Protecting the myocardium: A role for the �2 adrenergic receptorin the heart
Andrew J. Patterson, MD, PhD; Weizhong Zhu, MD, PhD; Amy Chow; Rani Agrawal, MS; Jon Kosek, MD;Rui Ping Xiao, MD, PhD; Brian Kobilka, MD
T he �1 adrenergic receptors(�1ARs) and �2ARs are closelyrelated receptor subtypes thatmediate the primary cardio-
vascular effects of catecholamines in themammalian heart. Both receptors are Gprotein coupled and possess seven trans-membrane helixes. Both receptors havebeen shown to enhance heart contractil-ity (1, 2) as well as relaxation (1). Inaddition, they share many common ago-nists and antagonists.
Despite these similarities, �1AR and�2ARs differ in many ways. For instance,their gene loci are entirely different. The�1AR gene is located on chromosome 10whereas the gene for the �2AR resides onchromosome 5. The receptor subtypesdiffer in size: The �2AR is 477 aminoacids in length whereas the �1AR is 413amino acids (3, 4). Also, recent investiga-tions suggest that �1ARs and �2ARs haveunique signaling properties. �2ARs incultured rat and mouse ventricular myo-cytes couple to both stimulatory (Gs) andinhibitory (Gi) G proteins whereas �1ARscouple only to Gs. Furthermore, in ratneonatal cardiac myocytes, �2AR activa-tion has been shown to deliver an anti-apoptotic signal to cardiac myocytesthrough a Gi-dependent coupling tophosphatidylinositol 3'-kinase and Akt(protein kinase B). In contrast, �1AR ac-tivation in adult rat ventricular myocyteshas been shown to increase apoptosis viaa cyclic adenosine monophosphate-dependent mechanism (5, 6).
Previous studies using transgenicmice suggest that when �1ARs and�2ARs are expressed at levels many timesgreater than normal, their effects are also
different. For example, five-fold trans-genic overexpression of �1ARs in the mu-rine heart causes cardiomyopathy (7),whereas overexpression of �2ARs, even atlevels as high as 60-fold above normal,improves cardiac contractile force with-out any cardiomyopathic consequences(8).
Although these data suggest dissimilarroles for cardiac �1ARs and �2ARs, thedegree to which they reveal differencesbetween the two receptor subtypes dur-ing chronic activation is unclear. The ef-fects of receptor overexpression may, infact, differ from those of continuousstimulation. For example, the extent towhich signaling elements are activatedmay be strongly influenced by variationsin receptor expression levels. �2ARs, forinstance, appear to operate within mi-crodomains (9). Signal transduction ele-ments within these domains may be fi-nite, and it is conceivable that at somelevel overexpression exceeds the capacityof these microdomains and leads to atyp-ical receptor signaling.
Our approach to the study of chronic�AR subtype activation has been to useknockout mice. Using this approach we
From the Department of Anesthesia (AJP, RA) andDepartments of Medicine and Molecular and CellularPhysiology (BK), Stanford University, Stanford, CA; Ger-ontology Research Center (WZ, RPX), National Instituteon Aging, Baltimore, MD; Stanford University MedicalSchool (AC), Stanford, CA; and Department of Pathol-ogy (JK), Veterans Administration Medical Center, PaloAlto, CA.
Supported, in part, by the Federation for Anesthe-sia Education and Research and Stanford UniversityDepartment of Anesthesia
Address requests for reprints to: Andrew J. Patter-son, MD, PhD, Department of Anesthesia, StanfordUniversity Medical Center, Stanford, CA 94305. E-mail:[email protected]
Objective: The sympathetic nervous system enhances cardiacmuscle function by activating � adrenergic receptors (�ARs).Recent studies suggest that chronic �AR stimulation is detrimen-tal, however, and that it may play a role in the clinical deterio-ration of patients with congestive heart failure. To examine theimpact of chronic �1AR and �2AR subtype stimulation individu-ally, we studied the cardiovascular effects of catecholamine in-fusions in �AR subtype knockout mice (�1KO, �2KO).
