Endothelial nitric oxide synthase overexpression attenuates congestive heart failure in mice Steven P.Jones",James J.M.Groor,Rien van Haperon',Dirk J.Dunckert,Rini de Cromts,and David 1 Lefer* and GenetiesaruanEperimental CardioloEramUnryMedical Cente Pterda300 DR.The Netherans Edited by Louis J lgnrro.University of Calitoris School of Medicine.Los Angeles CA,and approwes Febroary 21.2003 (receved toe revew 门c4mhr金01升 Congestive heart failure results in cardiowascular dysfunction and in ND availsbility in both normotensive and hypertensive rats diminishod vascular nitric oxide (NO)production.We hypothesized after acute myocardial infaretion. that ovrspression of endathelial ND syntha within the We are uniware of any previous studies of conzestive heart endathelium would reduce the axtent of contractil dysfunction in a tailure in any eNOS overexpressing mouse line.Soch studies are murine model of infartt-induced congestive heart fsilure.We gener imporlanl fur twu neuns.Fird,cndothclial wverexpnoion of ated transgenic [TG)mke overexpressing the human eNos gene.The eNOS would theoretically res in a relativdy low site-specific TG mice displayed significandy enhanced eNoS protein kwels and (physiological)level ot NO production.(verexpressoe ot eNos cNOS activity levels (10 to 12 fold greater)in the aorta and the msy mimie the:clinical servtian ofplormnlge apents (eg coronary vasculature.Non-TG (NTg)and eNOS TG mice wre sub statins and angiotensin-conwrting enzyme inhibitors)inducing the jected to permanent left anterior descending coronary artery ocrlu- prududinn of NO nosulting in cardinertion Uimlely,ths sion and then observed for 1 mo.We assessed cardiac function in wvo would clarify whether moderate ekvaticns in NO production by using echocardography and ultraminiature ventricular pressure coetined to the vascular endothelium atfect the pathogenesis of catheters.Myocardial infarct size was similar between study groups slive hexrt failure,In the pnsent slih,we smght lo ceeter- (70%of the rik zone).Survival wars incruased by 43%in the NOS mine whether genetic overexpresion of eNOS in the vascular TG mice compared with NTg (P 0.5).Fractional shortening and endothetuum ooald attect the seventy of congestive heart tailure cardiat cutput were alsn signifiranthy [P 005)gramer in the ANOS after the acute myoeandial infaretion model in mice. TG than in NTg.Interestingly.pulmonary edema was evident only in NTg mice,and no evidence of pulmonary cdema was observed in the Methods cNOS TG mice.Thus,targeted overexpression of the cNos pene Transgenic (T间Miee.A DNA tragment comt色inz the.监an within the varscular endotheium in mice atenuates both cardfac and NOS gene was isulslad frum a bomcmad human genomic ooemid pulmanary dysfunction and dramatically impemvs wrvival during library (15-17)by using eNOS cDNA (kindhy donated by S. severe congestive heart falure. Jansans.Univ.of Leuven.Leuwen,Belgium)(18)aa prob.In addition,the DNA fragment contained "6kb of5'natural tlankingg ardiovascular disease achersely affects the lives of millions od sequence,induding the native eNOS promnoter,and ~3 kh of 3' people.Often precpitated by myocardial intarction,con- soguence lo the guns.Thus,promder DNA clements were in- glive hesrl failure is charadcriad by wwre ventriobr dy duded that hine been shown to be essemtial in transcnptional function.remodeling.pulmonary edema,and decreased exercise regulation as wel i elements responsible for the tisue dstrihution capacity.Despite its pruvalunce,many of the mechanims asso- of cNOS cprosion (16 19).Ab,the cmiutbclium enhxnocr ciated with the development and pathophysiology of heart ekment that is located 49 kb upstream from the transcriptio start failure remain undetermined.One area bolding particular prom- site of the eNLS gene (161 i included m this constroct.Vector ic is the rle of nilnc oxide (NO)in cungedive hearl failure. saiencs were remived hy nsrietion enckimmclsces Awolutionaf NO i a diffusihk:highl resetive gs farmed hy theee NO 1-2 ug/ml DNA wrs ued tor micronjection of fertilizod oocytes synthae (NOS)isoforms:NOS