Diminished Cardioprotective Response to Inhibition of ...circres.ahajournals.org/content/circresaha/88/10/1072.full.pdf · (Acuson c256) as described previously.23 All studies were

Diminished Cardioprotective Response to Inhibition ofAngiotensin-Converting Enzyme and Angiotensin II Type 1

Receptor in B2 Kinin Receptor Gene Knockout MiceXiao-Ping Yang, Yun-He Liu, Dharmesh Mehta, Maria A. Cavasin, Edward Shesely,

Jiang Xu, Fang Liu, Oscar A. Carretero

Abstract—Using B2 kinin receptor gene knockout mice (B22/2), we tested the hypothesis that (l) lack of B2 receptors may

affect blood pressure and cardiac function and aggravate cardiac remodeling after myocardial infarction (MI), and (2)kinins partially mediate the cardiac beneficial effect of angiotensin-converting enzyme inhibitors (ACEi) or angiotensinII type 1 receptor antagonists (AT1-ant), whereas lack of B2 receptors may diminish this cardioprotective effect. Chronicheart failure (HF) was induced by MI, which was caused by coronary artery ligation in both B2

2/2 and 129/SvEvTacmice (wild-type control, B2

1/1). An ACEi (ramipril, 2.5 mg/kg/d) or AT1-ant (L-158809, 3 mg/kg/d) was given 1 weekafter MI and was continued for 12 weeks. Left ventricular (LV) ejection fraction, cardiac output (CO), diastolic LVdimension (LVDd), and LV mass were evaluated by echocardiography. Myocyte cross-sectional area and interstitialcollagen fraction were studied histopathologically. We found that basal blood pressure and cardiac function were similarin B2

1/1 and B22/2 mice. After MI, development of HF and remodeling were also similar between the 2 strains. The

ACEi improved cardiac function and remodeling in both strains; however, its effects were attenuated in B22/2 mice

(respective values for B21/1 versus B2

2/2 mice: overall increase in ejection fraction, 64610% versus 2165% [P,0.01];increase in CO, 69617% versus 2369% [P,0.01]; overall decrease in LVDd,22463% versus2764% [P,0.01]; anddecrease in LV mass,23863% versus2666% [P,0.01]). AT1-ant had a beneficial cardiac effect similar to thatproduced by ACEi, and this effect was also diminished in B2

2/2 mice (respective values for B21/1 versus B2

2/2 mice:overall increase in ejection fraction, 46610% versus 2569% [P,0.01]; increase in CO, 44614% versus 1565%[P,0.01]; overall decrease in LVDd,21464% versus2663% [P,0.01]; and decrease in LV mass,23364 versus21667% [P,0.01]). The effect of ACEi or AT1-ant on myocyte cross-sectional area was similar between strains;however, their effect on the interstitial collagen fraction was diminished in B2

2/2 mice. We concluded that (1) lack ofB2 kinin receptors does not affect cardiac phenotype or function, either under normal physiological conditions or duringthe development of HF; and (2) kinins acting via the B2 receptor play an important role in the cardioprotective effectof ACEi and AT1-ant. (Circ Res. 2001;88:1072-1079.)

Key Words: angiotensin-converting enzyme inhibitorsn AT1 receptor antagonistn heart failuren B2 kinin receptorsn mice

Chronic heart failure (CHF) is characterized by leftventricular (LV) pump dysfunction, chamber dilatation,

neurohormonal system activation, and exercise intolerance.The renin-angiotensin system (RAS) plays a central role inthis process.1–3 Over the past decade, clinical and laboratorystudies have provided evidence that interruption of the RASachieved by angiotensin-converting enzyme inhibitors(ACEi) improves cardiac function, regresses LV remodeling,and prolongs survival in patients with CHF.4–6 However, itremains unclear whether the benefits of ACEi are entirely dueto blockade of angiotensin II (Ang II) formation or partiallyderived from increased kinins, because ACE is also the majorkininase that degrades kinins to inactive fragments.7,8 We and

