Laboratory Investigation

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1 620 Lbortory Investigtion Atril Ntriuretic Peptide in Rt Model of Crdic Filure Atril nd Ventriculr mrna, Atril Content, Plsm Levels, nd Effect of Volume Loding H. Drexler, MD, J. Hnze, PhD, M. Finckh, MD, W. Lu, MD, H. Just, MD, nd R.E. Lng, MD With the technicl ssistnce of Evi Hblwetz Downloded from by guest on April 12, 2018 This study exmined the reltion between synthesis, tril storge, nd plsm levels of tril ntriuretic peptide (ANP), nd it exmined plsm ANP levels nd hemodynmic output in response to volume expnsion in rt model of myocrdil infrction nd filure. Arteril ANP concentrtions did not correlte linerly with infrct size, but they did show n brupt increse when infrct size exceeded 30% of the left ventricle, similr to the brupt increse of left ventriculr end-distolic pressure with infrct size greter thn 30%. Consequently, close reltion ws found between plsm ANP levels nd left ventriculr end-distolic pressure (n =23, r=0.89, p < 0.001). Atril ANP content per grm of tissue but not ANP content per pir of tri ws reduced in rts with lrge infrcts (> 40%,p < 0.05 vs. control nimls). ANP mrna level per pir of tri (relted to totl tril RNA), determined by liquid hybridiztion (controlled by northern blot nlysis), ws incresed by 38% in infrcted rts (p<o0.05 vs. controls), but the rtio of tril ANP mrna reltive to tril j3-ctin mrna levels ws not incresed. Right nd left ventriculr ANP mrna level incresed by 90% nd 380%, respectively, fr exceeding the concomitnt increse in f,-ctin mrna (+26% in the left ventricle). Plsm ANP incresed with volume loding in controls nd rts with moderte infrcts but not in rts with lrge infrcts despite similr increse in right tril pressure (compred with control nimls); thus, the reltion of AANP/Aright tril pressure exerted by volume loding decresed in rts with lrge infrcts. Similrly, the response of crdic output nd renl blood flow (determined by rdioctive microspheres) to volume loding ws ttenuted in rts with lrge infrcts. Thus, in this model of chronic crdic filure, the ctivtion of the ANP system is closely coupled with the increse in intrcrdic pressures without correlting linerly to the extent of myocrdil loss. Second, in severe crdic filure, dditionl stimultion such s volume loding my elicit only n ttenuted ANP secretion response, for exmple, due to sturtion of the ANP receptor sensing system or to limited trnsformtion rte of pro-anp. Third, the increse in tril ANP synthesis nd the increse in tril ANP gene expression seems limited; however, substntil specific ANP gene expression occurs in the ventricles, which, in turn, my contribute to incresed plsm ANP levels in chronic hert filure. (Circultion 1989;79: ) P revious studies showed tht n tril fctor tht induces ntriuresis nd diuresis is relesed from rt hert-lung preprtions in From Medizinische Klinik III (H.D., M.F., W.L., H.J.), University of Freiburg, Freiburg, nd the Phrmkologisches Institut (J.H., R.E.L.), University of Heidelberg, Heidelberg, FRG. Supported by the Deutsche Forschungsgemeinschft (DFG, Dr 148/3-2 nd SFB 320 A2). Address for correspondence: Helmut Drexler, MD, Medizinische Klinik III, Universitt Freiburg, HugstetterstrBe 55, 7800 Freiburg, FRG 0761/ Received July 21, 1987; revision ccepted November 18, response to high tril perfusion pressure.1 Right tril ppendectomy hs reduced the renl response to cute hypervolume in the rt,2 nd relese of immunorective tril ntriuretic peptide (ANP) from isolted rt herts in response to volume loding hs been shown.3 Plsm ANP concentrtions re elevted in ptients with pressure or volume overlod, such s hypertension, renl filure, or congestive hert filure4,5,6 nd from secondry to incresed tril stretch.7 Conversely, recent studies indicted tht crdic tri of the Syrin hmster with crdiomyopthy or

2 Downloded from by guest on April 12, 2018 rts with myocrdil infrction re deficient in ANP when mesured per grm of tissue.8 9 Furthermore, ANP messenger RNA ws recently shown to increse in rt model of circultory filure due to ortl-cvl shunt,10 indicting tht tril ANP synthesis nd turnover is incresed in hert filure. Additionl stimuli (such s volume loding) in this sitution my elicit only limited secretion of ANP into the circultion. However, significnt increses in ANP content nd ANP mrna in ventricles were observed in recent studies, suggesting tht the ventricle could be n importnt source of ANP in congestive hert filure.10"11 To investigte biosynthesis, storge, nd the mechnisms of ANP secretion in hert filure, we mesured tril nd ventriculr ANP mrna levels, tril content of ANP, plsm ANP levels, nd the effects of volume loding in rt model of myocrdil infrction nd filure.12"3 Specific ANP mrna nd f3-ctin mrna levels in tril nd ventriculr tissue were mesured simultneously to determine whether or not the chnges in ANP mrna levels re specific nd not merely mrkers of overll protein synthesis. Methods Experimentl Preprtions Infrction ws produced by left coronry rteril ligtion s described previously14 nd s modified by McLen et l.15 In mle Sprgue-Dwley rts, the left coronry rtery ws ligted pproximtely 2 mm from its origin with 6-0 suture. In the shmoperted rts, the suture ws tied loosely so s not to obstruct coronry flow. With this method, the 24-hour mortlity rte ws 40% for the infrction group nd 5% for the shm-operted group. The surviving rts were mintined on stndrd rt chow, nd subsequent experimentl procedures were strted 23 (rnge, 22-24) dys fter surgery. During this postsurgicl period, the mortlity rte in the infrction group ws pproximtely 15%. Instrumenttion Animls were nesthetized with hlothne (1% in oxygen), nd ctheters (PE 50) were inserted into the left ventricle through the right crotid rtery, til rtery, right jugulr vein, nd right femorl vein. Ctheter positions in the left ventricle were determined by pressure wve detection by Stthm P231D pressure trnsducers (Clevelnd, Ohio) nd displyed on recorder screen (Siemens Elem Mingogrf, Erlngen, FRG). After closure, nimls were llowed to recover for minimum of 3 hours before experimentl procedures were initited. This recovery period is of sufficient durtion to ensure return to stedy-stte conditions in the rt.16 Regionl Blood Flow Mesurements Rdioctive microspheres (`41Ce, 103Ru; New Englnd Nucler, Dreieich, FRG), 15±+5,um in Drexler et l ANP in Crdic Filure 621 dimeter, were used to mesure regionl blood flow nd crdic output with the reference smple technique17 s dpted for use in the rt.18 A detiled presenttion of the rdioctive microsphere technique used in this study hs been described.16 In brief, the microspheres suspension ws thoroughly mixed by gittion nd soniction (Brnson Sonifier B-30, Dnbury, Connecticut) immeditely before ech injection. Approximtely 250,000 microspheres (0.5 ml) ws then injected into the left ventricle during 15-second period followed immeditely by 0.5-ml heprinized sline flush during 15-second period. To obtin the reference smple, rteril blood ws withdrwn (beginning 15 seconds before nd continuing during the injection of microspheres) through the til rtery t rte of 0.35 ml/ min mintined by Hrvrd constnt flow pump (Model 906, South Ntick, Msschusetts). The totl blood withdrwl period ws 1.5 minutes for ech injection nd ws used s the blood flow reference smple. Immeditely fterwrd, 0.5 ml blood ws withdrwn for mesuring plsm ANP levels. At the end of the study, nimls were killed by phenobrbitl injection in the ventricle, nd orgns nd tissue smples were removed. All smples were immeditely blotted, weighed, nd trnsferred to two-chnnel Gmm scintilltion counter (Kontron 480 AR, Munich, FRG) for mesuring rdioctivity levels. A Digitl Equipment Professionl 350 computer ws used to clculte regionl blood flow nd totl crdic output.19 Hemodynmic Output Trcings from the left ventriculr, right tril, nd cudl ctheter were recorded continuously during the experimentl protocol nd were used to obtin hert rte, left ventriculr pek systolic pressure (LVSP), left ventriculr end-distolic pressure (LVEDP), men right tril pressure (RAP), nd men rteril pressure (MAP) (by electronic integrtion). The pulstion pressure trce ws displyed periodiclly to check for proper undmped pulse trce, prticulrly from the right tril ctheter. Stroke volume ws clculted from crdic output nd hert rte. Totl vsculr resistnce ws determined from crdic output nd men rteril pressure dt. With the exception of crdic output, hemodynmic dt were collected immeditely before the microsphere injection. Determintion of Infrct Size The left ventricle nd the ventriculr septum were seprted, weighed, nd fixed in 10% formlin nd subsequently cut in eight trnsverse slices (5,um) from pex to bse nd stined with vn Gieson's stin nd mounted.20 With plnimeter Digitl Imge Anlyzer (Leitz, Wetzlr, FRG), the endocrdil nd epicrdil circumference of the infrcted nd noninfrcted portions of the left ventricle were mesured. The infrcted men circumference (men