I (neuronal).NOS 2 (inducibl). fm FVB ckmnor mi:a Irmplanld inl the vidc af and NOS 3 (endothelinl).NO is constituthely produced by the pseudopreznant B10XCBA mice.Founder moce and offspring were vioc.lar endochelium and serves to prnmnte vsculir homecstasis genotyped by PCR on DNA isalated from tail biopsies.Primers (1).NO generated by NUS 3 mantaies normal viscular tone (1,2). sal were S'-GTOCTGCAGACCGTGCAGC-()and S. regale leakoeyte-endthdial ocll intemetions (3)inhiirs plate- GGCTOTTGGTGTCTGAGCCG-3'(antisense).Mice were hackcrossed to C47HL/6 tor at least seven genemtions (99 kt aperegation (4),limits smooth musck oell proliferation (5)and S7B图6.TG行mice were criginally penerated in Rottendm and yfan击k1lefn(6)Gih5n physioic profik,it is likely that NO therapy my prove bene hine boen ustensicly charactericcd previously (16% An equal number of male and female mice (3 4 mo old)were ficial in a number ot dsease states Recenty,inhalntion at esoge used in the NIe and TG groups in the present study.The indivklual ns NO grs has he小wn ta he:nhig与eve th:1gkr performing all esperiments深插niea网oeme3lye infant respiratory distress syndrome in bumans (7刀.Since its ide色 t日all dbata wen:fully sm小心l.Ales:in女kmal lificalion ax snothelium-derivad rclarsing Gdor in 1951)(8).NO Louisiana State Univensity Health Sciences Center conformod to has been implicated m mmerous pathologx sequclae as both a benefical ned a deleterous woiety. state and tederal regulations regarding animal experaentation and to American Physinlogcal Secety Guiddlines Several studies have provided evidence that NO production by endothelial NO5 feNOS)is markedl diminished in congestive Western Blotting and Immunohistochemistry.Acrtas were freshhy heart failure (9-14).Palicnts with hcart faiure have diminishod collected and homogenized in 50 mM Tris-HCI,pH 7.4,con- furctional eNOS capacity compared with normal humans both at rest and during submaximal exercise (9).It was also shown that serm collected from heart failure patients causes endothelial cell chsfunction b doun-repulating eNOS activiry (10).Wiemer e af.(11)repurted dercasod NO bicuvailability in a rodcnt 0ea甲4 K TG tras好nic NTo namtrangenie LV,ltt wenericl;区noccrHa时i model of infrel-inluoeil hesrt fxilure andl fouml this ckererent Tfewvrnoa-prakto-derfdle. NAS Apri 15,2001 vaL to0 ns.B 4181-4896
Endothelial nitric oxide synthase overexpression attenuates congestive heart failure in mice Steven P. Jones*, James J. M. Greer*, Rien van Haperen†, Dirk J. Duncker†‡, Rini de Crom†§, and David J. Lefer*¶ *Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA 71130; and Departments of †Cell Biology and Genetics, §Vascular Surgery, and ‡Experimental Cardiology, Erasmus University Medical Center, Rotterdam 3000 DR, The Netherlands Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved February 21, 2003 (received for review December 6, 2002) Congestive heart failure results in cardiovascular dysfunction and diminished vascular nitric oxide (NO) production. We hypothesized that overexpression of endothelial NO synthase (eNOS) within the endothelium would reduce the extent of contractile dysfunction in a murine model of infarct-induced congestive heart failure. We generated transgenic (TG) mice overexpressing the human eNOS gene. The TG mice displayed significantly enhanced eNOS protein levels and eNOS activity levels (10- to 12-fold greater) in the aorta and the coronary vasculature. Non-TG (NTg) and eNOS TG mice were subjected to permanent left anterior descending coronary artery occlusion and then observed for 1 mo. We assessed cardiac function in vivo by using echocardiography and ultraminiature ventricular pressure catheters. Myocardial infarct size was similar between study groups (70% of the risk zone). Survival was increased by 43% in the eNOS TG mice compared with NTg (P < 0.05). Fractional shortening and cardiac output were also significantly (P < 0.05) greater in the eNOS TG than in NTg. Interestingly, pulmonary edema was evident only in NTg mice, and no evidence of pulmonary edema was observed in the eNOS TG mice. Thus, targeted