others have previously reported that ACEi attenuated thedeterioration of LV function and remodeling in animals withCHF due to myocardial infarction (MI) and that this effectwas either blocked by a B2 kinin receptor antagonist (B2-ant)9,10 or blunted in rats with kininogen deficiency due tospontaneous mutation of the kininogen gene,11 indicating thatkinins play an important role in the cardioprotective mecha-nism of ACEi. However, it remains controversial whetherkinins play an essential role in regulating blood pressure (BP)and cardiac function under physiological conditions or in thepathophysiology of CHF. It has recently been reported thatdisruption of the bradykinin B2 receptor gene in mice (B22/2

mice) increased BP, heart weight, and LV chamber dimen-

Original received January 5, 2001; revision received March 26, 2001; accepted March 28, 2001.From the Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Mich.Correspondence to Xiao-Ping Yang, MD, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 West Grand Blvd, Detroit, MI

48202. E-mail [email protected]© 2001 American Heart Association, Inc.

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sion.12,13 However, we previously found that blockade of theB2 kinin receptor or genetic kinin deficiency neither alteredBP nor aggravated cardiac remodeling and LV dysfunction,although it did partially block the cardioprotective effect ofACEi.9,11,14 We also showed that in B22/2 mice, BP and theseverity of ischemia/reperfusion injury did not differ fromtheir wild-type controls (B21/1).15 However, it is not knownwhether the chronic maladaptive response to MI (such as LVhypertrophy, chamber dilatation, and dysfunction) is en-hanced in B22/2 mice.

Despite treatment with ACEi, some patients still experi-ence worsening symptoms and deterioration of LV function,which may be related to incomplete inhibition of Ang IIformation or continued activation of the RAS. Thus, it hasbeen proposed that blockade of the RAS at the receptor levelmay provide an additional advantage over ACEi. However,our previous study in rats showed that an Ang II type 1 (AT1)receptor antagonist (AT1-ant) had a cardioprotective effectsimilar to that of ACEi, and that this effect was partiallyblocked by a B2-ant or Ang II type 2 (AT2) receptorantagonist (AT2-ant),9 indicating that (1) at least in this ratmodel of heart failure (HF), AT1-ant are not superior to ACEi,although it is not certain whether combined treatment withACEi and AT1-ant would provide a better effect than eitherdrug alone; and (2) activation of the AT2 receptor during AT1inhibition might be partially responsible for the cardioprotec-tive effect of AT1-ant either directly or via stimulation ofkinins and/or NO and cGMP.16–18

To further test the hypothesis that kinins mediate thecardioprotective effect of ACEi and AT1-ant, we producedCHF in B2

1/1 and B22/2 mice by ligating the left anterior

descending coronary artery (LAD) and studied whether (1)lack of kinin B2 receptors aggravates cardiac remodeling andLV dysfunction, and (2) the cardioprotective effect of ACEior AT1-ant is diminished or absent in B2

2/2 mice.

Materials and MethodsAnimalsB2

2/2 mice were derived from a breeding pair of homozygous miceon a 129/SvEv genetic background19 and are currently being bred inour Mutant Mouse Facilities. Wild-type 129/SvEvTac mice (B2

1/1)purchased from Taconic Farms (Germantown, NY) served as con-trols. Animals were housed in an air-conditioned room with a12-hour light/dark cycle, received standard mouse chow, and dranktap water. The Henry Ford Hospital Care of Experimental AnimalsCommittee approved the present study.

Surgical ProceduresMale mice aged 10 to 12 weeks were anesthetized with sodiumpentobarbital (50 mg/kg IP), intubated, and ventilated with room airusing a positive-pressure respirator. A left thoracotomy was per-formed via the fourth intercostal space, the heart was exposed, andthe pericardium opened as described previously.20 The LAD wasligated with a 9-0 silk suture near its origin between the pulmonaryoutflow tract and the edge of the left atrium. MI was deemedsuccessful when the anterior wall of the LV became cyanotic and theECG showed obvious ST-segment elevation. The lungs were inflatedby increasing positive end-expiratory pressure, and the thoracotomysite was closed. Sham-operated mice were subjected to the sameprocedure, except that the suture around the LAD was not tied.Animals were kept on a heating pad until they were awake.

