Roos, Srecko Gajovic, Francisco E. Baralle and Andrés F. MuroMartín L. Marro, Oscar U. Scremin, Maria C. Jordan, Ly Huynh, Fabiola Porro, Kenneth P.

Deficient Mice−-AdducinβHypertension in

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Hypertension in b-Adducin–Deficient MiceMartın L. Marro,pOscar U. Scremin,pMaria C. Jordan, Ly Huynh, Fabiola Porro, Kenneth P. Roos,

Srecko Gajovic, Francisco E. Baralle, Andres F. Muro

Abstract—Polymorphic variants of the cytoskeletal protein adducin have been associated with hypertension in humans andrats. However, the direct role of this protein in modulating arterial blood pressure has never been demonstrated. Toassess the effect ofb-adducin on blood pressure, ab-adducin–deficient mouse strain (2/2) was studied and comparedwith wild-type controls (1/1). Aortic blood pressure was measured in nonanesthetized, freely moving animals with theuse of telemetry implants. It is important to note that these mice have at least 98% of C57Bl/6 genetic background, withthe only difference from wild-type animals being theb-adducin mutation. We found statistically significant higher levelsof systolic blood pressure (mm Hg) (mean6SE values:2/2: 126.9461.14, n55;1/1: 108.0662.34, n56;P#0.0001),diastolic blood pressure (2/2: 83.5461.07;1/1: 74.8762.23;P#0.005), and pulse blood pressure (2/2: 43.3261.10;1/1: 33.1961.96;P#0.001) in b-adducin–deficient mice. Western blot analysis showed that as a result of theintroduced genetic modification,b-adducin was not present in heart protein extracts from2/2 mice. Consequently, thisdeficiency produced a sharp decrease ofa-adducin and a lesser reduction ing-adducin levels. However, we foundneither cardiac remodeling nor modification of the heart function in these animals. This is the first report showing directevidence that hypertension is triggered by a mutation in the adducin gene family.(Hypertension. 2000;36:449-453.)

Key Words: hypertension, geneticn adducinn mice, knockoutn echocardiographyn cytoskeletonn telemetryn electrocardiography

The adducin protein family is composed of 3 membersencoded by closely related genes:a-, b-, and

g-adducin.1,2 Thea andb subunits, but not theg subunit, arefound in membrane cytoskeletons of human erythrocytes atthe actin-spectrin junctions as a mixture of heterodimers andheterotetramers. Combinations ofa/b anda/g oligomers arefound in the actin cytoskeleton at cell-cell contact sites inother cells.2,3

Adducin, an unexpected cytoskeletal player as blood pres-sure (BP) modulator, has been the subject of several associ-ation studies that attempted to link adducin variants tohypertension. Since the publication of the first positiveassociation of adducin polymorphism with human hyperten-sion,4 contradictory results have appeared in the literature. Infact, the humana-adducin gene polymorphism (G460W) hasbeen found to be significantly associated with hypertensionand salt sensitivity in certain patient groups,4–8 although thisobservation was not confirmed by other groups.9–14

The attention to adducin in hypertension was originallydrawn by a rat model (Milan hypertensive strain [MHS]),after our characterization of thea- and b-adducin–specificvariants associated with high BP levels.15 These mutatedadducins lead to a higher level of filamentous actin, enhancedactin bundling in cell-free systems, and increased Na-K pump

activity when transfected into kidney epithelial cells.16 How-ever, in both humans and rats, there has been no proof fordirect involvement of any of the adducins in the modulationof BP. Furthermore, adducin polymorphism cosegregationanalysis and kidney cross-transplantation experiments in theMHS rat model showed that, in a manner similar to thatobserved in humans,4,5,7adducin accounted for only a portionof the hypertension.15 Therefore, the simultaneous action ofother genes and other organs may be involved in the primarycause of hypertension in both human and rats.15,17

The knockout technology allows elimination of all differ-ences in genetic background except for the desired mutation.Taking advantage of this possibility, we have created ab-adducin–deficient mouse strain by targeted disruption ofthe b-adducin gene.18 The shape and osmotic fragility of redblood cells (RBCs) of homozygous mutant animals werealtered, and consequently the mice suffered from a mildanemia with compensated hemolysis, similar to humanspherocytic hereditary elliptocytosis.18

Our knockout model also gave us the opportunity to studythe direct association betweenb-adducin and hypertension ina well-defined genetic background. Therefore, we studied theeffect of b-adducin deficiency on arterial BP, and we foundthat b-adducin–mutant animals developed hypertension. We

Received February 1, 2000; first decision February 28, 2000; revision accepted April 10, 2000.From the International Center for Genetic Engineering and Biotechnology, Trieste, Italy (M.L.M., F.P., S.G., F.E.B., A.F.M.); Department of

Physiology, School of Medicine, University of California at Los Angeles (O.U.S., M.C.J., L.H., K.P.R.); Veterans Affairs GLA Healthcare System, LosAngeles, California (O.U.S., L.H.); and Department of Histology and Embryology, School of Medicine, University of Zagreb, Croatia (S.G.).

pDr Marro and Dr Scremin contributed equally to this work.Correspondence to A.F. Muro, ICGEB, Padriciano, 99, 34012 Trieste, Italy. E-mail muro@icgeb.trieste.it© 2000 American Heart Association, Inc.