overexpression of the eNOS gene within the vascular endothelium in mice attenuates both cardiac and pulmonary dysfunction and dramatically improves survival during severe congestive heart failure. Cardiovascular disease adversely affects the lives of millions of people. Often precipitated by myocardial infarction, congestive heart failure is characterized by severe ventricular dysfunction, remodeling, pulmonary edema, and decreased exercise capacity. Despite its prevalence, many of the mechanisms associated with the development and pathophysiology of heart failure remain undetermined. One area holding particular promise is the role of nitric oxide (NO) in congestive heart failure. NO is a diffusible highly reactive gas formed by three NO synthase (NOS) isoforms: NOS 1 (neuronal), NOS 2 (inducible), and NOS 3 (endothelial). NO is constitutively produced by the vascular endothelium and serves to promote vascular homeostasis (1). NO generated by NOS 3 maintains normal vascular tone (1, 2), regulates leukocyte–endothelial cell interactions (3), inhibits platelet aggregation (4), limits smooth muscle cell proliferation (5), and may affect cardiac myocyte function (6). Given this favorable physiological profile, it is likely that NO therapy may prove beneficial in a number of disease states. Recently, inhalation of exogenous NO gas has been shown to be a highly effective therapy for infant respiratory distress syndrome in humans (7). Since its identification as endothelium-derived relaxing factor in 1980 (8), NO has been implicated in numerous pathologic sequelae as both a beneficial and a deleterious moiety. Several studies have provided evidence that NO production by endothelial NOS (eNOS) is markedly diminished in congestive heart failure (9–14). Patients with heart failure have diminished functional eNOS capacity compared with normal humans both at rest and during submaximal exercise (9). It was also shown that serum collected from heart failure patients causes endothelial cell dysfunction by down-regulating eNOS activity (10). Wiemer et al. (11) reported decreased NO bioavailability in a rodent model of infarct-induced heart failure and found this decrement in NO availability in both normotensive and hypertensive rats after acute myocardial infarction. We are unaware of any previous studies of congestive heart failure in any eNOS overexpressing mouse line. Such studies are important for two reasons. First, endothelial overexpression of eNOS would theoretically result in a relatively low site-specific (physiological) level of NO production. Overexpression of eNOS may mimic the clinical observations of pharmacologic agents (e.g., statins and angiotensin-converting enzyme inhibitors) inducing the production of NO resulting in cardioprotection. Ultimately, this would clarify whether moderate elevations in NO production confined to the vascular endothelium affect the pathogenesis of congestive heart failure. In the present study, we sought to determine whether genetic overexpression of eNOS in the vascular endothelium could affect the severity of congestive heart failure after the acute myocardial infarction model in mice. Methods Transgenic (TG) Mice. A DNA fragment containing the human eNOS gene was isolated from a homemade human genomic cosmid library (15–17) by using eNOS cDNA (kindly donated by S. Janssens, Univ. of Leuven, Leuven, Belgium) (18) as a probe. In addition, the DNA fragment contained 6 kb of 5 natural flanking sequence, including the native eNOS promoter, and 3 kb of 3 sequence to the gene. Thus, promoter DNA elements were included that have been shown to be essential in transcriptional regulation as well as elements responsible for the tissue distribution of eNOS expression (16, 19). Also, the endothelium enhancer element that is located 4.9 kb upstream from the transcription start site of the eNOS gene (16) is included in this construct. Vector sequences were removed by restriction endonucleases. A solution of 1–2 gml DNA was used for microinjection of fertilized oocytes from FVB donor mice and transplanted into the