Design: Prospective, randomized, experimental study.Setting: Animal research laboratory.Subjects: �1KO and �2KO mice and wild-type controls.Interventions: The animals were subjected to 2 wks of continuous
infusion of the �AR agonist isoproterenol. Analyses of cardiac func-tion and structure were performed during and 3 days after comple-tion of the infusions. Functional studies included graded exercisetreadmill testing, in vivo assessments of left ventricular function
using Mikro-Tip catheter transducers, right ventricular pressuremeasurements, and analyses of organ weight to body weight ratios.Structural studies included heart weight measurements, assess-ments of myocyte ultrastructure using electron microscopy, and insitu terminal deoxynucleotidyl transferase-mediated biotin-dUTPnick-end labeling staining to quantitate myocyte apoptosis.
Measurements and Main Results: We found that isoproterenol-treated �2KO mice experienced greater mortality rates (p � .001,chi-square test using Fisher’s exact method) and increased myo-cyte apoptosis at 3- and 7-day time points (p � .04 and p �.0007, respectively, two-way analysis of variance).
Conclusion: The results of this study suggest that in vivo �2ARactivation is antiapoptotic and contributes to myocardial protec-tion. (Crit Care Med 2004; 32:1041–1048)
have investigated the effects of chroni-cally stimulating individual �AR subtypesexpressed at physiologic levels. Our pilotstudies (mortality rate and exercise ca-pacity studies using small numbers ofanimals) suggested that mice lacking�1ARs (�1KO and �1�2KO) might beresistant to isoproterenol-induced car-diac injury. They also suggested that�2KO mice might be more susceptible tothe detrimental effects of isoproterenolinfusion than wild-type animals. Basedon these preliminary findings, we focusedour investigation on �2KO mice. We ex-amined the cardiovascular effects ofchronic �1AR activation in the absence of�2AR stimulation in vivo.
METHODS
Mice. These experiments were reviewedand approved by our institution’s Subcommit-tee on Animal Studies and were in accordancewith the provisions of the Animal Welfare Act,the Public Health Service Guide for the Careand Use of Laboratory Animals. The genera-tion of �1KO, �2KO, and �1�2 adrenergicreceptor double knockout (�1�2dKO) micehas been previously described (10–12). Allmice used in these experiments were malemice between 25 and 35 g. The �2KO miceand their strain-specific wild-type control an-imals were on congenic FVB/N backgrounds.
We infused isoproterenol or saline into�2KO and strain-specific wild-type control an-imals for 2 wks. The study groups were care-fully matched in terms of age, weight, gender,and living environment. We studied 30 � 3 gmale mice between 12 and 14 wks of age.Within genotypes, littermates were dividedevenly and randomly assigned to isoproterenolor saline infusion, rather than assigning entirelitters to one treatment or the other.
Reagents. (�)-Isoproterenol bitartrate(120 �g·g�1·day�1; Sigma Chemical, St.Louis, MO) was infused subcutaneously intoeach study animal via miniosmotic pumps(Alza Corporation, Palo Alto, CA). Control an-imals received infusions of sterile 0.9% so-dium chloride (Abbott Laboratories, NorthChicago, IL).
Surgery and Anesthesia. Miniosmoticpumps were placed subcutaneously in eachmouse under general anesthesia. We used 3%isoflurane anesthesia for induction of anesthe-sia; 1.5% isoflurane was used for maintenance.Each animal was allowed to breathe spontane-ously throughout the procedure. A small sub-cutaneous pouch was created on each animal’sflank using blunt-end forceps. The osmoticpumps were inserted, and the skin wasclosed using two 7.5 � 1.75 mm skin staples(Michel).
Mortality Investigation. The mortality in-vestigation included mice that resided in indi-vidual cages for the duration of the study after
undergoing infusion pump insertions. Theseanimals were not included in any physiologyexperiments. Two separate mortality investi-gations were conducted. The data were pooledfor analysis. In total, 36 isoproterenol-treated�2KO, 33 isoproterenol-treated wild-type, 22saline-treated �2KO, and 30 saline-treatedwild-type animals were studied.