3 Downloded from by guest on April 12, Circultion Vol 79, No 3, Mrch 1989 of epicrdil nd endocrdil circumferences) of ll eight slices ws summed nd then expressed s rtio of the summed men circumference of the left ventricle. For ll nimls with infrction, myocrdil infrction ws trnsmurl with mture scr locted t the left nterior free wll. Are mesurements of infrct size were not mde becuse these hve underestimted infrct size s result of resorption of necrotic tissue nd subsequent wll thinning20 nd lte development of compenstory myocrdil hypertrophy.21 Determintion of Plsm Levels nd Atril Content of Immunorective Atril Ntriuretic Peptide Blood smples were collected into plstic tubes contining disodiummethylenediminetetrcetic cid (NEDTA). Plsm concentrtions of ANP (determined s immunorective tril ntriuretic fctor) were determined by direct rdioimmunossy without extrction. Fifty microliters of plsm were dded to 350 btl 0.1 M Tris buffer, ph 7.4, contining 0. 1% geltin nd ntibody t finl dilution of 1:80,000. The chrcteristics nd specificity of this ntibody hve been described.3 Trcer (1251 rt ANP, Amershm, Brunschweig, FRG) ws dded fter overnight incubtion (4,000 cpm/tube), nd the incubtion continued for nother 24 hours. Seprtion of free from ntibody bound 1251-peptide ws chieved by precipittion with second ntirbbit immunoglobulin ntibody. Synthetic ranp ws used to construct stndrd curves. To correct for possible interference of plsm proteins with binding, ll smples of the stndrd curve contined 50 gl plsm from rt plsm pool tht ws pssed previously through SepPk crtridges for removing endogenous ANP. With this procedure, the lowest concentrtion of ANP yielding binding significnt from tht in the bsence of stndrd t the 95% confidence intervl ws 2 pg/tube. The 50% intercept ws t 58 pg/tube. The interssy vrition ws 15%, nd the intr-ssy vrition ws 7% (n= 10). For mesuring tril ANP content, the tril tissue of right nd left tri ws minced nd boiled for 15 minutes in 0.1 M HCl to void proteolysis. The cooled tissue ws homogenized subsequently with Polytron tissue homogenizer (Kinemtik, Luzern, Switzerlnd) nd centrifugted for 30 minutes t 3,000g. The resultnt superntnt ws extrcted with ODS-silic crtridges s described recently.3 The recoveiy of ANP ws between 85% nd 90%.3 Protocol 1 Hemodynmic output including RAP, hert rte, LVESP, LVEDP, nd MAP were recorded continuously until remining unchnged (t lest 20 minutes), nd 0.5 ml rteril blood ws withdrwn through the ctheter in the til rtery (infrcted group, n = 23; shm-operted group, n =15). Nineteen of these nimls (infrcted group, n =11, 401 ± 22 g body weight; shm-operted group, n = 8, 389 ± 17 g body weight) successfully underwent the following volume-loding procedure. Rts were excluded if ctheter problems occurred; for exmple, insufficient blood withdrwl from the til rtery or unrelible ctheter position during volume loding. Four infrcted nimls died suddenly before nd during volume loding. Five milliliters 0.9% i.v. sline ws dministered during 1-minute period (through the ctheter in the ven femorlis), nd blood ws withdrwn 1, 3, nd 5 minutes fter the sline infusion for mesuring plsm ANP concentrtions. The nimls were killed by n overdose of phenobrbitl, nd the hert ws quickly removed; the right nd left tri were weighed, frozen, nd stored t - 70 C until mesuring ANP content. Tissue ANP levels were expressed per microgrm of tissue nd per pir of tri. The left ventricles were fixed in formlin s described bove. In seprte group (infrcted, n = 9; shm-operted, n = 8), the nimls were used solely for mesuring infrct size nd tril ANP mrna level per pir of tri. RNA blot hybrtidiztion nd hybrtidiztion ssy. For prepring n ANP-specific complementry RNA hybridiztion probe, 580-bses Pst I-restricted ANP-cDNA frgment, obtined from K. Bloch nd J. Seidmn, Boston, Msschusetts, ws subcloned in psp64 plsmid.22 The Eco Rl-linerized plsmid served s templte for ANP-cRNA trnscription with 100 gci -32P UTP (400 Ci/mmol, Amershm) nd the SP 6-Polymerse-promotor systems described by Melton et l.23 Totl RNA ws prepred by the LiCl-ure technique.24 After quntifiction by bsorption t 260 nm, predetermined mount of RNA ws dentured in glyoxl, loded on 1% grose gel, frctionted electrophoreticlly, nd blotted to nitrocellulose pper. Hybridiztion ws done in 50% formmid, 50 mm Nphosphte, ph 6.8, 1 M NCI, 200 jug/ml herring sperm DNA, 5xDenhrdt, 0.1% sodium dodecyl sulfte (SDS), nd 10 mm NEDTA for 18 hours t 650 C. Liquid hybridiztion ws done with modified procedure described by Durnm nd Plmiter.25 Hybridiztion totl RNA ws dissolved in 30,ul hybridiztion buffer [40% formmid, 40 mm piperzine-n,n'-bis(2-ethnesulfonic cid), 0.4 M NCl, 1 mm NEDTA, nd 1,000 cpm ANP-cRNA/,ul] nd incubted for 18 hours t 700 C. After RNAse tretment (dilution in 10-fold volume of 0.3 M NCI, 10 mm Tris buffer, ph 7.4, 5 mm NEDTA, incubted for 1 hour t 30 C), intct crna-mrna hybrid ws precipitted with 10% trichlorocetic cid, the precipitte ws collected in nitrocellulose filter, nd rdioctivity ws counted in,l-scintilltion counter. The mount of hybridized mrna ws clculted from the rdioctivity of the precipitte nd the specific ctivity of the crna probe. Results were then expressed s picogrm of mrna per microgrm of totl RNA.