oviducts of pseudopregnant B10CBA mice. Founder mice and offspring were genotyped by PCR on DNA isolated from tail biopsies. Primers used were 5-GTCCTGCAGACCGTGCAGC-3 (sense) and 5- GGCTGTTGGTGTCTGAGCCG-3 (antisense). Mice were backcrossed to C57BL6 for at least seven generations (99% C57BL6). TG mice were originally generated in Rotterdam and have been extensively characterized previously (16). An equal number of male and female mice (3–4 mo old) were used in the NTg and TG groups in the present study. The individual performing all experiments was blinded to the mouse genotype until all data were fully analyzed. All experiments performed at Louisiana State University Health Sciences Center conformed to state and federal regulations regarding animal experimentation and to American Physiological Society Guidelines. Western Blotting and Immunohistochemistry. Aortas were freshly collected and homogenized in 50 mM TrisHCl, pH 7.4, conThis paper was submitted directly (Track II) to the PNAS office. Abbreviations: NOS, NO synthase; eNOS, endothelial NOS; LAD, left anterior descending coronary artery; TG, transgenic; NTg, nontransgenic; LV, left ventricle; NS, not significant. ¶To whom correspondence should be addressed. E-mail: dlefer@lsuhsc.edu. www.pnas.orgcgidoi10.1073pnas.0837428100 PNAS April 15, 2003 vol. 100 no. 8 4891–4896 PHARMACOLOGY
A kDa B NTg -200 97.4 -68 eNOS Ta -43 NTg Tg Fig.1.Representarive Wesem blot from aorta of NTg and eNOs TG mice.180 Representative photomicrograph of efos immunohitochenistry taining 1 mM EDTA,01.25 M sucrose.and 20 mM 3(3- at-risk and myocardial infarct size per area at risk and lett cholamidopropyl)dimethylammonio-1-propanesultonate. wentricle (LV)were determined m ench animal as described 23). Wesern hllting was perfurmed as dered previously (2). Twenty-the micrograms ot protein (BCA protein assany kit, Evalation of Arterial and Left Yentricular Hemodynamics.A 14 Picr)was applied to each lane.Ami-NOS w oblained from French Millr Tnrumenls (SPR-671:Housn)pressure Irams Santa Cruz Rinterhnalogy.This antihodly was sko ued far duction catheter was inserted simar to methods described (22. immunohistocbcmistry cxperimentx,whkh were performed ac 23)Data for cach animal were caleulatod frum at least 10 scc of conling lo Rakker el ai.(21). chart recording (arithmetic mean ot at least 5 cardiar cycles) eNOS Activity Assay.Aotas were freshly collected and bomoge Ecocardiographic Assessment of Left Yentricular Function.Irans- nized in 50 mM TrisHCl,pll 7.4,coctaining 1 mM EDTA.0.25 tharacie echocardinerspln of the I.V using a 15-MHz linear M socrose.and 20 mM 3((3-cholamidopropyl)dimethylammo- array transducer (1518)interfaced with a Soquoia C256(Acu- nio]-1-proparesultonate.eNS activity assays were pertormed con,Monlain Viw,CA)wios performed as tscribod (22).All by measuring 1-arginine to L-citrulline comversion with a NOS data were calrulated from 10 cardiac cycles per expenment. assay kit (Calhiochemj nccording to the mamfacturer's instruc. lioex In acklilioral esperimentx.cNO5 emymtie xdiviry was Pumonary Edema.Aocumalation of pulmonary thaid was assessed abo measured in freshly collected hearts.For mocardial eNOS hy weigh鱼gt也ewet)lungs from里ice suh句ected to myocardial aclivily,a membrane-enriched fradtion uars usod imlead of a intarction (or sham).The hangswere then placed in a drying oven tissue homogenate.All measurements of eNOS activity were normaliaed for total protein content.Proecin content was mca- sured by using the BC protein assay kit (ercel. 30 Myocardial Infarction Protocol.Liantion oc the lett anteroe descend 25 igyE(们AD)客sT金lim2 r to methixk described (22).Brlefly.mice were fully anesthetized with Lp. inediomns of ketamine (50 mg/kg)amd penllurhilal sdlium (50 me/ke).The animeks were then attached to surgiend hoard wich heir ventral side up.A modian sicrnutomy u都pcTf和rmed,and the prexim LADviscmplelely ligaled hy potxing a 7-0 sik suture mountod on a tapered neede (BV-1,Ethicon. 5 Somgrvilk.NJ)oompllely around the artery and fily ligating the LAD.The ocdusion remained intact throughout the 4 wk prococol. 0 In additioeal experiments 6 per group),both NTg and NTg eNOS Tg eNUS 'TG mice were subjected to permanent LAD ligation tor 24 h to determine the extent of acute myocardial infarction.Mice shs TO mke.vi!wasdulerind by monur ing the correurion af were alloeed to fully recover after LAD ligation and were -io i-chrullne (pmncl/q/mn).TG mica dnp wyed ( reanesthetized at 24 h after LAD occlusion.Myocardial area P0D10 nhanca过gTtw山y tompined wth Hiy con山ete p2】ww裤ona.op./cg/付e/10193s0374223 的“鞋时L