Exercise Capacity. Several mice were chal-lenged individually with a graded treadmillexercise protocol on a Simplex II rodent tread-mill (Columbus Instruments, Columbus, OH).The animals were studied 1 day before and 17days after osmotic pump insertion (3 days af-ter the pump contents had been completelyinjected). Treadmill activity was initiated aftereach mouse had equilibrated in the exercisechamber for 10 mins. Stepwise increases intreadmill speed (2.5 m/min) and inclination(2°) were made every 3 mins until eachmouse stopped running from exhaustion.Exercise capacity was calculated as the totaldistance run by the animal during the exer-cise protocol.
Left Ventricle Change in Pressure OverTime (dp/dt). Each animal studied was anes-thetized using isoflurane as previously de-scribed. The animal’s neck was shaved andprepped in sterile fashion, and a midline neckskin incision was made. The right commoncarotid artery was isolated using a microscope.A suture was tied around the vessel approxi-mately 0.25 cm below the skull base. Approx-imately 0.75 cm proximal to this point, bloodflow was disrupted using a vascular clamp.This provided a site for insertion of a Mikro-tipcatheter transducer (Millar Instruments,Houston Texas). The contralateral carotid ar-tery was allowed to remain patent, maintain-ing adequate cerebral blood flow on the rightside of the brain by perfusion through thecircle of Willis. Proximal to the point of ca-rotid artery ligation, a small arterotomy inci-sion was made using a curved 25-gauge nee-dle. The Mikro-tip catheter was introducedinto the artery and advanced under waveformguidance into the left ventricle. A pressurewaveform was generated using an amplifier(Gould Instrument Systems, Valley View, OH),a Transducer Control Unit TC-510 (Millar In-struments), a Dell Optiplex GX300 Computer(Dell Computer, Austin, TX), and WinDaq Ac-quisition Software version 2.43 (DATAQ In-struments, Akron, OH). The ventricular pres-sure was measured before and afterintraperitoneal injection of propranolol (30mg/kg), which was used to block the effects ofendogenous catecholamines that might have agreater impact on dp/dt in mice of one geno-type relative to the other. To calculate dp/dtbased on the left ventricle waveform, WinDaqWaveform Browser software (DATAQ Instru-ments) was used.
Right Ventricle Mean Systolic Pressure.Each animal studied was anesthetized usingisoflurane as previously described. The ani-mal’s chest was shaved and prepped in sterilefashion. A 22-gauge needle was placed into the
right ventricle using a transcutaneous subxi-phoid approach. Using a Transpac IV Monitor-ing Kit (Abbott Critical Care Systems, NorthChicago, IL), an SN-E8748 Interface Cable(Fogg System, Aurora, CO), a Gould 13-6615Transducer (Gould Instrument Systems,Babylon, N Y), a PowerLab 14SP (ADInstru-ments, Mountain View, CA), an Apple Power-Mac G4 Computer running OS9.1 OperatingSystem (Apple Computer, Cupertino, CA), andChart version 3.6.3/s software (ADInstru-ments), the right ventricle pressure waveformwas transduced and recorded for 5 secs. At alater time, the peak of each pressure waveformwas identified manually and all the peak sys-tolic pressures over the course of 5 secs wereaveraged to generate a right ventricle meansystolic pressure.
Noninvasive (Tail Cuff) Blood Pressureand Heart Rate. A BP 2000 Physiologic Re-search Instrument (Visitech Systems, Apex,NC) was used to determine systolic blood pres-sure at two time points before osmotic pumpinsertion and at nine time points after osmoticpump insertion for several animals. Before thestudy, each animal underwent 2 wks of bloodpressure measurement training. For training,each animal underwent 20 tail cuff blood pres-sure measurements every other day. At eachtime point during the study, each animal un-derwent ten practice measurements followedby ten actual measurements. A mean heartrate and systolic blood pressure were deter-mined by the BP 2000 instrument for eachanimal at each time point.
Electrocardiography. Each animal studiedwas anesthetized using isoflurane as previ-ously described. The animal’s chest wasshaved and prepped in sterile fashion. Usingtranscutaneous needle electrodes, a PhysioTelTA10EA-F20 Transmitter (Data Sciences In-ternational, St. Paul, MN), a PhysioTel Re-ceiver RPC-1 (Data Sciences International), aBCM100 Consolidation Matrix Device (DataSciences International), a Powerlab 14SP(ADInstruments), an Apple PowerMac G4Computer (Apple Computer), and Chart ver-sion 3.6.3/s software, an eight-lead electrocar-diogram (ECG) was performed before and atseveral time points after initiation of saline orisoproterenol infusion. All ECGs were inter-preted by a board eligible cardiologist.