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also studied cardiac remodeling and heart function throughpostmortem analysis and in vivo dynamic assessment of leftventricular dimensions and electrocardiography to discoverany possible pathology associated with hypertension or apossible effect of theb-adducin deficiency on excitable cellmembrane ion channels.

Methodsb-Adducin–Deficient MiceThe b-adducin gene was disrupted (knocked out) as describedpreviously.18 This strain was crossed with C57Bl/6 mice for 5generations to obtain a homogeneous background (theoretically98%). Mice used in this study (wild-type and2/2) were 8 to 10months old.

BP DeterminationsArterial BP was measured in 52/2 and 61/1 nonanesthetized,freely moving animals with the use of telemetry implants (PA-C20BP device, Data Sciences International) for a period of 6 hours. BPdeterminations started 30 minutes after discontinuation of halothaneanesthesia. The first hour was considered from 30 minutes afterdiscontinuation of anesthesia until 59 minutes later, the second hourwas from 60 minutes after discontinuation of anesthesia until 119minutes later, and so on every 60 minutes. During this time,abdominal aortic BP waveforms were continuously sampled, and thevalues are means of 5-second periods every 3 minutes (a total oftwenty 5-second periods per hour, except that in the first hour onlyten 5-second periods were considered). The device calculated a meanof all individual pulses ('40 pulses) during that time. Therefore,every hourly mean includes'800 pulses.

ElectrocardiographyTwo platinum needle electrodes were implanted in the subcutaneoustissue overlying the right scapula and the apex of the heart in 52/2and 71/1 halothane-anesthetized mice. Halothane was then discon-tinued; the ECG was recorded for 30 minutes with the use of ICM-01amplifiers on a Gilson polygraph and digitized with a LabmasterA-D board at 10 KHz. Analysis of ECG parameters was performedwith Axotape software (Axon, Inc).

Heart Morphology and MassMeasurements of heart mass and volume of ventricular walls weremade by 2 methods: planimetry of histological section images andweight of dissected ventricles. In the first (62/2 and 51/1 mice),hearts were removed, flash-frozen in methylbutane at dry icetemperature, embedded in OCT compound (Miles Laboratories),serially cut in 20-mm slices perpendicular to their long axis on aMicrohm cryostat (Carl Zeiss), and stained with hematoxylin-eosin,and images of slices were digitized. Thickness and total area of the

ventricular walls were measured with IPPLUS software (MediaCybernetics, Inc). Volume of ventricular walls was calculated fromslice areas and interslice distances.

In a separate series of animals (72/2 and 81/1 mice), heartswere dissected to eliminate the great vessels and atria. The remainingleft and right ventricles were opened to discard any blood contentsand weighed.

EchocardiographyMice (6 1/1 and 5 2/2) were sedated with Avertin (2,2,2-tribromethanol, 2.5% solution, 0.016 mL/g body mass, AldrichChemical Co). Two-dimensionally guided M-mode and Dopplerimages were obtained with a 7.5-MHz probe and 10-mm standoff onan ATL Apogee CX200.19,20 Animals were positioned in the supineor left decubitus position. The images were digitized for analysis(SigmaScan software, Systat, Inc) of left ventricular dimensionsduring systole and diastole. Ejection times were determined fromflow velocity profiles of the aorta.

Western Blot AnalysisHeart homogenates were electrophoresed on a 10% Laemmli gel,blotted onto nitrocellulose membranes, probed with rabbit anti–a-,b-, andg-adducin polyclonal antibodies (1:500, 1:1000, and 1:1000dilution, respectively), and analyzed by densitometry, as previouslydescribed.18 The experiments were repeated, and identical resultswere obtained with 2 independent protein preparations.

Creatinine and Kidney Weight AnalysisFresh blood from wild-type andb-adducin2/2 mice (10 mice pergroup) was collected in Eppendorf tubes. Creatinine levels wereanalyzed according to standard methods. For kidney weight analysis,mice were killed (9 mice per group) and weighed; then the kidneyswere removed, and left and right kidneys were weighed separately.Data of kidney weight correspond to the sum of both kidneys and areexpressed as percentage of total animal weight.