4 Downloded from by guest on April 12, 2018 Protocol 2 Hemodynmic output (similr to protocol 1) ws monitored during 30 minutes, nd rdioctive microspheres were injected t the end of this bseline period (infrcted group: n = 11; body weight, g; shm-operted group: n = 12; body weight, 367 ± 18 g). Arteril blood ws withdrwn from the til rtery for mesuring crdic output s described bove, nd second smple ws obtined for mesuring plsm ANP levels (pproximtely 2 minutes fter injection of microspheres). Three minutes fter strting the sline infusion (identicl to protocol 1), the injection of microspheres ws repeted for mesuring crdic output nd regionl blood flow. This short time lg between sline infusion nd injection of microspheres ws selected becuse erlier experiments3 showed tht plsm ANP should rise to mximl levels within 5 minutes.3 Injection of microspheres nd 2-minute time period before blood smpling, similr to the protocol for obtining control mesurements, resulted in mximl rise of ANP levels during the nticipted time period of 5 minutes. The nimls were killed by injecting phenobrbitl into the left ventricle, nd the tissues including hert nd kidneys were removed for rscintilltion nlysis (Kontron 480 AR). The primry objective of this second protocol (opposed to protocol 1) ws to evlute the systemic nd regionl vsculr effects fter incresed plsm ANP levels induced by volume loding. Withdrwl of 0.5 ml blood for mesuring ANP plsm levels nd removl of blood in the ctheter ded spce mkes up to 1 ml of blood, which my influence regionl vsculr blood flow by ctivting neurohumorl effects. Therefore, blood for mesuring ANP plsm levels ws withdrwn fter injecting microspheres (which includes withdrwl of 0.5 ml of blood for mesuring crdic output), lthough it ws nticipted tht the volume lod ssocited with the injection of microspheres would increse the plsm ANP concentrtion. Protocol 3 The herts of seven shm-operted nimls (body weight, 409±14 g) nd 11 infrcted nimls (body weight, 424±17 g) were excised 41±3 dys fter surgery. Atri (right nd left tri together) nd the right nd left ventricles were seprted, weighed, nd frozen in liquid nitrogen. Specil ttention ws pid to the seprtion of tril nd ventriculr myocrdium. Therefore, fter weighing the tissues, the edges of the bse of right nd left ventricles were cut to exclude contmintion of right or left ventriculr myocrdium with tril tissue. The scr tissue of the left ventricle ws seprted, nd only vible myocrdium of the left ventricle (hypertrophied inferior wll nd septum) ws used for mesuring mrna. The infrct size of the left ventricle ws estimted bsed on the totl weight of the left ventricle nd the size of the scr tissue (infrcted re). Although less ccurte thn histologic mesurement of infrct size, this method yields infrct Drexler et l ANP in Crdic Filure 623 sizes similr to histologic plnimetry (unpublished observtion from this lbortory). The ANP-specific crna hybridiztion probe ws prepred s described bove. For preprtion of ctin-specific crna hybridiztion probe, 640- bse Sl I-EcoRI frgment of,b-ctin-like pseudogene ws subcloned into pgem4 plsmid.26 The Hind III-linerized plsmid served s templte for trnscription with SP6 Polymerse s described for ANP. Totl RNA ws prepred by the LiClure technique.24 After quntifiction by bsorption t 260 nm, 2.5,ug RNA ws dentured in 1 M glyoxl, ph 5, 50% dimethyl sulfoxide, 0.01 M N-phosphte, ph 7, loded on 1.2% grose gel, frctionted electrophoreticlly, nd blotted to nytrn 13N membrne. Hybridiztion ws done in 50% formmid, 50 mm N-phosphte, ph 7, 1 M NCl, 200,ug/ml slmon sperm DNA, 200 pig/ml yest RNA, 5 x Denhrdt, 0.2% SDS, nd 10 mm NEDTA for 3 hours t 700 C. Then, 2 x 10 cpm/ml /3-ctin nd ANP probe, respectively, were dded to the hybridiztion mix, nd incubtion ws continued for further 18 hours. The membrnes were wshed in 0.1 x 15 mm NCl nd 1.5 mm N-citrte, 0.1% SDS t room temperture nd t 700 C nd then exposed to rdiogrphic film with intensifying screens t - 70 C. The specific bnds of,b-ctin nd ANP were cut, nd rdioctivity ws mesured in,/-scintilltion counter. In the cse of the tri blots, ANP nd f8-ctin hybridiztion ws performed seprtely becuse of very strong ANP signl compred with the /3-ctin signl. Sttisticl Anlysis Dt re reported s men ± SEM. Student's t test nd nlysis of vrince followed by Student- Newmn-Keuls test were used to determine sttisticl significnce of differences. Results Weights of right ventricle, lungs, nd tri of three infrcted rts used for microsphere studies were mesured nd were significntly elevted compred with shm-operted control rts (tri: shmoperted group/infrcted group, 0.25 ± 0.03/0.61 ± 0.05 g/kg body weight; right ventricle, 0.57±0.04/ 0.81±0.05 g/kg body weight; lungs, 3.43±0.3/ 6.22±0.9 g/kg body weight; for ll three orgn weights, p<0.02). Infrct size of these nimls verged 38.4±2% of the left ventricle. Crdic output nd renl blood flow were significntly decresed in infrcted nimls (p <0.05, Tble 1), wheres LVEDP (infrcted group, 25±2 mm Hg; shm-operted group, 8±1 mm Hg; p <0.01) nd RAP (p<o0.05, Tble 1) were significntly elevted. Thus, depressed crdic performnce indicting chronic hert filure is documented by hemodynmic output nd confirmtory signs of hert filure such s tril nd right ventriculr hypertrophy or incresed lung weights.