Organ Weight to Body Weight Ratios. Themice that underwent exercise treadmill test-ing were anesthetized using 0.5 mg/g of bodyweight (about 0.5 mL) Avertin administeredby injection into the peritoneum. Each animalwas weighed and then killed using cervicaldislocation. A clam-shell bilateral thoracot-omy incision followed by a midline abdominalincision was immediately performed, and theheart, lungs, liver, spleen, and kidneys wereremoved. Each organ was rinsed in saline,blotted dry, and immediately weighed. Organweight to body weight ratios were then calcu-lated for each animal using these values.
Electron Microscopy and Analyses. Tissuewas fixed for �8 hrs at room temperature in
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1.5% glutaraldehyde (cacodylate buffer, pH7.4); sucrose was added to bring the millios-molarity to 300. The tissue was stored at 4°Cand dehydrated in ethanol, transferred to pro-pylene oxide, and embedded in Epon 12 resin.Polymerized blocks were sectioned at 0.5–1�m, and the sections were stained with tolu-idine blue for examination by light micros-copy. Selected areas were sectioned at 0.05�m, enhanced with uranyl acetate and leadcitrate, and examined with a Philips 201 elec-tron microscope. A quantitative analysis ofmyocyte injury was performed by a pathologistsubspecializing in cardiac pathology. Blindedwith regard to genotype and treatment group,the pathologist scored 113 midventricle im-ages based on myofiber integrity, intracellularfluid collection, and mitochondrial degenera-tion. The grading scale ranged from 0 (noinjury) to 4 (severe injury) (13).
Apoptosis. Mice were killed for cardiac ap-optosis studies 3, 7, or 10 days after initiationof isoproterenol or saline infusion. Each ani-mal was anesthetized by an intraperitonealinjection of Avertin. Each animal was thenkilled using cervical dislocation. The heart wasimmediately exposed using a bilateral clam-shell incision. A 20-gauge needle was insertedinto the beating right ventricle to serve as avent. Six milliliters of chilled (4°C) saline wasinjected into the beating left ventricle via a20-gauge needle. Immediately after saline in-jection, 30 mL of chilled (4°C) 4% paraformal-dehyde fixative was injected into the left ven-tricle. Each animal’s heart was then removedand stored in 4% paraformaldehyde at 4°Cuntil it was sliced into three sections perpen-dicular to the long axis of the ventricle. Sec-tions were then stored in 10% neutral bufferedformalin for 24 hrs and then embedded inparaffin. Slide sections (5 �m) from the mid-ventricular level were stained by in situ termi-nal deoxynucleotide transferase-mediateddUTP nick-end labeling (TUNEL) using an insitu apoptosis detection kit (TACS 2TdT,Trevigen, Gaithersburg MD) (14). TUNEL-positive nuclei stained blue in this assay. Tocount the number of TUNEL-positive nuclei,the entire section was scanned at �100 bylight microscopy. When TUNEL-positive cellswere detected, magnification was changed to�400 to assess whether staining occurred inmyocytes. Only TUNEL-positive myocyteswere counted in each slide. Any nuclei thatwere ambiguous were not counted. Round ho-mogeneous dark dots (considered to be arti-fact) and stained cell debris were carefullyeliminated from counting. Brownish nuclei-like deposits, considered to be Formalin crys-tals, were not counted. The number of apopto-tic nuclei was normalized to apoptotic nucleiper 10,000 cardiac myocyte nuclei. This wasdone using the morphometric method. Fromthe positive control of each section, 12 lightmicroscopic fields (400�) were chosen tocount the number of myocyte nuclei. Endo-cardial, midmyocardial, and epicardial areaswere chosen from the septum, lateral, ante-
rior, and posterior walls of the left ventricle.The number of cardiac myocyte nuclei in eachmicroscope field was counted under a gridcovering a total area of 0.0625 mm2. Onlynuclei that were completely contained withinthe myocyte contours were counted. Thenumber of nuclei within 12 fields was aver-aged. The number of TUNEL-positive cells per10,000 cardiomyocyte nuclei in each slide wascalculated as the total number of TUNEL-positive cells in each slide divided by the esti-mated total of myocyte nuclei in each slidemultiplied by 10,000.