Statistical MethodsData analysis was performed with the use of the software packageStata, version 6.0 (StataCorp, 1999). The data were summarized withthe mean as a measure of central tendency and the standard error asa measure of dispersion. The difference in BP between the wild-typeand mutant mice was assessed by a repeated-measures ANOVAaccording to a split-plot factorial design. AP value of 0.05 waschosen as the limit of statistical significance. Student’st test wasused for the rest of the analyzed parameters.

ResultsWe studied the effect ofb-adducin deficiency on arterial BPby comparing wild-type andb-adducin knockout mice. Themutant strain was backcrossed with C57Bl/6 mice for 5

Figure 1. BP determinations (mean and SE) inb-adducin–mutant mice. Every variable wasrecorded over 6 hours after discontinuation ofanesthesia, as described in Methods ('5400measurements per mouse). Inset, Means ofpulse BP (systolic2diastolic; left) and heart rate(right). 2/2 indicates b-adducin knockout mice;1/1, wild-type mice; SYST, systolic; andDIAST, diastolic. *P#0.005; **P#0.001;***P#0.0001.

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generations to obtain a homogeneous genetic background(theoretically 98%). Means of systolic, diastolic, and pulseaortic BPs, as well as heart rate, were calculated from thepressure waveforms provided by the telemetry system (seeMethods) in awake, free-roaming mice. BP was stable duringthe recording period, indicating steady state conditions (Fig-ure 1). Statistically significant increases in systolic BP(mm Hg) (mean6SE values:2/2: 126.9461.14, n55;1/1:108.0662.34, n56;P#131024; 17.47% increase), diastolicBP (2/2: 83.5461.07;1/1: 74.8762.23,P#0.005; 11.58%increase), and pulse BP (2/2: 43.3261.10; 1/1:33.1961.96;P#0.001; 30.51% increase) were observed inb-adducin–deficient mice with the use of repeated-measures

ANOVA (Figure 1). However, heart rate was not signifi-cantly higher in mutant mice (Figure 1, inset). Repeated-measures ANOVA of the BP data showed that there was anonsignificant effect for time and for the interactionmutation-by-time in BP determinations (data not shown),except for a significant effect for time in the case of pulse BP.Further data analysis showed that the differences between thetime point at hour 1 and the time points from hours 3 to 6account for the overall significant effect of time for pulse BP.In fact, if the data between hours 2 to 6 are considered, thereis no effect of time for pulse BP (Figure 1), suggesting that itmight be generated by a residual effect of anesthesia in thefirst hour after its discontinuation.

We analyzed the expression levels of the different adducinsubunits (a, b, and g) in heart protein extracts by Westernblot. No b-adducin (normal or deleted form) was detected intotal heart homogenates ofb-adducin2/2 mice (Figure 2A).By using an anti–a-adducin antibody, we detected a sharpdecrease (70%) in the amount of this subunit in2/2 mice(Figure 2B). In addition, when we used an anti–g-adducinantibody, we observed a downregulation (50%) ofg-adducinlevels in these mice (Figure 2C). Although adducin mRNAshad been previously found in hearts of some organisms,2,21,22

the presence ofa-, b-, andg-adducin protein in this tissue hasnever been reported.

Qualitative histological analysis showed no obvious mor-phological differences between hearts from wild-type andmutant mice (not shown). The quantitative histological studyof frozen hearts did not show any difference in dimensions ofventricular walls (Figure 3A and 3B). This result was con-firmed by the measurement of left and right combinedventricular masses in a separate series of animals used forECG recording (0.40160.019% and 0.41860.086% of bodymass for2/2 and1/1 mice, respectively). In addition, thein vivo M-mode echocardiography study did not revealdifferences among the 2 strains in septum and posterior left

Figure 2. Western blot analysis of a-, b-, and g-adducin inheart protein extracts. A, Heart protein extracts (20 mg) fromadult wild-type (1/1) and mutant (2/2) mice were analyzedwith a rabbit anti-mouse b-adducin polyclonal antibody18 (lanes1 to 3). Lanes 4 to 6 are an overexposure of the blot shown inlanes 1 to 3. The b-adducin bands corresponding to the wild-type mice (lanes 1 and 4) and the recombinant deletedb-adducin18 (lanes 3 and 6) are marked by black arrows. B andC, The same protein extract was analyzed with anti–a- andg-adducin polyclonal antibodies (50 and 100 mg of heart extract,respectively). Bact. pur. indicates the recombinant b-adducinD9-13 product.18

Figure 3. A, Measurements of left ventricu-lar wall thickness (mean and SE). Fourlocations1–4 were measured in frozen sec-tions of the heart. B, Volume of ventricularwalls (mean and SE; mm3/g). Volume ofright ventricular muscle was measured forthe free wall only. C, Echocardiography.Heart rates (mean6SE) were as follows:1/1: 51765.7 bpm; 2/2: 52267.7 bpm.Units of measure are as follows: ventricularseptum thickness (VST), posterior wallthickness (PWT), end-diastolic diameter(EDD), and end-systolic diameter(ESD), mm; left ventricular fractional short-ening (LVFS), %; ejection time (ET), ms;velocity of circumferential shortening (VCF),1/s. D, Electrocardiography. ECG waveformintervals (mean6SE), measured fromb-adducin deficient (2/2) and wild-typecontrol (1/1) mice, are shown. Heart rates(mean6SE) were as follows: 1/1:575645.3 bpm; 2/2: 601680.9 bpm.Body temperatures were as follows: 1/1:37.260.11°C; 2/2: 36.760.35°C. None ofthe differences among group means werestatistically significant.

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ventricular wall thickness, end-diastolic and end-systolicdiameters, and left ventricular fractional shortening (Figure3C). The Doppler echocardiography study also showed sim-ilar values of ejection time in both strains (Figure 3C). ECGdemonstrated ECG waveforms characterized by a QRS com-plex with a small or absent Q wave, a prominent R wave, andan S wave followed immediately by a T wave with nodiscernible ST interval in both mice strains. Values of QRScomplex duration and of PR and QT intervals did not differamong the 2 strains (Figure 3D). These ECG data indicatethat the absence ofb-adducin does not produce significantchanges in the muscle cell membrane ion channels of theheart to induce changes in the ECG parameters. It is note-worthy that despite the marked changes in adducin expressionobserved, no alterations of heart morphological, mechanical,or electric properties have been detected in this model,suggesting a lack of involvement of these proteins in heartfunction.

DiscussionStudies in humans and rats have associated adducin withhypertension, but the direct role of this protein in BP hasnever been demonstrated. To investigate the role ofb-adducin in BP regulation, we generated mice homozygous(2/2) for a targeted disruption of theb-adducin gene andtransferred this gene mutation to a homogeneous geneticbackground (theoretically, 98% identical to that of the controlanimals). This is the first report showing strong evidence thathypertension is triggered by a mutation in theb-adducingene.

One of the clear consequences of the absence ofb-adducinin some tissues (References 18 and 22 and data not shown) isthe concomitant modification ofa- and g-adducin levels,supporting the hypothesis that the abnormal adducin complexassembly might be an important determinant of BP.23 Hence,b-adducin deficiency could be directly responsible for BPchanges, or it may be acting through the consequent modifi-cation ofa- andg-adducin levels or other as yet undetectedeffects.b-Adducin–deficient mice have a lower hematocritthan wild-type animals (43.5 versus 46.1;P#0.005),18 andthis cannot explain the change in BP.

Although in hearts of2/2 mice, b-adducin deficiencyproduced altereda- and g-adducin levels, we have foundneither cardiac remodeling nor modification of the heartfunction in these animals. This phenomenon is probablyrelated to the moderate magnitude of the change in arterial BPpresent and/or the relatively young age of the animals understudy. However, we cannot exclude the involvement ofadducin on heart function under different types of load andstress.

The involvement of the kidney in the sequence of eventsconnecting adducin polymorphism to the development andmaintenance of hypertension was shown in the MHS strain ofrats17,24 and was suggested in humans.6,8 However, prelimi-nary analysis of kidneys of ourb-adducin–deficient miceshowed no difference by gross histological analysis with1/1mice, and the kidney weight of2/2 mice was similar to thatof 1/1 animals (0.5660.09% and 0.5860.06% of animalweight for1/1 and2/2 animals, respectively [mean6SD];

n59 per group;P50.226, Student’st test). Moreover, serumcreatinine levels showed no difference between2/2 and1/1 animals (0.41660.044 mg/dL and 0.41960.025 mg/dLfor 1/1 and2/2 animals, respectively [mean6SD]; n510per group;P50.853, Student’st test).

The interest of our work for clinical research liesprimarily in the definitive proof of the direct involvementof adducin in BP modulation. Contrary to the partialcontribution of adducin genes to hypertension in bothdifferent human populations and MHS rats, the mutation ofthe b-adducin gene in the knockout mouse is the only oneresponsible for BP changes in this strain. Considering thisand the very high degree of sequence homology betweenmouse and human adducins (Reference 18 and A.F. Muro,unpublished data, 1999), it is possible that the molecularmechanisms of hypertension in these mice are relevant tohumans. Furthermore, since in our mouse model RBCabnormalities were present, causing mild hemolytic ane-mia, it will be of great interest to search for adducinpolymorphisms in selected groups of patients with elevatedBP accompanied by erythrocyte changes similar to thosefound in our mouse model.

AcknowledgmentsWe are grateful to G. Lunazzi and M. Sturnega for animal care andto M. Bovenzi for help and advice regarding statistical analysis.

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