5 624 Circultion Vol 79, No 3, Mrch 1989 Downloded from by guest on April 12, 2018 TABLE 1. Crdic Output, Right Atril Pressure, Plsm ANP, nd Regionl Blood Flow Before nd After Volume Loding Hemodynmic Shm-operted (n = 12) Infrcted (n = 1 1) vlue Control Volume loding Control Volume loding Crdic output (ml/min/kg) ±35* 310± ±24 Right tril pressure (mm Hg) 0.4 ± ± 0.6* 2.6 ± 1.Ot 6.9 ± 1. 1 Plsm ANP (pg/ml) 1259 ± ± 198* t Regionl blood flow (ml/min/g) Kidney 4.6± ± 0.4* 3.6 ± 0.5t 3.5 ±0.4 Left ventricle 6.9± ±1.2* 4.2 ±0.3t * Right ventricle 5.7± ± ± ± 1.3* Intestines 3.2± ± 0.4* 2.9 ± ±0.5 Brin ±0.1* 1.1± ±0.1 Skin 0.07± ± 0.009* 0.04 ± 0.004t 0.05 ±0.01 Liver 0.27± ± ±0.06 Dt re men ± SEM. ANP, tril ntriuretic peptide. *p<0.05 vs. corresponding control; tp<0.05 vs. shm-operted control; ::p<0.01 vs. shm-operted control. Figure 1 (top pnel) shows the reltion of LVEDP to infrct size, indicting tht LVEDP is consistently incresed when infrct size exceeds the 30% level. Arteril ANP concentrtions did not correlte linerly with infrct size but showed the sme brupt increse (compred with LVEDP) when the infrct size exceeded 30% (Figure 1, middle pnel). However, the rteril plsm ANP levels were closely correlted with LVEDP (Figure 1, bottom pnel). In 12 infrcted rts, in which relible mesurements of right tril pressure were obtined, the correltion between rteril ANP levels nd RAP yielded r= 0.612, p<0.05. Atril immunorective ANP concentrtion per grm of tissue ws decresed in nimls with lrge infrcts (>40%), wheres no difference ws found between control rts nd rts with infrcts less thn 30% (Figure 2). However, when tril ANP content ws expressed per totl weight of both tri, no differences were found between rts with lrge infrcts nd shmoperted rts. In Figure 3, the tril ANP mrna per totl RNA (determined by liquid hybridiztion) is plotted ginst infrct size. The vlidity of the liquid hybridiztion dt ws controlled by northern blotting in smller number of herts, yielding similr results. The tril ANP mrna content ws incresed (by more thn 2 SD of the men of the control group) only in rts with lrge infrcts ( > 40%), which corresponds to the reduced tril ANP content per pir of tri in these nimls (Figure 2). In seprte group of nimls, tril ANP mrna per totl RNA ws incresed in infrcted rts compred with shm-operted rts (+ 38%, p < 0.05) (Figure 4). Infrct size of the ltter group verged 33 ±4% (rnge, 25-40%) of the left ventricle, nd the weights of tri nd right ventricle were significntly higher thn in the corresponding shm-operted nimls (tri, 0.29±0.04 vs. 0.45±0.07 g; right ventricle, 0.62 ± 0.05 vs ± 0.06 g; p < 0.05 shm-operted vs. infrcted). The tril 13-ctin mrna per totl RNA of shm-operted nd infrction groups did not differ significntly, lthough slight increse occurred in the infrction group (+ 13%) (Figure 4). However, the rtio of tril ANP mrna to f8-ctin mrna ws not significntly elevted in the infrction group (10.1 vs. 8.9), indicting tht the increse in ANP mrna reltive to fl-ctin mrna my not be specific in this group of nimls (Figure 5). In contrst, the rtio of ANP mrna to f3-ctin mrna ws substntilly incresed in both ventricles in the infrction group compred with the shm-operted group (Figure 5). The right nd left ventriculr ANP mrna levels were 1.9 nd 3.8 times higher (+90% nd 380%) in the infrction groups thn in shm-operted nimls (Figures 4 nd 6). In contrst, right nd left ventriculr /3-ctin mrna levels of infrction nd shmoperted groups did not differ significntly (Figures 4 nd 6). Thus, despite significnt ventriculr hypertrophy nd incresed overll protein synthesis in this setting, mrked specific increse in ANP mrna ws found in both ventricles in this model of myocrdil infrction nd filure. The plsm ANP levels of nimls with moderte infrcts ( < 30%) were higher thn in control nimls; however, the difference ws not significnt becuse of the wide rnge of the ANP concentrtions of this infrcted group. However, the plsm levels in nimls with lrge infrcts were substntilly nd significntly higher thn in control nimls nd in nimls with moderte infrcts (Figure 7). After volume loding, the plsm concentrtions of ANP did not increse in nimls with lrge infrcts. In rts with moderte infrcts (<30%) nd in the control group, significnt increse in plsm

6 Drexler et l ANP in Crdic Filure T LVEDP 30t 20t * m 10t 2SD U1 n U * ' infwrct size (x of LV) Downloded from by guest on April 12, 2018 AW petl 1400T ' 800' 600' 400' 200' o 1400T ( - : I ~ ~ ~ ~~~~~~~~9 I2S inferct size (I of LV) nw r p< _.~~~~~~ *y'v~~~~~ t 1- -i -0 _k LVEDP (Hg). FIGURE 1. Plots of reltions of left ventriculr enddistolic pressure (LVEDP) (top) nd plsm tril ntriuretic peptide (ANP) (middle) to infrct size expressed s percentge of the left ventricle nd of the reltion between plsm ANP levels nd LVEDP (bottom) (n =23 for ech). n = 15 for shm-operted nimls with infrct size=0. ANP concentrtions occurred. Of note, the increse in right tril pressure during volume loding ws similr in ll three groups (Tble 2). Bseline tril pressure ws higher in the group with lrge infrcts, lthough the difference ws not significnt, perhps becuse of the smll smple size (type II error; note, tht in two shm-operted nimls nd in nimls with moderte infrcts no relible mesurements of RAP were obtined nd, therefore, were excluded from RAP nlysis). However, RAP ws significntly incresed in group of nimls tht underwent blood flow studies (see below nd Tble 1). Considering the time course fter volume loding, plsm ANP concentrtions decresed fster (5 minutes fter volume loding, Figure 7) in the control group thn in the infrcted group with moderte infrcts. The decrese in plsm ANP levels between 1 nd 5 minutes fter volume loding ws significnt in the shm-operted group only (p < 0.05 by ANOVA). The difference between the shm-operted nd infrcted group with moderte infrcts for the plsm ANP vlues 5 minutes fter volume loding ws not significnt (p= by nonpired t test). Infrcted nimls (infrct size, %) did not increse significntly crdic output fter volume loding (Tble 1), which is in contrst to the control group. Similrly, renl blood flow ws incresed fter volume loding in the control group but not in the infrction group. Plsm ANP concentrtions did rise significntly with volume loding only in the control group, not in the infrction group, lthough the increse in RAP chieved with volume loding ws similr for both groups (Tble 1). When