Statistics. All data were expressed as mean� SEM. Most of the statistical analyses involvedtwo-way analysis of variance (ANOVA), withpredictors for drug treatment and genotype aswell as an interaction term. An exception wasthe comparison of left ventricular contractility(dp/dtmax). In this experiment, isoproterenol-treated �2KO mice were compared with iso-proterenol-treated wild-type animals, and aStudent’s t-test was used for the statisticalanalysis. The saline-treated �2KO group wasstudied simply for comparison with publishednormal values to validate our experimentaltechnique.
Several of the experiments described wereaffected by mortality rate among the isoprot-erenol-treated animals. We investigatedwhether mortality rate affected the statisticalsignificance of the results by censoring thecohorts where fewer mice died. We repeatedthe statistical analyses withholding the high-est values and then withholding the lowestvalues from these cohorts. In only one casewas the effect of mortality rate significant:liver weight to body weight ratio. When highvalues were removed, we observed a statisti-cally significant interaction between genotypeand treatment (p � .006; two-way analysis ofvariance).
RESULTS
Mortality. Mortality was defined asdeath within 17 days of drug infusiononset. On day 17, all animals were killedfor analysis. Statistical analysis revealedsignificantly greater mortality rateamong isoproterenol-treated �2KO micecompared with isoproterenol-treatedwild-type mice (p � .001, chi-squaredtest using Fisher’s exact method). Wefound that 14 of 36 isoproterenol-treated�2KO mice died during two mortalitystudies, whereas two of 33 isoproterenol-treated wild-type animals died (no saline-treated �2KO or wild-type mice died).
Exercise Capacity. To assess whetherisoproterenol infusion affected overallcardiovascular function of �2KO or wild-type animals, we performed graded exer-cise treadmill studies. Our data revealed adecrease in exercise capacity after isopro-terenol infusion for both wild-type and
�2KO animals. Although the drug effectwas found to be significant, the genotypeeffect was not significant (Fig. 1).
Cardiac Function. An increase in lungweight reflects left ventricular dysfunc-tion due to pulmonary congestion as theleft ventricle fails. We used lung weightto body weight ratios as an indirect mea-sure of left ventricular cardiac function in�2KO and wild-type mice. Analysis ofdata from this study revealed a significantdrug effect (p � 0.0001, two-way ANOVA)but no genotype effect (p � .7843, two-way ANOVA).
Using Mikro-Tip catheter transducers,we performed direct measurements of leftventricular function in isoproterenol-treated �2KO and wild-type animals. Wealso studied saline-treated �2KO miceand compared their left ventricle dp/dtmeasurements to previously reported val-ues from wild-type mice to validate ourexperimental technique. The dp/dt valuesmeasured in our saline-treated �2KO an-imals were comparable to values previ-ously reported for wild-type mice (15,16). Our data did not reveal significantdifferences in dp/dtmax or �dp/dt whenisoproterenol-treated �2KO and isoprot-erenol-treated wild-type mice were com-pared (p � .377 and p � .306, respec-tively, Student’s t-test) (Fig. 2).
When the right ventricle becomes dys-functional, central venous pressures in-crease. An increase in liver weight mayreflect right ventricular dysfunction be-cause this organ can become more con-gested when venous return to the heart isimpeded by rising central pressures. Anincrease in liver weight may also occurfor reasons unrelated to cardiac function.We determined liver to body weight ratiosand found a significant genotype effect(p � .028, two-way ANOVA; Fig. 3). Whenthe data were censored to correct formortality by removing the highest liverweight values from both cohorts, the in-teraction between isoproterenol treatmentand genotype was significant (p � .006,two-way ANOVA).