Measurement of BP and Cardiac Function

Systolic BPSystolic BP (SBP) was measured in conscious mice by use of anoninvasive computerized tail-cuff system (BP-2000, Visitech Sys-tems) as described previously.21,22Briefly, the mice were trained for7 days by measuring SBP daily, after which SBP was recordedweekly. Three sets of 10 measurements were obtained during eachrecording; a set was accepted if the computer identified.6 success-ful readings out of 10 measurements.

EchocardiographyCardiac geometry and function were evaluated with a Dopplerechocardiographic system equipped with a 15-MHz linear transducer(Acuson c256) as described previously.23 All studies were performedon awake mice before MI and periodically thereafter. The followingparameters were obtained: (1) LV chamber dimensions and wallthickness; (2) LV mass, which is equivalent to 1.055[(IVSd1LVDd1PWTd)32(LVDd)3], where 1.055 is the specificgravity of the myocardium, IVSd is interventricular septum thick-ness, LVDd is diastolic LV dimension, and PWTd is diastolicposterior wall thickness (LV mass was normalized for body weightand expressed as mg/10 g); (3) ejection fraction (EF), which isequivalent to [(LVAd2LVAs)/LVAd]3100, where LVAd is LVdiastolic area and LVAs is LV systolic area; and (4) cardiac output(CO), which is equivalent to SV3HR, with SV5CSA3VTI andCSA5[(AoD/2)2]p, where SV is stroke volume, HR is heart rate,CSA is aortic cross-sectional area, VTI is the aortic flow velocity-time integral, and AoD is aortic diameter (CO was normalized forbody weight and expressed as mL/min/10 g).

All primary measurements, such as LV wall thickness, dimen-sions, and CSA, were traced manually and digitized by goal-directed,diagnostically driven software installed within the echocardiograph.Three beats were averaged for each measurement.

Histopathological Study

Heart Weight, Lung Wet Weight, and Infarct SizeMice were killed after 12 weeks of MI, and their hearts and lungswere weighed. The LV was sectioned transversely into 3 slices fromapex to base, rapidly frozen in isopentane precooled in liquidnitrogen, and then stored at270°C. For infarct size, 6-mm sectionsfrom each slice were stained with Gomori trichrome to identifyfibrous tissue (infarction). Infarct size was calculated as the ratio ofinfarct length to the circumference of both endocardium andepicardium.24

MCSA and ICFSections (6-mm) were cut from each slice and double-stained with(1) fluorescein-labeled peanut agglutinin to delineate the myocytecross-sectional area (MCSA) and interstitial space, and (2)rhodamine-labeledGriffonia simplicifolia lectin I to show the cap-illaries.9 Four radially oriented microscopic fields were selected fromeach section and photographed at a magnification of3100. MCSAwas measured by computer-based planimetry (Jandel). For theinterstitial collagen fraction (ICF), the total surface area (microscop-ic field), interstitial space (collagen plus capillaries), and areaoccupied by the capillaries alone were measured with computer-assisted videodensitometry and calculated as per cent total surfacearea occupied by the interstitial space minus per cent total surfacearea occupied by the capillaries. Average MCSA and ICF werecalculated for each mouse.

Experimental ProtocolsProtocol 1 involved comparing the cardiac phenotype between B2

1/1

and B22/2 mice before and after MI and determining whether the

development of cardiac dysfunction and LV remodeling was moresevere or accelerated in B2

2/2 mice. Each strain was subjected toeither coronary ligation (HF-vehicle) or sham MI and was followedup for 12 weeks.