7 626 Circultion Vol 79, No 3, Mrch 1989 Downloded from by guest on April 12, 2018 tril ANP content ug 20mr let 12- SHAM MI<30% MI>40% FIGURE 2. Br grph of tril immunorective tril ntriuretic peptide (ANP) concentrtions in shmoperted nimls (n = 8), in rts with infrct size less thn 30% (n =6) nd more thn 40% of the left ventricle (n = 5). Open brs, vlues per grm of tissue; htched brs, vlues per pir of tri. *p < 0.05 vs. corresponding shm-ope, subgrour signiflcr ANP Pg4jg totl mrna/ RNA FiGURE 3. (ANP) me infrct sil proportiot shm-ope, * Blood flows (other thn to the renl bed) before nd fter volume loding re given in Tble 1, indicting increses in blood flow to most circultory beds in the shm-operted group nd to the hert in the infrction group. Plese note tht blood flow dt for the left ventricle represent the composite of vlues for flows to norml tissue, infrcted tissue, nd intermedite regions of residul ischemic tissue becuse the left ventricle of these nimls ws used for mesuring infrct sizes (trnsverse slices) fter mesuring blood flow. Figure 8 depicts the reltion between infrct size nd AANP/LXRAP elicited by volume loding. The increse in plsm ANP relted to the increse of RAP (exerted by volume loding) vries widely in shm-operted nimls; nevertheless, the bility to increse plsm ANP levels for given increse in RAP seems ttenuted in nimls with lrge infrcts. This is evident lso from Tble 2 where RAP nd ANP vlues of shm-operted rts nd of rts with moderte nd lrge infrcts re given seprtely. The plsm ANP vlues in shm-operted rts in Tble 1 were considerbly higher thn in the groups in Figures 1 nd 7 becuse these blood smples were obtined fter injecting microspheres, which, in itself, is ssocited with moderte volume lod. Discussion 'rted vlue. This niml model of myocrdil infrction nd filure yielded wide rnge of left nd right yen- infrcted nimls tht hd n increse triculr dysfunctions in proportion to infrct size, subset offfive in crdi, c output ws nlyzed, no consistent similr to recent reports from Pfeffer et l'2 nd increse in renl flow ws observed. The bseline Tsunod et l.9 However, threshold phenomenon vlues olf RAP (1.7 ± 0.9 mm Hg) of the ltter five rther thn close liner reltion ws observed nimls were lower compred with the group s between LVEDP nd infrct size. Pfeffer et l whole,, t:)ut these ltter nimls hd experienced reported tht LVEDP ws consistently incresed similr iincreses in RAP fter volume loding when infrct size exceeded 46% of the left (5.8±1.3 mm Hg). Plsm ANP incresed in this ventricle.12 Although the threshold for infrct size,but this increse ws not sttisticlly ws lower in the present study, perhps becuse of nt (1,690± 130 vs. 2,142± 182 pg/ml). the different method of mesuring infrct size or 1. n. -_ different rt strin or both, we observed similr brupt rise in LVEDP, when infrct size pproched 30-35% of the left ventricle (Figure 1). A similr threshold phenomenon ws observed when infrct size ws plotted ginst plsm ANP concentrtions; consequently, ANP plsm levels showed close liner reltion to the LVEDP s shown in Figure 1. A similr reltion ws observed between plsm ANP levels nd RAP; lso, this correltion ws less close. RAP my correlte more closely to right ventriculr or pulmonry rtery ANP levels u, ~~~thn in rteril blood smples mesured in the D The threshold phenomenon between infrct size 0102D 3D 40 SD ~~~nd tril ANP levels my indicte tht the regionl INFARCT SIZE (% OF LV) wll motion bnormlity represented by the moder- Plot of reltion of tril ntriuretic peptide te infrcts re compensted by crdic mech-?ssenger RNA per microgrm of totl RNA nd nisms, which do not include the (tril) ANP sysze. Note tht ANP mrna incre-ses out of tem. If tril ntriuretic peptide ws ctivted under n only in rts with lrge infrcts. n =8 for these circumstnces, one would expect stimulted,rted nimls with infrct size=0. synthesis nd turnover, possibly combined with

8 Drexler et l ANP in Crdic Filure 627 ATRIA 250 OT T mrna 2.5,ug totl RNA TL SHAM INFARCT 5000 ol ACTIN Downloded from by guest on April 12, 2018 mrna per 2.5 MJg totl RNA RIGHT VENTRICLE ANP 1SHAM INFARCT FIGURE 4. Br grphs of messenger RNA of /3-ctin nd tril ntriuretic peptide (ANP) (cpm/2.5 mg totl RNA) of tri-pir of tri, right nd left ventricle in shmoperted (n= 7) nd infrcted (n = 11) rts. Note tht the tril 3-ctin mrna levels re not comprble to the ventriculr -ctin mrna vlues (see "Methods"). *p < vs. corresponding shm-operted vlue; **p < vs. corresponding shmoperted vlue. LEFT VENTRICLE ** mrna per 2.5 g totl RNA shm /ielinfarct ACTIN

9 628 Circultion Vol 79, No 3, Mrch 1989 ANP mrna/ctin rtio mrna L ll SHAM //// INFARCT ** * Downloded from by guest on April 12, 2018 ATRIA RV LV FIGURE 5. Br grph of rtio of tril ntriuretic peptide (ANP) messenger RNA to 13-ctin mrna in tri nd right nd left ventricles (RV, LV) of shm-operted (n = 7) nd infrcted (n = 11) rts. The rtio incresed by 2.5 times for RV nd L V. *p < vs. corresponding shm-operted vlue; **p < vs. corresponding shm-operted vlue. reduced tril content of tril ntriuretic peptide. However, no significnt difference in tril ANP content nor in plsm ANP concentrtions ws found between rts with moderte infrctions nd shm-operted nimls. In ccord with recent studies,8'9 however, tril content of ANP per grm of tissue ws reduced in nimls with severe left ventriculr dysfunction. However, totl tril ANP content per pir of tri ws not reduced even in rts with lrge infrcts due to substntil tril hypertrophy. This finding is in keeping with recent study investigting tril ANP content (per grm nd pir of tri) of the Syrin hmster.27 The tril ANP mrna relted to totl tril RNA ws incresed by 38% in infrcted rts compred with controls, which is consistent with recent observtions.10 Totl tril RNA, itself, increses with elevted intrcrdic pressures possibly with proportionl increses in ANP mrna. This would indicte tht the increses in tril ANP mrna my not be specific for the ANP gene s suggested by Mendez et 128 but is merely mrker for incresed totl mrna nd protein synthesis. These investigtors reported similr reltive tril ANP mrna levels (tril ANP mrna to 18S RNA) in rts with infrct size greter thn 30% compred with controls.28 To ddress this issue of whether or not these chnges in ANP mrna re specific, both tril ANP mrna nd /3-ctin mrna levels were simultneously mesured. In contrst to tril ANP mrna, there ws only minor nd insignificnt increse in tril,3-ctin mrna level in this model. However, the rtio of ANP mrna to /3-ctin mrna, lthough slightly incresed (10 vs. 8.9), did not differ significntly for the shmoperted nd infrcted groups. Thus, the increse in tril ANP mrna my not be specific in this group with moderte infrct size (33%); however, with very severe crdic filure, ANP mrna my increse out of proportion s suggested by the substntilly incresed ANP mrna levels observed in rts with infrct sizes greter thn 40%. Nevertheless, the increses in tril ANP mrna levels re moderte, t best, compred with the mrkedly incresed ventriculr ANP gene expression. As suggested by Frnch et l,11 the ANP gene my be expressed in norml tri ner its mximl rte.'1 Incresed demnds due to elevted pressure nd stretch7 nd stimulted secretion then would led to reduced tril ANP storge (tril ANP per milligrm of tissue) s observed in nimls with lrge infrcts in the present study s well s in severl previous studies.27'29 The present finding of substntil increses in ventriculr ANP mrna is consistent with recent reports, lthough the mgnitude of chnges in ANP mrna seems different in the hmster nd in the spontneously hypertensive rt (perhps becuse of different time course of the disese). Frnch et ll' reported tht ventriculr ANP mrna levels were seven nd 13 times higher in hmsters with moderte nd severe hert filure, respectively, thn in control nimls. ANP mrna levels in ventricles of spontneously hypertensive rts were seven times higher thn in those of control Wistr-Kyoto rts.30 Dy et 131 reported fold increse in reltive ANP mrna levels of the left ventricle fter ortic bnding nd left ventriculr hypertrophy. The ltter