taining 1 mM EDTA, 0.25 M sucrose, and 20 mM 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate. Western blotting was performed as described previously (20). Twenty-five micrograms of protein (BCA protein assay kit, Pierce) was applied to each lane. Anti-eNOS was obtained from Santa Cruz Biotechnology. This antibody was also used for immunohistochemistry experiments, which were performed according to Bakker et al. (21). eNOS Activity Assay. Aortas were freshly collected and homogenized in 50 mM TrisHCl, pH 7.4, containing 1 mM EDTA, 0.25 M sucrose, and 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. eNOS activity assays were performed by measuring L-arginine to L-citrulline conversion with a NOS assay kit (Calbiochem) according to the manufacturer’s instructions. In additional experiments, eNOS enzymatic activity was also measured in freshly collected hearts. For myocardial eNOS activity, a membrane-enriched fraction was used instead of a tissue homogenate. All measurements of eNOS activity were normalized for total protein content. Protein content was measured by using the BCA protein assay kit (Pierce). Myocardial Infarction Protocol. Ligation of the left anterior descending coronary artery (LAD) was performed similar to methods described (22). Briefly, mice were fully anesthetized with i.p. injections of ketamine (50 mgkg) and pentobarbital sodium (50 mgkg). The animals were then attached to a surgical board with their ventral side up. A median sternotomy was performed, and the proximal LAD was visualized and completely ligated by passing a 7–0 silk suture mounted on a tapered needle (BV-1, Ethicon, Somerville, NJ) completely around the artery and fully ligating the LAD. The occlusion remained intact throughout the 4-wk protocol. In additional experiments (n 6 per group), both NTg and eNOS TG mice were subjected to permanent LAD ligation for 24 h to determine the extent of acute myocardial infarction. Mice were allowed to fully recover after LAD ligation and were reanesthetized at 24 h after LAD occlusion. Myocardial areaat-risk and myocardial infarct size per area at risk and left ventricle (LV) were determined in each animal as described (23). Evaluation of Arterial and Left Ventricular Hemodynamics. A 1.4 French Millar Instruments (SPR-671; Houston) pressure transduction catheter was inserted similar to methods described (22, 23). Data for each animal were calculated from at least 10 sec of chart recording (arithmetic mean of at least 50 cardiac cycles). Echocardiographic Assessment of Left Ventricular Function. Transthoracic echocardiography of the LV using a 15-MHz linear array transducer (15L8) interfaced with a Sequoia C256 (Acuson, Mountain View, CA) was performed as described (22). All data were calculated from 10 cardiac cycles per experiment. Pulmonary Edema. Accumulation of pulmonary fluid was assessed by weighing the (wet) lungs from mice subjected to myocardial infarction (or sham). The lungs were then placed in a drying oven Fig. 1. (A) Representative Western blot from aorta of NTg and eNOS TG mice. (B) Representative photomicrograph of eNOS immunohistochemistry demonstrating increased abundance of eNOS protein in the endothelium of eNOS TG mice compared with NTg. Fig. 2. Assessment of aortic eNOS enzymatic activity in eight NTg and nine eNOS TG mice. eNOS activity was determined by measuring the conversion of L-arginine to L-citrulline (pmolgmin). TG mice displayed significantly (**, P 0.01) enhanced eNOS enzyme activity compared with NTg control mice. 4892 www.pnas.orgcgidoi10.1073pnas.0837428100 Jones et al.