Protocol 2 involved determining whether the effect of ACEi orAT1-ant was diminished or absent in B2

2/2 mice. One week after the

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operation, each strain was divided into (1) HF-vehicle, (2) HF-ACEi(ramipril, 2.5 mg/kg per day in drinking water, provided by Upjohn),and (3) HF-AT1-ant (L-158809, 3 mg/kg/d in drinking water,provided by Merck). Treatment was continued for 11 weeks. Wehave previously shown that ramipril at 2.5 mg/kg per day signifi-cantly inhibited the vasopressor effect of exogenous Ang I at 12.5,25, 50, and 100 ng per mouse and that L-158809 at 3 mg/kg per daysignificantly inhibited the vasopressor effect of exogenous Ang II at12.5, 25, 50, and 100 ng per mouse.25

Data AnalysisData were expressed as mean6SE. Two-way repeated-measuresANOVA was used to detect differences within each strain. Forcomparison between strains, repeated-measures ANOVA was usedwith a test of interaction to determine whether the average changeafter treatment (from week 2 to week 12) was different betweenB2

1/1 and B22/2 mice, takingP,0.05 as being statistically signifi-

cant. One-way ANOVA was used for heart and lung weight andhistopathological data. The Simes method was used to adjust formultiple comparisons.

Results

MortalityThe mortality rate was similar between the 2 strains. Earlymortality (within 24 hours after surgery) was 15.8% in B2

1/1

mice and 13.5% in B22/2 mice. During the first week of MI,40% of the B2

1/1 mice and 23% of the B22/2 mice died, mostlyfrom cardiac rupture. During weeks 2 to 12, only 1 B2

1/1

mouse and 2 B22/2 mice died. None of the B21/1 or B22/2 mice

that underwent the sham procedure died during or after theoperation.

Body, Heart, Lung, and Liver Weight andInfarct SizeThere was no significant difference in any of these parametersbetween strains in sham-ligated groups (Table). In the HF-vehicle groups, heart and lung weight increased similarly inboth strains. ACEi or AT1-ant reduced heart weight to asimilar extent in both strains but had no effect on lung weight.Liver weight was increased only in B2

1/1 mice, and drugtreatment had no effect on it.

SBP and HRBasal SBP and HR were similar for both strains in all groups.After MI, SBP in the B2

1/1 HF-vehicle group decreasedsignificantly, which was not seen in the B2

2/2 group. ACEi orAT1-ant did not influence SBP in B21/1 but did reduce SBP inB2

2/2 (Figure 1, top). There was a slight increase in HR afterMI, but it did not reach statistical significance. Drug treat-ment had no effect on HR (Figure 1, bottom).

Cardiac Function and RemodelingThere was no difference between sham-ligated B2

1/1 andB2

2/2 mice with regard to EF, CO, LVDd, and cardiac mass(Figure 2). MI caused a significant reduction in EF and COand elevation in LVDd and LV mass, occurring as early as 1week after MI and progressing similarly over time in bothstrains (Figure 2). ACEi significantly increased EF and CO(Figures 3 and 4) and decreased LVDd and LV mass (Figures3 and 5) in both strains with HF; however, the effect of ACEi

Body, Heart, Lung, and Liver Weight and Infarct Size in B22/2 and B2

1/1 Mice

Treatment

B21/1 Mice B2

2/2 Mice

Sham(n513)

HF

Sham(n513)

HF

Vehicle(n510)

ACEi(n510)

AT1-ant(n58)

Vehicle(n516)

ACEi(n513)

AT1-ant(n512)

BW, g 30.260.6 31.260.3 29.560.7 28.561.3 29.360.6 30.060.7 30.960.8 30.960.7

Atria, mg 10.060.6 21.161.3* 14.161.2‡ 15.061.5‡ 11.660.8 20.361.4* 16.861.4‡ 16.261.4‡

RV, mg 25.960.7 34.962.7* 27.761.5‡ 28.562.1‡ 23.660.9 32.463.0* 27.061.1‡ 30.061.6

LV, mg/10 g 34.060.7 51.864.0* 37.461.0‡ 37.662.3‡ 32.661.2 47.162.5* 37.261.3\ 38.961.7\

Lungs, mg/10 g 48.261.3 59.063.7* 56.662.2 56.564.8 49.262.5 57.362.9* 61.162.3 57.663.3

Liver, mg/10 g 34469 384624† 404613 393619 367623 368616 38368 34267

IS, % z z z 39.863.7 34.462.8 36.161.7§ z z z 42.062.2 35.863.2 44.562.5

Values are mean6SE. BW indicates body weight; RV, right ventricular weight corrected for body weight; LV, LV weight corrected for body weight; and IS, infarctsize.