10 Drexler et l ANP in Crdic Filure 28s lbs ACTIN ANF..E..jl-,:i; E - 1* 2* 4* 5* 6* 7* 8* 3* 9 10 R.VENTRICLE FIGURE 6. Northern blot nlysis of tril ntriuretic peptide (ANP) messenger RNA nd 13-ctin mrna from right (R) nd left (L) ventricle of ll 18 herts. Numbers with sterisks denote rts with infrction nd filure. 28s Downloded from by guest on April 12, 2018 ACTIN ANF t ' 188 1' 2* 3* 28s 4* 5* L,VENTRICLE R.VENTR ICLE - lts ACT IN ANF -6* 7* 8* * L.VENTRICLE is close to the 3.8 times increse in left ventriculr ANP mrna levels observed in the present study. Similr to tril ANP mrna, this increse in ventriculr ANP mrna level could reflect n overll increse of protein synthesis during right nd left ventriculr hypertrophy. In fct, substntil right ventriculr hypertrophy is documented by significntly incresed weight of the right ventricle in infrcted nimls. Compenstory left ventriculr hypertrophy in this model hs been reported by severl investigtors.21'32'33 In contrst to the slight increse in left ventriculr f3-ctin mrna, left ventriculr ANP mrna levels were mrkedly elevted in the infrction group compred with the shmoperted group. Consequently, the rtio of ventriculr ANP mrna to /3-ctin mrna in infrcted rts ws 2.5 times tht of shm-operted nimls. Thus, the increse in ventriculr ANP gene expression is specific for the ANP gene beyond n increse in protein nd mrna synthesis in response to stimulus to hypertrophy. Nevertheless, this incresed expres- l * 18* sion of ventriculr ANP gene my be relted (to some extent) to ventriculr hypertrophy; for exmple, both the development of hypertrophy nd ANP gene expression my be secondry to incresed wll stress. In fct, this ugmented expression of the ANP gene my operte s self-compenstory mechnism of the hert s suggested by Ari et l.311 Considering the close reltion between LVEDP nd rteril plsm ANP levels in the present study, it is tempting to speculte tht the left ventricle serves s n importnt source of ANP, which, in turn, my contribute to reduction of fterlod nd left ventriculr wll stress. The combined mesurements of ANP mrna, tril ANP content, nd plsm ANP levels show tht incresed intrcrdic pressures cuse longterm stimultion of ANP synthesis nd secretion of ANP into the circultion. If this long-term stimultion is pronounced, tril ANP content (per grm of tissue) eventully decreses. The plsm concentrtions before nd fter volume loding re consistent

11 630 Circultion Vol 79, No 3, Mrch 1989 Downloded from by guest on April 12, 2018 ANP pg/mi TIME (min) with this hypothesis. If left ventriculr dysfunction is only moderte, the response to volume loding would be similr to the response in control nimls. Similrly, preliminry results were reported recently from ptients with moderte crdic filure when secretion of ANP ws stimulted by volume expnsion.34 The reltively slow decrese in ANP plsm levels fter volume loding (in nimls with moderte infrcts, see Figure 7) my be explined by the time course of tril pressure in these nimls. Compred with the control nimls, RAP required longer time period to rech the bseline vlues. In nimls with lrge infrcts, plsm ANP concentrtions were mrkedly elevted; however, volume loding ppers to elicit no significnt further increse (i.e., ttenuted response) under these circumstnces. Similrly, reduced response to cute volume loding ws observed in spontneously hypertensive rts.35 In contrst, recent report by Chien et 136 suggested tht incresed ANP secretion fter volume loding is preserved in rts with hert filure. However, this conclusion ws bsed on six rts with infrcts less thn 20% FIGURE 7. Plot of effect of volume loding (5 ml sline during 1-minute period) on plsm immunorective tril ntriuretic fctor (ANP) levels in rts with moderte infrctions (open dimonds, n=6, <30% of the left ventricle), lrge infrctions (closed tringles, n =5, >40%) compred with shm-operted nimls (closed squres, n = 8). *p < vs. shmoperted rts during bseline; **p < vs. corresponding control vlues before volume loding (for both the shmoperted nd the infrcted group with moderte infrct size); +p<0.05 vs. the corresponding shm-operted group t 5 minutes; + +p<0.05 (ANOVA) vs. 1- minute time point of the shm-operted group. nd four nimls with infrcts greter thn 20%. In the present study, n ttenuted secretion of ANP fter volume loding ws noted only in rts with lrge infrcts (>40%, see Figure 7), wheres the response of rts with moderte infrcts (< 30%) did not differ from control nimls, which is similr to the observtions of Chien et l. Furthermore, nd of note, our infusion rte ws twice s fst s the one used by those investigtors. Our results obtined in nimls with lrge infrcts suggest tht ctivtion of ANP ws ner mximl t bseline, nd therefore, dditionl stimultion such s volume loding my exert only n ttenuted ANP secretion response, for exmple, one tht is due to sturtion of the ANP receptor sensing system or to limited trnsformtion rte of pro-anp. In support of the ltter hypothesis, Grci et 137 recently observed tht most of the relesed ANP during blood volume expnsion in the rt corresponded to high moleculr weight peptide. This suggests tht the processing enzymes re not ble to trnsform pro-anp into the shorter peptide during volume chllenge. Although some dysfunction of the sensing mech- TABLE 2. Increse in Right Atril Pressure nd Atril Ntriuretic Pressure With Volume Loding in Rts With Moderte or Lrge Infrcts or Without Infrcts Shm-operted (n=6) Myocrdil infrction <30% (n=4) Myocrdil infrction >40% (n=5) Right tril pressure (mm Hg) Before volume loding - 1 min -0.33± ± ±0.52 After volume loding 1 min 4.20± ± ± min ± 0.42* * 5 min 0.21± ± ARight tril pressure (mm Hg) kAtril ntriuretic peptide (pg/ml) 1, Dt re men ± SEM. *p<0.05 vs. corresponding dt of the shm-operted group t 3 minutes.