B D 200- 974- 68- NTg Tg NTg NTg Tg Tg Nontransgenic eNOS Transgenic aorta heart enhanced in the sorta and myocardium of eNOSTG mice (51 Representative immunohistochem cal staining of reyocardial tksue haresed from NTg and eNOs T mive sNs prolein in the of uNDS TO mis tompu'ud with the NTy mite. (Econotherm Laboratory Oven,Precision Systems,Natick.MA) We observed no differences in the area at risk per LV or in the for 7 d at 4rC.The lungs were weighed and the dry weights infarct size per area at risk or infarct sixe per LV.Thus,the reconded.The srithmelie differenee between the wet and dry everiry of seute mocandial infaretion was similar in the NTg wcighls yiclded the pulmonary fluid aceumulation values. and eNO5 TG hefore the onset of congestive heart failure. Statistical Analyses.Data were anahzed by ANOVA with post boc Effect af NOS Oveewapreasian an Ventricular Dimensions and Fune- anbsis by using STATVIEW (SAS Cary,NC)sflwan.Dala ane tion.Overexpression of eNOS did not siznificantly affect cardiac rcported as mcans slandard errur of the mean with differenocs function or LV dimensions in control mice under baseline accepted as significant when P=0.05. oonditions (Tale 1).Specifically,I.V diameters,wall thick- nexses,and fractional shortening were similar hetween the NTg Resufts and eNOS TG groups [P-not significunt (NS)]. Characterization of eNOS TG Mice.TG overuxpression uf cNOS Survival During Congestie Heart Fallure.eNOS TG and NTg mice resulted in signifcant incruases in eNOS protein lovels according experienced a significant (P<0.01)degree of mortality com to Wester baot analysis (Fig.14)and immunohistochemistry pared with sham-operated mice during the coegestive heart (Fg 18).This increase in protein levels was also reflected hy an ailure protocol (Fie 5).However,overexpresion of eNOS increase in eNOS activiry (Fig 2).In addition,we observed significantly (P<0.05)improved survival compared with NTg increased eNOS protein levels in the myocardium of eNOS TG mice Survival was ineresed by 43 in the eNOS TG group mice by using Western blotting (Fig.3)and immunostaining at I mo after myocardial infaretion. technigues (Fig.38). Hamedynamies and NOS Overexpresian Daring Congestive Heart Severity of Myocardial Infarction in eNOS TG Mice.The extent of Failare.In NTe and eNOS TGi mice suhjected to myocardial myocardial infarction was measured in NTg and eNOS TG mice infarction,there were no significant differences in systemic at 24 h after permanent LAD coronary artery occlusion (Fig.4). 160 ■g 90 n情0，T白物■线Ta桶 F:NS 0- 80 70 70 60 知 50 40 30 20 0 8 011 10 0248810121418182022242到28 Days post NI AARLV INFMAR INEALV Fig.5.Kaplan-Meier wurviwal turw n sham NTq NTa and aNas TG min Fig.4.Myocardal infarct size in NTg and eNO5 TG mice subjected to pu mununt mytcardal小un.Tha area-eo业风A0 LV wi3MmI aignilkannt(d.)ttrninsurral compatud mihahum mkv after 1 mo AAR and IV w形milor in the NT)3 nd eNOS TG groups(F=5n=年er geaver survival compared with NTg diring te congestiee heart falure group e陌N N5|A15.03|阳l0|e.8|4射
(Econotherm Laboratory Oven, Precision Systems, Natick, MA) for 7 d at 40°C. The lungs were weighed and the dry weights recorded. The arithmetic difference between the wet and dry weights yielded the pulmonary fluid accumulation values. Statistical Analyses. Data were analyzed by ANOVA with post hoc analysis by using STATVIEW (SAS, Cary, NC) software. Data are reported as means standard error of the mean with differences accepted as significant when P 0.05. Results Characterization of eNOS TG Mice. TG overexpression of eNOS resulted in significant increases in eNOS protein levels according to Western blot analysis (Fig. 1A) and immunohistochemistry (Fig. 1B). This increase in protein levels was also reflected by an increase in eNOS activity (Fig. 2). In addition, we observed increased eNOS protein levels in the myocardium of eNOS TG mice by using Western blotting (Fig. 3A) and immunostaining techniques (Fig. 3B). Severity of Myocardial Infarction in eNOS TG Mice. The extent of myocardial infarction was measured in NTg and eNOS TG mice at 24 h after permanent LAD coronary artery occlusion (Fig. 4). We observed no differences in the area at risk per LV or in the infarct size per area at risk or infarct size per LV. Thus, the severity of acute myocardial infarction was similar in the NTg and eNOS TG before the onset of congestive heart failure. Effect of eNOS Overexpression on Ventricular Dimensions and Function. Overexpression of eNOS did not significantly affect cardiac function or LV dimensions in control mice under baseline conditions (Table 1). Specifically, LV diameters, wall thicknesses, and fractional shortening were similar between the NTg and eNOS TG groups [P not significant (NS)]. Survival During Congestive Heart Failure. eNOS TG and NTg mice experienced a significant (P 0.01) degree of mortality compared with sham-operated mice during the congestive heart failure protocol (Fig. 5). However, overexpression of eNOS significantly (P 0.05) improved survival compared with NTg mice. Survival was increased by 43% in the eNOS TG group at 1 mo after myocardial infarction. Hemodynamics and eNOS Overexpression During Congestive Heart Failure. In NTg and eNOS TG mice subjected to myocardial infarction, there were no significant differences in systemic Fig. 3. (A) Aortic and myocardial eNOS protein levels as measured by Western blot analysis in NTg and eNOS TG mice. eNOS protein expression is significantly enhanced in the aorta and myocardium of eNOS TG mice. (B) Representative immunohistochemical staining of myocardial tissue harvested from NTg and eNOS TG mice demonstrating increased eNOS protein in the myocardium of eNOS TG mice compared with the NTg control mice. Fig. 4. Myocardial infarct size in NTg and eNOS TG mice subjected to permanent myocardial occlusion. The area-at-risk (AAR) per LV was similar (P NS) in the NTg and eNOS TG groups. In addition, the infarct size (INF) per AAR and LV was similar in the NTg and eNOS TG groups (P NS). n 6 per group. Fig. 5. Kaplan–Meier survival curve in sham NTg, NTg, and eNOS TG mice during the 4 wk after myocardial infarction. Both NTg and eNOS TG exhibited significant (P 0.01) decreases in survival compared with sham mice after 1 mo of myocardial infarction. eNOS TG mice demonstrated significantly (P 0.05) greater survival compared with NTg during the congestive heart failure protocol. Jones et al. PNAS April 15, 2003 vol. 100 no. 8 4893 PHARMACOLOGY
Table 1.in vivo left ventrioular dimensions of NTg and eNOS TG mice not subjected to any surgery IVrhamher IV antreinr wall IV pnuerior wal L5% D与分oe,mm Systole,mm Dlastole,mm Systole.mm Na式oe,mm Systole,mm NTg 250+1.1 15+01， 之6满+0.16 nRJ+001 1.15+G01 0.5+Q5 1.12-005 eNOS TG 26.4±21 3.53=015 253±0.17 088±0.03 120±0.03 0.9±Q.0明 1.13=0.05 There were nn ugrificant dHerences betwveen the twn g'oupsin amy of the parameten hman.LV5,left wertricular frac士onulshoriening方。topr可3up hemodynamics measured at 4 wk atter myocardial intarctiony So±2sl/min/g）mice compared with幼hum.Cardiac oatput commparod with sham (Tabl 2).Allhough LV systolic prossure in eNOS TG was abo significantly (P 005)higher than NTg was not signiticantly ditterent ansong the three experimental groups,LV cnd diastolic prussure was simnificantly (P005) Pumonary Edema Pulmonary oduma is assucitod with deprcsud higher in the eNOS'ICi and NI'g hearts compared with sham.In cardiac function in the serting of infaret-induced Ly failure. addition,LV devuloped prussure was significantly (P<005) Pulmonary cdema (Fig.7)is expreesed as the absolute fluid kower in the NIg and eNOS Tti hearts compared with sham. weight in the lungs od sham NTg,N'Tg and eNOS IG arter 4 wk of myocardial infarctin.Sham (1022 mg)fuid weight was not Cardiac Hypertrophy.Both NIg and eNOS'TG henrts demonstmted signiticantly ditterent from eNOS TCi (105+3 mg).However. significant (P00l)increases in heart-to-body-weight ratios I mo NTg(l2±3g}palmoeary fluid accumlation wa都signi after coroeary artery occlusion when compared with sham (Table cantly (U.II)bigher than sham NTg mice. 2).'The extent ot cardiac bypertroph was not significanty dtterent betwoen the NTg and eNOS TG groups of hearts. Discussion We provide compelling evidence for a beneticial role of en- Left Ventrioular Dimensions.In additicn to hypertrophy,the present hanced eNOS-derived NO in congestive heart failure.Improve. model of comgestive heart faure induces profound ettecis an ment in survival of congestive heart failure in the eNCS TCi ventricular dimensores (Fig 6).Namely,left veatricular chamber group is the paramount finding of the present study.In addition diametersare significanrly()greaer in diastole and systole to improvements in mortality;mice overexpressing eNOS by 10- in the NTg5.64±024m5.24±029 