Elevated pulmonary artery pressurecan lead to right ventricle dysfunction byforcing the right ventricle to eject againsta greater resistance. To determinewhether the absence of �2ARs in the pul-monary vasculature might lead to higherpulmonary artery pressure in �2KOmice, we evaluated right ventricle meansystolic pressure. Right ventricular meansystolic pressure reflects the pulmonaryartery pressure because the pulmonaryvalve is open during this phase of the
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cardiac cycle. Mice without infusionpumps as well as animals that had re-ceived saline or isoproterenol infusionswere studied. We failed to detect a signif-icant genotype effect on right ventriclemean systolic pressure (p � .488, two-way ANOVA).
Cardiac Structure. We employed three
techniques for a study of cardiac struc-ture: heart weight to body weight ratioanalysis, assessment of myocyte ultra-structure using electron microscopy, andquantitative measurement of myocyte ap-optosis using in situ TUNEL staining.
Heart weight to body weight ratioanalysis is a simple technique for identi-
fying cardiac injury (17–19) or enlarge-ment (20). We detected a drug treatmenteffect but no genotype effect when heartweight to body weight ratio comparisonswere made (Fig. 4).
Electron microscopy can be used toidentify subtle structural changes in theheart. Since these changes may occur inthe absence of cardiac enlargement, elec-tron microscopy can distinguish differ-ences in heart structure that might notbe identified using heart weight to bodyweight ratio analysis.
We provided a pathologist 113 elec-tron microscopies from midventricularsections of isoproterenol-treated �2KOmice, isoproterenol-treated wild-typemice, saline-treated �2KO mice, and sa-line-treated wild-type mice. Blinded tothe genotype and treatment group ofeach sample, we asked the pathologist toevaluate the electron microscopies interms of myofiber integrity, organelleseparation, and mitochondrial integrity.We asked him to provide an overall injuryscore ranging from 1 to 4 (1 � little or noinjury; 4 � severe injury) (13). He failedto identify a statistically significant geno-type effect (p � 0.315, two-way ANOVA;Fig. 5).
Myocyte apoptosis is thought to play arole in the progression of cardiac injuryafter a variety of insults, such as ischemiaand drug toxicity (21, 22). Using in situTUNEL staining, we found that after 3and 7 days of isoproterenol infusion,�2KO mice demonstrated significantlymore apoptosis than wild-type mice (ge-notype effect: p � .04 and p � .0007,respectively, two-way ANOVA). Saline-treated mice served as control animals.Evaluation of the data from this studyalso suggested that myocyte apoptosis in-creased progressively over the course ofthe study, increasing at 3 days, peaking at7 days, and then declining again by 10days (Fig. 6). We believe that the declinein myocyte apoptosis at 10 days reflectsthe elimination of apoptotic cells fromthe myocardium over time.
In a separate experiment, we com-pared myocyte apoptosis in animals with-out infusion pumps to myocyte apoptosisin mice at 7 days of saline and isoproter-enol infusion (Fig. 7). We observed a sig-nificant genotype effect (p � .0001, two-way ANOVA) and saline and isoproterenoltreatment effects (p � .03 and p � .0001,respectively; two-way ANOVA). How-ever, the genotype-treatment interac-tion terms were not significant (F �3.87 and F � 1.92, respectively, Bonfer-
Figure 1. Cardiac function in wild-type and �2 adrenergic receptor knockout (�2KO) mice afterisoproterenol infusion: graded exercise treadmill testing. Using graded exercise treadmill testing 3 daysafter completion of a 14-day isoproterenol infusion, we indirectly evaluated cardiac function. The datarevealed a decrease in exercise capacity after isoproterenol infusion for both wild-type and �2KOanimals. Although the drug effect was found to be significant (p � .0001, two-way analysis of variance),the genotype effect was not significant (p � .248, two-way analysis of variance).
Figure 2. In vivo assessment of left ventricle contractile function. Mikro-Tip catheter transducers wereused to measure left ventricle maximum change in pressure over time (dp/dtmax). We administeredintraperitoneal DL-propranolol (30 mg/kg) 30 secs before the onset of data collection to minimize theeffects of � adrenergic receptor stimulation. The mean dp/dtmax values for isoproterenol-treatedwild-type and �2 adrenergic receptor knockout (�2KO) mice were not statistically significant before(not shown) or after propranolol administration (p � .377, Student’s t-test).