12 Drexler et l ANP in Crdic Filure 631 5OOT El 0 l A ANP/A RAP 300-0o K cl 0 m FIGURE 8. Plot of reltion between infrct size nd the Aincrese oftril ntriureticpeptide (ANP) vs. Aincrese of right tril pressure (RAP) (pg/ml mm Hg); n = 6 for shm-operted nimls with infrct size = ' i Downloded from by guest on April 12, 2018 INFARCT SIZE (X OF LV) nism for the relese of ANP cnnot be totlly excluded from the present dt, this interprettion seems unlikely from our nlysis of the reltion between /ANP nd ARAP in these nimls (Figure 8, Tble 2). Tken together, synthesis, tril content, nd plsm ANP concentrtions pper to respond ppropritely to wide rnge of crdic dysfunction (ccording to the increses in intrcrdic pressures). During continued stimultion of ANP secretion (due to mrkedly elevted LVEDP in the presence of lrge infrction), the storge of ANP in the tri is decresed; for exmple, the storge of ANP my equilibrte t lower level during incresed synthesis nd turnover. Previous histologic studies on tri hve indeed shown tht grnules in tri were not restored under long-term stimultion.38 Nevertheless, plsm ANP concentrtions t bseline do correlte with the degree of crdic dysfunction (s estimted by LVEDP). This indictes tht despite reduced tril ANP storge, the cpbility for ANP secretion into the circultion is not impired. However, ANP secretion in response to further stimultion by volume loding my be limited in severe crdic filure nd mrkedly elevted LVEDP. We hve shown recently tht the bility of ANP to reduce crdic filling pressures nd to improve renl perfusion is ttenuted in this rt model of crdic filure, 39 which is consistent with findings in the dog40 nd in ptients with congestive hert filure.41 One my speculte, therefore, tht the cute regultory role of ANP is limited under these circumstnces. This does not preclude ANP from hving role in the erly phse of hert filure or from exerting sustined humorl long-term effects or both. ANP hs inhibited renin relese42 nd ldosterone production.43 Incresed plsm ANP levels during the development of crdic filure my counterblnce the vsopressor systems, such s the renin-ngiotensin nd the sympthetic nervous systems. Indeed, n increse in plsm ANP ws observed in the erly phse of crdic filure in cnine model without stimultion of the reninngiotensin-system.4 Moreover, sustined hypotensive response ws observed in srlsin-sensitive two-kidney, one-clip rts during long-term ANP infusion, which my be ttributed to n inhibitory effect on renin relese.45 Furthermore, endogenous ANP hs modulted sympthetic ctivity by inhibiting epinephrine relese nd broreceptor reflexes.46 Whether or not these ctions of ANP persist in chronic hert filure remins to be seen. Recent studies from this lbortory using monoclonl ANP ntibodies suggest tht the elevted endogenous ANP plsm levels do exert vsodiltory ction indicting crdiocircultory role of ANP in this rt model of crdic filure.47 In summry, the present studies suggest tht ctivtion of the ANP system is directly coupled with the increse of intrcrdic pressures without correlting linerly to the extent of myocrdil loss (s estimted by infrct size). In severe crdic filure, the bility of ANP secretion in response to further stimultion seems limited, which is similr to the ttenuted response of trget orgns to ANP under these circumstnces. Wheres the increse in tril ANP synthesis nd the increse of tril ANP gene expression my be limited in severe chronic hert filure, substntil specific ANP gene expression emerges in the ventricles. This ventriculr biosynthesis of ANP, in turn, my contribute to incresed plsm ANP levels in chronic hert filure nd-by reducing fterlod nd left ventriculr wll stress-my constitute self-compenstory mechnism in this setting. Acknowledgments We re indebted to Nikolus Freudenberg, PhD, nd Hnnes Winterer, BS, for skillful mesure-

13 Downloded from by guest on April 12, Circultion Vol 79, No 3, Mrch 1989 ments of infrct size nd K. Bloch nd J. Seidmn (Boston, Msschusetts) for providing the ANP cdna frgment necessry for crna hybridiztion. The,-ctin probe ws kindly provided by M. Moos, PhD, Institute of Neurobiology, University of Heidelberg, Heidelberg, FRG. References 1. Dietz JR: Relese of ntriuretic fctor from rt hert-lung preprtion by tril distension. Am J Physiol 1984; 247:R1093-R Veress AT, Sonnenberg H: Right tril ppendectomy reduces the renl response to cute hypervolemi in the rt. Am J Phiysiol 1984;247:R610-R Lng RE, Tholken H, Gnten D, Luft FC, Ruskoho H, Unger T: Atril ntriuretic fctor A circulting hormone stimulted by volume loding. Nture 1985;314: Gutkowsk J, Horky K, Lchnce C, Rcz K, Grci R, Thibult G, Kuchel 0, Genest J, Cntin M: Atril ntriuretic fctor in spontneously hypertensive rts. Hypertension 1986;8(suppl I):I Rscher W, Tulssy T, Lng RE: Atril ntriuretic peptide in plsm of volume-overloded children with chronic renl filure. Lncet 1985;2: Shenker Y, Sider RS, Ostfin EA, Grekin RJ: Plsm levels of immunorective tril ntriuretic fctor in helthy subjects nd in ptients with edem. J Clin Invest 1985; 76: Edwrds BS, Zimmermn RS, Schwb TR, Heublein DM, Burnett JC: Atril stretch, not pressure, is the principl determinnt controlling the cute relese of tril ntriuretic fctor. Circ Res 1988;62: Chimoskey JE, Spielmn WS, Brndt MA, Heidemnn SR: Crdic tri of BIO 14, 6 hmsters re deficient in ntriuretic fctor. Science 1984;223: Tsunod K, Hodsmn GP, Sumithrn E, Johnston CI: Atril ntriuretic peptide in chronic hert filure in the rt: A correltion with ventriculr dysfunction. Circ Res 1986; 59: Lttion A-L, Michel J-B, Arnuld E, Corvol P, Soubrier F: Myocrdil recruitment during ANF mrna increse with volume overlod in the rt.am JPhysiol 1986;251:H890-H Frnch HA, Dixon RAF, Bline EH, Siegl PKS: Ventriculr tril ntriuretic fctor in the crdiomyopthic hmster model of congestive hert filure. Circ Res 1988;62: Pfeffer MA, Pfeffer JM, Fishbein MC, Fletcher PJ, Spdro J, Kloner RA, Brunwld E: Myocrdil infrct size nd ventriculr function in rts. Circ Res 1979;44: Drexler H, Toggrt EJ, Glick MR, Held J, Flim SF, Zelis R: Regionl vsculr djustments during recovery from myocrdil infrction in rt. JAm Coil Crdiol 1986;8: Drexler H, Depenbusch JW, Truog AG, Zelis R, Flim SF: Effects of diltizem on crdic function nd regionl blood flow t rest nd during exercise in conscious rt preprtion of chronic hert filure (myocrdil infrction). Circultion 1985;71: Mclen D, Fishbein MC, Brunwld E, Mroko PR: Longterm preservtion of ischemic myocrdium fter experimentl coronry rtery occlusion. J Clin Invest 1978;61: Flim SF, Nellis SH, Toggrt EJ, Drexler H, Knd K, Newmn ED: Multiple simultneous determintions of hemodynmics nd flow distribution in conscious rt. J Phrmcol Methods 1984;11: Heymnn MA, Pyne BD, Hoffmn JIE, Rudolph AM: Blood flow mesurements with rdionuclide-lbeled prticles. Prog Crdiovsc Res 1977;20: Flim SF, Morris ZQ, Kennedy TJ: Dextrn s rdioctive microsphere suspending gent: Severe hypotensive effect in rt. Am J Physiol 1978;235:H587-H Wgner HN, Rhodes BA, Sski Y, Ryn JP: Studies of the circultion with rdioctive microspheres. Invest Rdiol 1969;4: Fishbein MC, Mclen D, Mroko PR: Experimentl myocrdil infrction in the rt. Am J Pthol 1978;90: Rubin SA, Fishbein MC, Swn HJC: Compenstory hypertrophy in the hert fter myocrdil infrction in rt. JAm Coll Crdiol 1983;1: Seidmn C, Duby AD, Choi E, Grhm RM, Hber E, Homcy C, Smith JA, Seidmn JG: The structure of rt preprotril ntriuretic fctor s defined by complementry DNA clone. Science 1984;225: Melton DA, Krieg PA, Rebgliti MR, Mnitis T, Zinn K, Green MR: Efficient in vitro synthesis of biologiclly ctive RNA nd RNA hybridiztion probes from plsmids contining bcteriophge SP6 promotor. Nucleic Acids Res 1978; 12: Auffry C, Rougeon F: Prifiction of mouse immunoglobulin hevy-chin messenger RNAs from totl myelom tumor RNA. Eur J Biochem 1980;107: Durnm D, Plmiter RD: A prcticl pproch for quntitting specific mrnas by solution hybridiztion. Anl Biochem 1983;131: Moos M, Gllwitz D: Structure of two humn /3-ctin-relted processed genes one of which is locted next to simple repetitive sequence. EMBO J 1983;2: Frnch HA, Cllhn LT, Bline EH: Plsm nd tril content of tril ntriuretic fctor in crdiomyopthic hmsters. Life Sci 1986;39: Mendez RE, Pfeffer JM, Ortol FV, Bloch KD, Anderson S, Seidmn JG, Brenner BM: Atril ntriuretic peptide trnscription, storge, nd relese in rts with myocrdil infrction. Am J Physiol 1987;253:H1449-H Edwrds BS, Ackermnn DM, Lee ME, Reeder GS, Wold LE, Burnett JC: Identifiction of tril ntriuretic fctor within ventriculr tissue in hmsters nd humns with congestive hert filure. J Clin Invest 1988;81: Ari H, Nko K, Sito Y, Morii N, Sugrw A, Ymd T, Itoh H, Shiono S, Mukoym M, Ohkubo H, Nknishi S, Imur H: Augmented expression of tril ntriuretic polypeptide gene in ventricles of spontneously hypertensive rts (SHR) nd SHR-stroke prone. Circ Res 1988; 62: Dy ML, Schwrtz D, Wiegnd RC, Stockmn PT, Brunnert SR, Toluny HE, Currie MG, Stndert DG, Needlemn P: Ventriculr triopeptin: Unmsking of messenger RNA nd peptide synthesis by hypertrophy or dexmethsone. Hypertension 1987;9: Roberts CS, Mclen D, Brunwld E, Mroko PR, Kloner RA: Topogrphic chnges in the left ventricle fter experimentlly induced myocrdil infrction in the rt. Am J Crdiol 1983;51: Anvers P, Loud AV, Levicky V, Guideri G: Left ventriculr filure induced by myocrdil infrction: I. Myocyte hypertrophy. Am J Physiol 1985;248:H876-H Uretsky BF, Vrblis JG, Murli S, Vldes AM, Kolesr JA, Reddy PS: Cn tril ntriuretic peptide secretion be further stimulted in ptients with severe congestive hert filure (bstrct)? JAm Coll Crdiol 1987;9:33A 35. Ruskoho H, Leppluoto J: Immunorective tril ntriuretic peptide in ventricles, tri, hypothlmus, nd plsm of geneticlly hypertensive rts. Circ Res 1988;62: Chien YW, Brbee RW, McPhee AA, Frohlich ED, Trippodo NC: Incresed ANF secretion fter volume expnsion is preserved in rts with hert filure. Am J Physiol 1988; 254:H185-H Grci R, Cntin M, Thibult G: Role of right nd left tri in ntriuresis nd tril ntriuretic fctor relese during blood volume chnges in the conscious rt. Circ Res 1987; 61: Mrie JP, Guillemot H, Hytt PY: Le degre de grnultion des crdiocytes uriculires: Etude plnimetrique u cours de differents pports d'eu et de sodium chez le rt. Pthol Biol 1976;24: Drexler H, Finckh M, Hoing S, Toth M, Just H, Lng RE: Systemic nd regionl vsculr effects of tril ntriuretic