*P,0.01 and †P,0.05 vs sham within strains; ‡P,0.05 and \P,0.01 vs vehicle within strains; and §P,0.05 vs AT1-ant between strains.

Figure 1. SBP and HR in sham-operated mice (sham) and micewith HF treated with vehicle, ACEi, or AT1-ant. Basal indicatesbefore surgery; 1w, 1 week after surgery without treatment; and2–12w, combined data during treatment period (2 to 12 weeks).

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was significantly attenuated in B22/2 mice compared with

B21/1. The bar graphs inFigures 4 and 5 show the average per

cent increase in EF and CO and decrease in LVDd and LVmass from 2 to 12 weeks of treatment between the 2 strains.

The overall increase in EF after ACEi was 64610% in B21/1

and 2165% in B22/2 (P,0.01), and the increase in CO was69617% in B2

1/1 and 2369% in B22/2 (P,0.01). The overallreduction in LVDd was22463% in B2

1/1 versus2764% inB2

2/2 (P,0.01), and the reduction in LV mass was23863 inB2

1/1 and2666% in B22/2 (P,0.01). AT1-ant had a benefi-

cial cardiac effect similar to ACEi; this effect was alsodiminished in B2

2/2 mice. The overall increase in EF withAT1-ant was 46610% in B21/1 and 2569% in B22/2

(P,0.01), and the increase in CO was 44614% in B21/1 and

1565% in B22/2 (P,0.01). The overall reduction in LVDd

was21464% in B21/1 and2663% in B2

2/2 (P,0.01), andthe reduction in LV mass was23364% in B2

1/1 and21667% in B2

2/2 (P,0.01) (Figures 4 and 5). Although theACEi appeared to have a better protective effect, the differ-ence between ACEi and AT1-ant did not reach statisticalsignificance.

Myocyte Size and ICFMCSA and ICF were similar in sham-operated B2

1/1 andB2

2/2 mice and increased similarly after MI in both strains(Figures 6 and 7). ACEi and AT1-ant significantly decreasedMCSA in both the B2

1/1 and B22/2 groups, and no statistical

difference between strains was detected (Figure 7, top).However, the effect of ACEi and AT1-ant on ICF wasobserved only in B21/1 mice and was absent in B2

2/2 (Figure7, bottom).

DiscussionWe found that basal SBP and cardiac function as well asmorphological and histological parameters were no differentin B2

2/2 mice compared with B21/1. Development and severityof cardiac dysfunction after MI were also similar in B2

2/2 andB2

1/1, suggesting that kinins acting on the B2 receptor maynot play an essential role in the regulation of BP and cardiacfunction, either under normal physiological conditions orduring the development of HF. Inhibition of ACE or blockadeof the AT1 receptor improved cardiac function and remodel-ing, as evidenced by increased EF and reduced LV chamberdimension, mass, and interstitial collagen deposition; theseeffects were attenuated in B2

2/2 mice, indicating that kininsare at least partially responsible for the therapeutic effect ofACEi and AT1-ant in HF.

Kinins are vasodilator polypeptides released from low- andhigh-molecular-weight kininogens by plasma and tissue kal-likreins and hydrolyzed mainly by ACE (also called kininaseII). The biological action of kinins is mediated by activationof at least 2 known subtypes of G-protein–coupled receptors,B1 and B2.8,26The B1 receptor is only weakly expressed underphysiological conditions but is strongly induced under patho-logical conditions, such as inflammation or tissue injury,27,28

and is sensitive to des-Arg9-bradykinin, a metabolite ofbradykinin. B2 receptors, which are constitutively expressedin most tissues, are sensitive to bradykinin and kallidin andare responsible for most known effects of bradykinin.8

Although the role of endogenous kinins in the regulation ofBP and cardiac hemodynamic homeostasis as well as in thepathophysiology of HF has been studied extensively, the dataremain controversial. Emanueli et al13 reported that disruption

Figure 2. Comparison of EF, CO, LVDd, and LV mass betweenB2

2/2 mice and B21/1 mice without HF (sham coronary artery

ligation) or with HF induced by coronary artery ligation (CL)before surgery (basal) and after surgery (1 to 12 weeks).