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roni posttests), suggesting that a base-line difference might exist between�2KO and wild-type mice in terms ofmyocyte apoptosis.
Electrophysiology. We evaluated fiveisoproterenol-treated �2KO mice and fivesaline-treated �2KO animals using ECG.No evidence of myocardial injury was de-tected in isoproterenol-treated �2KO an-
imals by ECG. No abnormalities in termsof rate, rhythm, intervals, or axis wereobserved either at baseline or after iso-proterenol or saline infusions (23).
Effects of Isoproterenol Infusion onBlood Pressure and Heart Rate. After-load reduction is an indirect means bywhich peripheral �2AR activation mightafford protection to the hearts of wild-
type animals relative to �2KO mice. Ac-tivation of �2ARs in the peripheral vas-culature has been shown to mediatevasodilation (24, 25). Using a noninvasiveblood pressure measurement device, weperformed a series of experiments to de-termine whether differences in bloodpressure existed between �2KO and wild-type mice. We found no significant differ-ences in systolic blood pressure betweenisoproterenol-treated �2KO and wild-type mice (p � .09, Bonferroni/Dunn posthoc analysis, repeated-measures ANOVA;Fig. 8).
Tachycardia-induced cardiac injuryhas been well documented (26, 27). Todetermine whether heart rate differencesexisted between isoproterenol-treated�2KO and wild-type mice, we comparedheart rates in the mice during the infu-sions. Results of this experiment sug-gested no significant differences betweenthe heart rates of isoproterenol-treated�2KO and isoproterenol-treated wild-type animals (p � .99, Bonferroni/Dunnpost hoc analysis, repeated-measuresANOVA).
DISCUSSION
Our results provide evidence that�2ARs mitigate some of the deleteriouseffects of chronic �1AR stimulation. Inmice subjected to a chronic infusion ofthe nonselective �AR agonist isoprotere-nol, we observed higher mortality rate in�2KO mice than in wild-type animals.Moreover, we observed more apoptosis inthe hearts of �2KO mice.
The results of our study indicate that�2KO and wild-type mice are more dis-similar than we initially believed. Previ-ous investigations suggested that �2KOanimals were normal in size and demon-strated heart rates and blood pressuressimilar to wild-type control animals. Onlyduring significant stress were differencesbetween �2KO and wild-type mice appar-ent. �2KO mice were found to becomehypertensive in response to exercise.However, they also demonstrated en-hanced exercise capacity and more effi-cient oxygen utilization during exercise(lower respiratory exchange ratio at agiven workload) (12). Our data suggestthat even modest stress (such as salineinfusion) can elicit differences in myocyteapoptosis between �2KO and wild-typemice, indicating that the absence of �2ARsignaling is important even when physi-ologic differences are not apparent.
Figure 3. Liver weight to body weight ratio analysis after isoproterenol infusion. Analysis of data fromthis experiment revealed a significant genotype effect on liver weight to body weight ratio (p � .028,two-way analysis of variance). When data were corrected for mortality by removing the highest liverweights from each group, the interaction between isoproterenol treatment and genotype was found tobe significant (p � .006, two-way analysis of variance). �2KO, �2 adrenergic receptor knockout mice.
Figure 4. Heart weight to body weight ratio analysis after isoproterenol infusion. Analysis of the datarevealed a significant treatment effect but no significant genotype effect in terms of heart weight tobody weight ratio (treatment p � .0001, genotype p � .35, two-way analysis of variance). �2KO, �2adrenergic receptor knockout mice.
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A growing body of evidence suggeststhat �2AR stimulation is protective (6, 9,28). However, the mechanisms by which�2AR-mediated protection is conferred
remain unclear. �2AR-mediated reduc-tion in myocyte apoptosis by a Gi-dependent signaling pathway is likely onefactor. The capacity of �2ARs but not
�1ARs to couple to Gi represents an im-portant point of divergence between�1AR and �2AR signaling, and other Gi-dependent signaling pathways may alsocontribute to the protective effect of�2AR stimulation.