14 Drexler et l ANP in Crdic Filure 633 peptide in rt model of chronic hert filure. Bsic Res Crdiol 1987;82: Scriven RA, Burnett JC: Effects of synthetic tril ntriuretic peptide on renl function nd renin relese in cute experimentl hert filure. Circultion 1985;72: Cody RJ, Atls SA, Lrgh JH, Kubo SH, Covit AB, Rymn KS, Shknovich A, Pondolfino K, Clrk M, Cmrgo MJF, Scrborough RM, Lewicki JA: Atril ntriuretic fctor in norml subjects nd hert filure ptients. J Clin Invest 1986; 78: Burnett JC, Grnger JP, Opgenorth TJ: Effects of synthetic tril ntriuretic fctor on renl function nd renin relese. Am JPhysiol 1984;247:F863 -F Cmpbell WB, Currie MG, Needlemn P: Inhibition of ldosterone biosynthesis by triopeptins in rt drenl cells. Circ Res 1985;57: Holmer SR, Riegger AJG, Notheis WF, Kromer EP, Kochsiek K: Hemodynmic chnges nd renl plsm flow in erly hert filure: Implictions for renin, ldosterone, norepinephrine, tril ntriuretic peptide nd prostcyclin. Bsic Res Crdiol 1987;82: Grci R, Gutkowsk J, Cntin M, Thibult G: Renin dependency of the effect of chroniclly dministered tril ntriuretic fctor in two-kidney, one clip rts. Hypertension 1987;9: Holtz J, Sommer 0, Bssenge E: Inhibition of sympthodrenl ctivity by tril ntriuretic fctor in dogs. Hypertension 1987;9: Drexler H, Hirth C, Morich F, Trub C, Mio G: Vsodiltory ction of endogenous ANP in chronic hert filure s determined by monoclonl ANP-ntibodies. Circultion 1987; 76(suppl IV):IV-134 KEY WORDS * infrct size * tril ntriuretic peptide. mrna * hemodynmic output * volume expnsion. hert filure Downloded from by guest on April 12, 2018

15 Atril ntriuretic peptide in rt model of crdic filure. Atril nd ventriculr mrna, tril content, plsm levels, nd effect of volume loding. H Drexler, J Hänze, M Finckh, W Lu, H Just nd R E Lng Downloded from by guest on April 12, 2018 Circultion. 1989;79: doi: /01.CIR Circultion is published by the Americn Hert Assocition, 7272 Greenville Avenue, Dlls, TX Copyright 1989 Americn Hert Assocition, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this rticle, long with updted informtion nd services, is locted on the World Wide Web t: published Permissions: Requests for permissions to reproduce figures, tbles, or portions of rticles originlly in Circultion cn be obtined vi RightsLink, service of the Copyright Clernce Center, not the Editoril Office. Once the online version of the published rticle for which permission is being requested is locted, click Request Permissions in the middle column of the Web pge under Services. Further informtion bout this process is vilble in the Permissions nd Rights Question nd Answer document. Reprints: Informtion bout reprints cn be found online t: Subscriptions: Informtion bout subscribing to Circultion is online t: