKY versusage.

o 702

0 3 5 7 a 11 13 IS 17 IS 21 23

AQE Iraki)

FIGURE 2. Mean corpuscular volume (MCV) of erythrocytesfrom SHR and WKY versus age.

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ERYTHROCYTE DEFORMABILITY IN HYPERTENSION/Chabanel et al. 605

TABLE 1

Age(wk)

3

4

5

7

8

10

14

19

23

Values*p<0

sum test)

Elastic Modulus of RBC Membrane

Elastic(io-3

WKY

6.5±0.85.0±0.3

4.8±0.3

7.3±0.6

5.1±0.3

3.6±0.2

4.3 + 0.5

5.2±0.5

2.8±0.3

are means ±05, compared

modulusdyn/cm)

SHR

9.0±l.l*

7.5±0.5*

6.4±0.6*7.3±0.53.7 + 0.24.2±0.23.8±0.35.4±0.5

2.6±0.2

SEM.with values for

No.of

cells

20

28

26

22

20

15

12

10

12

WKY (by

SHR/WKYratio

1.4±0.1

1.5±0.1

1.3±0.1

1.0±0.0

0.8±0.2

1.2±0.1

0.9±0.3

1.0±0.1

0.9±0.1

Wilcoxon rank

'Eo>.

T3

bX

IC

MO

DU

LI

1-

8.0-

A

6.0-

T4.o i r*~

2.0-

Tn

4 weeks old

u • ~

^ 1 ~~ 2 3

B 8 weeks ok6.0-

are the means of two or three experiments performedwith different sets of rats on different dates. For eachof these experiments the results exhibited the sametrend as that of the means (Figure 3). Comparisons ofthe values for the elastic modulus between SHR andWKY were made at each age group. In Figure 4 theratio of the elastic modulus for the erythrocyte mem-brane of SHR to the value of the corresponding WKYcontrol is plotted against the age of the rats. Compari-son of these ratios not only facilitates the assessment ofthe difference between SHR and WKY at differentages but also minimizes any possible experimental er-rors (e.g., those due to variations in the radius of thepipette used in the different experiments), since thesame pipette was used for individual experiments onboth groups. The results of Figure 4 show that themembranes of erythrocytes from the young SHR weremore rigid than those of the age-matched WKY andthat this difference disappeared at about 7 weeks ofage.

DiscussionNo consensus exists as to the cause and effect rela-

tionship between the elevation in blood pressure andthe associated rheological and microcirculatory distur-bances during the evolution of the hypertensive syn-drome. Blood and plasma viscosities are increased inpatients with essential hypertension, even in the earlyphase of borderline elevation of arterial pressure.2 Inthe animal model of hypertension, the SHR, markedcardiovascular alterations occur in the neonatal stage.7

These findings suggest that changes begin in the prehy-pertensive stage and that some genetic factor may beinvolved. Our finding of a significant increase of RBCmembrane rigidity in the SHR before the definitiveestablishment of the hypertension indicates that achange in the membrane of the erythrocyte presagesthe established hypertension. The viscoelasticity of theRBC membrane is determined mainly by the proteinskeleton lining the cytoplasmic side of the membrane.We did not find any significant difference in the RBCmembrane protein profile between SHR and WKY, as

4.0-

2.0n

FIGURE 3. Results of individual experiments (numbered 1, 2,and 3). Membrane elastic modulus of erythrocytes from WKY(shaded bars) and SHR (open bars) at 4 (A) and 8 weeks of age(B). On average 10 cells were tested in each rat group for everyexperiment. For each of these experiments, the results exhibitedthe same trend as that of the means (see Table 1).

determined by sodium dodecylsulfate gel electropho-resis of the RBC membrane (data not shown). Thus,proteins of the membrane skeleton are present in equalamounts in SHR and WKY. The major protein of theRBC membrane skeleton is spectrin, and the state ofspectrin self-association appears to be of importancefor the viscoelasticity of the membrane.8 We thereforeused nondenaturing gel electrophoresis to determinethe state of protein oligomerization in the membrane.9

We did not find any difference between SHR andWKY at ages 3, 5, 7, and 23 weeks (data not shown).

Abnormalities of calcium binding to the RBC mem-brane have been detected in the SHR.10 A number ofcalcium abnormalities have been observed in the plas-ma membrane of various cell types, even in younganimals before the rise in blood pressure.5" Thesechanges may increase the amount of ionized calciumavailable for cross-linking of membrane proteins andconsequently influence the rigidity of the RBC mem-brane skeleton.12

In our study the SHR, in comparison with WKY,had a smaller MCV and a greater RBC membranerigidity prior to 7 to 9 weeks of age, the developmentalstage of arterial hypertension (see Figures 1-4), sug-

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606

FIGURE 4. Membrane elastic modulus of

erythrocytesfrom SHR versus age. The elasticmodulus is expressed as the ratio of the valuefor SHR to that for the normotensive WKY.