Figure 3. Two-dimensional M-mode echocardiographs of B22/2

mice and B21/1 mice with sham coronary ligation (sham) or HF.

IS indicates interventricular septum; DD, LV diastolic dimension;and PW, LV posterior wall.

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of the B2 receptor led to high BP, LV dilatation, andfunctional impairment, suggesting that kinins are essential forfunctional and structural preservation of the heart. However,we found that BP, cardiac performance, and histology inkininogen-deficient rats or B22/2 mice are no different fromtheir wild-type controls.11,14,29In the present study, we furtherdemonstrated that lack of B2 kinin receptors neither alters BPor cardiac phenotype nor aggravates cardiac remodeling afterMI, indicating that either (1) kinins may not play an importantrole in regulation of BP and function, or (2) there is acompensatory mechanism whereby metabolites of bradykininact on the B1 receptor to assume some of its vasoactiveproperties. Tschöpe et al30 recently showed that both B1 andB2 receptors are upregulated after MI. It has also been shownthat hindlimb ischemia in mice induced B1 gene overexpres-sion accompanied by an increase in muscular capillarydensity, and that this angiogenesis was blunted by a B1

receptor antagonist but not affected by B2 blockade.31 Fur-

thermore, Duka et al32 recently reported that the B1 receptoris upregulated in B22/2 mice and that these mice had ahypotensive response to a selective B1 agonist and a hyper-tensive response to a selective B1 receptor antagonist, indi-cating a compensatory function of the B1 receptor in main-taining hemodynamic homeostasis when the B2 receptor isabsent.

Despite the fact that the hemodynamic and cardiac pheno-types are similar in B22/2 and control mice, we found thatB2

2/2 mice had a diminished response to ACEi and AT1-ant.This agrees with our previous findings that ACEi and AT1-antimproved LV function and structural remodeling in Lewisinbred rats and that these effects were partially blocked by akinin receptor antagonist,9 suggesting that the cardioprotec-tive effects of ACEi are not solely attributable to inhibition ofAng II formation. In fact, ACE not only converts angiotensinI to Ang II but also degrades kinins to inactive fragments.Furthermore, the affinity of ACE for kinins is higher than for

Figure 4. Effect of ACEi and AT1-ant on EF andCO in B2

2/2 mice and B21/1 mice with HF induced

by CL before ligation (basal) and after ligation (1to 12 weeks). Veh indicates treatment with vehi-cle. *P,0.01 vs HF-vehicle for both ACEi andAT1-ant. Bar graphs show average per centincrease from 2 to 12 weeks of treatment.

Figure 5. Effect of ACEi and AT1-ant on LVDd andLV mass in B2

2/2 mice and B21/1 mice with HF

induced by CL before ligation (basal) and afterligation (1 to 12 weeks). *P,0.01 vs HF-vehicle forboth ACEi and AT1-ant. Bar graphs show averageper cent decrease from 2 to 12 weeks oftreatment.

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angiotensin I. Thus, inhibition of kinin degradation, which inturn results in increased endogenous kinins, is also largelyresponsible for the cardioprotection seen with ACEi. Theprecise mechanism by which kinins protect the heart is notyet well defined. It is known that kinins are potent stimuli forthe release of endothelial NO and prostaglandins. Recently,Emanueli et al showed that local delivery of the human tissuekallikrein gene accelerated ischemia-induced hindlimb angio-genesis and preserved energy utilization of ischemic muscle31

and that this effect was blocked by the inhibition of cyclo-oxygenase or NO synthase,33 indicating a prostaglandin-and/or NO-mediated mechanism. It has also been shown thatkinins inhibit collagen gene expression and collagen produc-tion via stimulation of arachidonic acid metabolites, particu-larly prostaglandin I2.34 In addition, kinins and NO may beinvolved in myocardial energy metabolism. Zhang et al35

recently showed that incubation of coronary microvessels ormyocardial slices with ACEi or kininogen significantly in-creased NO production and decreased myocardial oxygenconsumption,35,36 both of which were blocked by a B2 kininreceptor antagonist. They also showed that bradykinin stim-ulated the release of NO from the mouse myocardium andthat this effect is absent in B2