Evidence now suggests that in cardiacmyocytes, receptor targeting and traffick-ing mediated by interactions with cellularscaffolding proteins dictate subtype-specific signaling. The PDZ domain bind-ing motifs at the carboxyl termini of�ARs differ significantly between �1ARsand �2ARs and mediate interactions withdifferent cellular scaffolding proteins(29–32). For instance, the �1AR PDZ mo-tif interacts with postsynaptic densityprotein-95 and related proteins. This in-teraction prevents agonist-induced inter-nalization (33) and appears to prevent�1AR coupling to Gi. Disruption of thismotif leads to efficient �1AR-Gi coupling
Figure 5. Assessment of myocyte ultrastructure after isoproterenol infusion using electron microscopy. A trained specialist (cardiac pathologist) assessedthe ultrastructure injury score to be 2.9 � 0.2 in isoproterenol-treated �2 adrenergic receptor knockout mice (�2KO), 2.0 � 0.2 in isoproterenol-treatedwild-type mice, 1.6 � 0.2 in saline-treated �2KO mice, and 2.0 � 0.1 in saline-treated wild-type mice. Although a statistically significant treatment effectwas identified (p � .001, two-way analysis of variance), a genotype effect was not observed (p � .315, two-way analysis of variance).
Figure 6. Quantitative determination of myocyte apoptosis using in situ terminal deoxynucleotide trans-ferase-mediated dUTP nick-end labeling (TUNEL) staining. Quantitative determination of myocyte apopto-sis was performed at three time points: 3, 7, and 10 days after isoproterenol pump insertion. *A significantgenotype effect was observed at 3- and 7-day time points (p � .04 and p � .0007, respectively). Saline-treatedmice served as control animals (data not shown). �2KO, �2 adrenergic receptor knockout mice.
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in neonatal cardiac myocytes (29). Incontrast, the �2AR PDZ motif interactswith different PDZ domain-containingproteins, such as ezrin-binding-50/sodium-hydrogen exchange regulatoryfactor family proteins, which are respon-sible for receptor sorting between recy-cling and degradative endocytic pathways(32). In vitro studies demonstrate thatmutation of the �2AR PDZ motif disrupts�2AR-Gi coupling possibly by interferingwith receptor recycling after endocytosis(30).
Our data suggest that �2AR activationreduces myocyte apoptosis in vivo.
Whether myocyte apoptosis contributeddirectly to the higher mortality rateamong isoproterenol-treated �2KO ani-mals is unclear. We did not observegreater impairment of cardiac function inisoproterenol-treated �2KO mice relativeto isoproterenol-treated wild-type mice.However, structural injury leading toevents such as lethal arrhythmias re-mains a possible cause of the higher mor-tality. �2KO mice may be more suscepti-ble to arrhythmias due to alteredsignaling in their myocytes. Other inves-tigators have reported that in a caninemodel, arrhythmogenicity increases dur-
ing �1AR (but not �2AR) activation byvirtue of a cyclic adenosine monophos-phate-dependent pathway (34). Other in-vestigators have also reported that �2ARactivation attenuates �1AR-mediated ar-rhythmias (35). We did not observe ar-rhythmias during our ECG analyses.However, this approach cannot be con-sidered an adequate assessment method.
Noncardiac events may also haveplayed a role in the higher mortality rateamong our isoproterenol-treated �2KOmice. For example, changes in the he-patic, renal, and/or central nervous sys-tems may have adversely affected the�2KO mice. In addition, isoproterenol in-fusion may have affected metabolic andneuroendocrine pathways differently in�2KO mice compared with wild-type an-imals.
Our data also revealed a significantgenotype effect on liver weight to bodyweight ratios. However, we have not de-termined why this phenomenon oc-curred. One possibility is that the livers of�2KO mice are more susceptible to directeffects of isoproterenol infusion. Anotherpossibility is that venous congestion oc-curs in the livers of �2KO mice due toimpaired right heart function, althoughwe did not obtain direct evidence of rightventricular dysfunction.
In summary, the results of this studysuggest that in vivo �2AR activation isantiapoptotic and contributes to myocar-dial protection.
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