HYPERTENSION

2.0 -,

1.5-

VOL 10, No 6, DECEMBER 1987

CO

_ J

O 1.02oCO

0.5 J

• i - -1-

9 11 13 15 17 19 21 23

AGE (weeks)

gesting an association between these parameters. Be-cause of their greater rigidity, the RBCs from the SHRmay have to exit from the bone marrow at a smallerMCV. This early abnormal membrane rigidity mayalso result in an abnormal blood rheology and mayaccount for the ventricular hypertrophy present in SHRat a very early age.13 In human essential hypertension,the left ventricular mass has been found to be signifi-cantly correlated with blood viscosity.14 The increasedrigidity of the RBC membrane of SHR does not seemto affect the survival of the cells, since the RBC half-life survival time is identical for SHR and WKY.15

Our finding provides new evidence in support of agenetic predisposition in the SHR. The later recoveryof RBC membrane elasticity may be an adaptive pro-cess similar to that found for arteriolar distensibility.Karr-Dullien et al.16 found an enhanced distensibilityof the arterioles of newborn SHR, whereas others haveobserved a decreased distensibility in adult SHR.17

Karr-Dullien et al.16 also proposed that the early in-crease in vessel wall distensibility may lead to arterio-lar wall thickening (adaptive response), which laterresults in decreased distensibility. An increased activ-ity of the sympathetic nervous system has been ob-served in young SHR, as shown in particular by theelevation of the plasma level of dopamine-/3-hydroxy-lase (D/3H). In contrast, the plasma level of D/3H isidentical in 14-week-old SHR and WKY.1819 Al-though the age-dependent release of D/3H in the plas-ma parallels the change in RBC membrane rigidity, theeffect of D/3H, or other factors, on RBC rigidity re-mains to be investigated.

Our results show the importance of studying theRBC membrane in the early stage of hypertension. Bycontributing to the increase in vascular resistance, anabnormal RBC membrane deformability may be acause of the initial increase in blood pressure. At a laterstage, however, the RBC membrane deformability isnormalized and does not continue to contribute to thesustained elevation of arterial pressure.

AcknowledgmentsThe authors thank Daniel Batista and Juan Rodriguez for their

patience and expert technical assistance.

8

10

11

12

References1. Yamori Y. Development of the spontaneously hypertensive rat

(SHR) and of various spontaneous rat models, and their impli-cations. In: de Jong W, ed. Experimental and genetic models ofhypertension. Amsterdam: Elsevier Science Publishers,1984:224-239 (Handbook of hypertension: vol 4)

2. Letcher RL, Chien S, Pickering TG, Laragh JH. Elevatedblood viscosity in patients with borderline essential hyperten-sion. Hypertension 1983;5:757-762

3. Chien S. Blood rheology in hypertension and cardiovasculardiseases. Cardiovasc Med 1977;2:356-360

4. Zannad F, Voisin P, Pointel JP, Schmitt C, Freitag B, StoltzJF. Effects of ketanserin on platelet function and red cell filter-ability in hypertension and peripheral vascular disease. J Car-diovasc Pharmacol 1985;7(suppl 7):S32-S34

5. Kowarski S, Cowen LA, Schachter D. Decreased content ofintegral membrane calcium binding protein (IMCAL) in tissuesof the spontaneously hypertensive rat. Proc Nat! Acad Sci1986;83:1097-1100

6. Chien S, Sung KLP, Skalak R, Usami S, Tozeren A. Theoreti-cal and experimental studies of viscoelastic properties of eryth-rocyte membrane. Biophys J 1978;24:463-487

7. Gray SD. Spontaneous hypertension in the neonatal rat. ClinExper Hypertens [A] 1984;6(4):755-781Chabanel A, Sung KLP, Rapiejko J, et al. Effect of alteredspectrin dimer self association on erythrocyte membrane vis-coelasticity [Abstract]. Blood 1985;66:29aLiu SC, Palek J, Prchal JT. Defective spectrin dimer-dimerassociation in hereditary elliptocytosis. Proc Nat! ACad SciUSA 1982;79:2072-2076Postnov YV, Orlov SN, Pokudin NI. Decrease of calciumbinding by the red blood cell membrane in spontaneously hy-pertensive rats in essential hypertension. Pflugers Arch1979;379:191-195Devynck MA, Pemollet MG, Nunez AM, Meyer P. Alteredcalcium binding by plasma membranes from various tissues inyoung SHR rats. Clin Exp Hypertens 1981;3:797-807Palek J, Liu SC. Dependence of spectrin organization in redblood cell membranes on cell metabolism: implications forcontrol of red cell shape, deformability, and surface area. Se-min Hematol 1979;16:75-93

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ownloaded from

ERYTHROCYTE DEFORMABILITY IN HYPERTENSION/CT^ane/ et al. 607

13. Cutilleta AF, Benjamin M, Culpepper WS, Oparil S. Myocar-dial hypertrophy and ventricular performance in the absence ofhypertension in spontaneously hypertensive rats. J Mol CellCardiol 1978; 10:689-703

14. Devereaux RB, Drayer JIM, Chien S, et al. Whole bloodviscosity as a determinant of cardiac hypertrophy in systemichypertension. Am J Cardiol 1984;54:592-595

15. Sen S, Hoffman GC, Stowe NT, Smeby RR, Bumpus FM.Erythrocytosis in spontaneously hypertensive rats. J Clin In-vest 1972;51:710-714

16. Karr-Dullien V, Blosnquist El, Beringer T, El-Bermani AW.

Flow pressure relationships in newborn and infant spontane-ously hypertensive rats. Blood Vessels 1981 ;18:245-252

17. Folkow B. Constriction-distension relationships of resistancevessels in normo- and hyper-tension. Clin Sci 1979;57:235-255

18. Grobecker H, Roizen MF, Weise V, Saavedra JM, Kopin IJ.Sympathoadrenal medullary activity in young, spontaneouslyhypertensive rats. Nature 1975;258:267-268

19. Nagatsu T, Ikuta K, Numata Y, et al. Vascular and braindopamine /3-hydroxylase activity in young spontaneously hy-pertensive rats. Science 1976; 191:290-291

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A Chabanel, D Schachter and S ChienIncreased rigidity of red blood cell membrane in young spontaneously hypertensive rats.

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1987 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/01.HYP.10.6.603

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