2/2 mice.37 Using NO synthase(NOS) inhibitors or endothelial NOS knockout mice, Tada etal38 recently reported that NO participates in the regulation ofmyocardial glucose, lactate, and fatty acid metabolism.38

Perfusing the ischemic heart with bradykinin increases theproduction of myocardial high-energy phosphates as well as

glycogen content, along with a reduction in lactate dehydro-genase and creatinine kinase activity.39,40 Taken together,these data suggest that kinins or NO may reduce oxygenconsumption and facilitate energy utilization, thereby contrib-uting significantly to the cardioprotective action of ACEi.

Two major Ang II receptor subtypes, AT1 and AT2, havebeen identified.41 Most known biological actions of Ang IIhave been attributed to the AT1 receptor, whereas the role ofthe AT2 receptor remains controversial. Recent evidencesuggests that AT2 activation may antagonize the vasopressor,hypertrophic, and fibrogenic effects of AT1.42–44Tsutsumi etal45 showed that in aortas from mice with overexpression ofthe AT2 receptor, Ang II caused a significant increase inkininogenase activity and cGMP production, which wasfurther enhanced by an AT1-ant but blocked by an AT2-ant,kinin antagonist, or NOS inhibitor, suggesting that AT2

activation stimulates kinin release, which further promotesNO/cGMP production in a paracrine manner and thus poten-tiates vasodilatation and regional blood flow regulation. Wepreviously reported that in a rat model of CHF induced byMI, AT 1-ant had a cardioprotective effect similar to ACEi andthat part of the effect of AT1-ant, such as reducing LV systolicand diastolic volume, was blocked by an AT2-ant or a B2 kininantagonist.9 In the present study, using B2

2/2 mice as a model,we further confirmed the role of kinins in the cardioprotectiveeffect of AT1-ant. It is possible that blockade of AT1 increasesthe level of Ang II, which in turn activates AT2. Activation ofAT2 may stimulate the release of NO either directly or viakinins, leading to cardioprotection. We have recently demon-strated that the cardioprotective effect of ACEi or AT1-ant

Figure 6. Representative slides showing MCSA and interstitialcollagen deposition (green staining) in B2

2/2 and B21/1 mice with

either sham coronary ligation (sham) or HF.

Figure 7. Effect of ACEi and AT1-ant on MCSA (top) and ICF(bottom) in B2

2/2 and B21/1 mice with sham coronary ligation

(sham) or HF.

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was diminished in endothelial NOS knockout mice with CHFinduced by MI (Y.-H. Liu, J. Xu, X.-P. Yang, F. Yang, E.G.Shesely, O.A. Carretero, unpublished data, 2001), which mayprovide further evidence that endothelium-derived NO playsan important role in the beneficial cardiac effect of ACEi andAT1-ant.

In summary, we have demonstrated that (1) kinins actingvia the B2 receptor do not seem to play an essential role incardiac hemodynamics, morphology, and function eitherunder normal physiological conditions or during the devel-opment of HF, inasmuch as none of these parameters differedbetween B22/2 and B2

1/1 mice, and (2) inhibition of ACEor blockade of the AT1 receptor improves cardiac functionand regresses remodeling in HF, and this therapeutic effectis partially mediated by kinins, since it was attenuated inB2

2/2 mice.

AcknowledgmentsThis work was supported by National Institutes of Health GrantHL-28982 and American Heart Association Grant 0030232.

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Yang et al Heart Failure in B2 Kinin Receptor Knockout Mice 1079

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Fang Liu and Oscar A. CarreteroXiao-Ping Yang, Yun-He Liu, Dharmesh Mehta, Maria A. Cavasin, Edward Shesely, Jiang Xu,

Kinin Receptor Gene Knockout Mice2and Angiotensin II Type 1 Receptor in BDiminished Cardioprotective Response to Inhibition of Angiotensin-Converting Enzyme

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