nm)and eN0gTG to 12-fold exhibited reductions in pulmonary edema and im (5.0±Q22m;4s±Q27m）hearts compared with sham provement in cardiac functiom.All of these inprovements oc- (3.86 009 me 2.80=0.09 mm).The differences between the curred in the eNOS TG mice without apparent differences in NTg and eNOS TCi mice were net statistically significant (P NS) hiseline ventrienlar morphology and function.However,eNOS Fractional shortening (Fig.68)wus also significantly (P<0.01) overexpression did not attenuate the extent of cardiac hyper kwer in hoth heart failure groups compared with sham (27.8 trophy or improe cardisc contractality.It is likely that the 1.2%).More importantly.eNOS TG(12.1.6%)heartsexhibited improvements in cardiac function and survival are a result of a a sienificanth (P<D05)greater extent of fractioml shortening decrease in vasculer resistanee in the eNOS TGi mice.Thee compared with NTg(75±16i findings support the idea that physiological level of NO exert Indices of Cardiac Function.At 4 wk of coronary artery occusion. Before the discovery that the endotbelial-derived relaxing sham NTe NTe and eNOS Ti mice were suhjected ta i ivo factoe w NO.it wis not appreciated that the primary mecha- left ventricular catheterization for determination of intracyclic nism of action of traditional nitrates was via the release of NO ventriclar pressures (Fig.)NTg 4,35 623 mmHa/see (1 and suhsequent vasodilarion.Nitrewasndilatoes have heen used mmlg 133 Pa)and eNOS TG (4,112 611 mmllg/sec) since at kast the mid-19th century (24)for the treatment of exhibited significantly (P 005)diminished maximum (+ angina pectori.Mare recemtly,nitnwodilators have been used dP/dt compared with sham (6.109491 mmlg/sec).Similarly. for the treatment of heart failure (25-27).Nitrovasodilators have minimum (-)dP/dt was significantly attenuated in NTg also been shown to improwe exercise tolerance in patients with (-4,145-58mmHe/x)an1eN05TG(4,225±-425 severe congestie heart Eailure (25,26).It is now known that the mmlla/sec)bearts compared with sham (-7055-662 nitrovasodilators'mechanism of action is via the release of NO mmH/see)Tere were no significant dlifferenees between NTe from the parent compound.Interestingly,adlministration of and eNOS TG in maximum or minimum dP/dt inhaled NO to cangestive heart faihure patients exerts beneficial By tsing Doppler fow analysis during cchcardiograpln effects witboul significantly alterng syslemic hemo:lyrmis cardiac output was measured in all groups of mie atter 4 wk of 29 Although systemic上eod小n2 mics were unch2 nged.t也e myocardial infaretion (Fig 6D)Cardiae oulput only in NTg mecism still appesred to he relsted to alterstions in pulmo- {421±28L/mn/》mice was s1ghic2sty'g01)redaced nary blood floo (Le.,decreased pulmonary vascular resistance compared with sham(S45±30l/Hi/g〉kw,fli In the present stuly,it is poxsle that oerespression of eNOS output was not signinicantly (NS)diminished in eNOS TG exerted beneticial regional ettects on pulmonary blood flow. Table 2.Systemic hemodynamies,vntriculr hamodymamics and cardiac hypertrophy in sham,NTg.and NOS TG mice subjected to 1 mo of coronary artery ocdusion HR bpm MA8即，mmHg SBP.mmHg DEP.mmHg LVSP,mmHg LVEDP,m-Hg HW/BW.mg/g Sh2m 393=12 73=4 86±4 E0±4 8g±3 1g1 4.43±Q.15 NTa 4n1-10 5+1 61+1 刷±4 10+4 625+024* eNOS TG 422±12 6所±5 78±5 56±4 81=5 7=5 6.46±Q.25*n H慑heart rab MAllP,mean arterial上ocd prewut;5园，yvoli blocd preu新EaR,diastolik blood premun需LV巧Rn作nincuar号dplit prewure;LV生De LV end diasolic pressure:HW/3W.heart weignt to body weight rado. P <0.05 vh shom:++P<0.01 vs.sham:otherwise P NS:n-10-12/group
hemodynamics measured at 4 wk after myocardial infarction compared with sham (Table 2). Although LV systolic pressure was not significantly different among the three experimental groups, LV end diastolic pressure was significantly (P 0.05) higher in the eNOS TG and NTg hearts compared with sham. In addition, LV developed pressure was significantly (P 0.05) lower in the NTg and eNOS TG hearts compared with sham. Cardiac Hypertrophy. Both NTg and eNOS TG hearts demonstrated significant (P 0.01) increases in heart-to-body-weight ratios 1 mo after coronary artery occlusion when compared with sham (Table 2). The extent of cardiac hypertrophy was not significantly different between the NTg and eNOS TG groups of hearts. Left Ventricular Dimensions. In addition to hypertrophy, the present model of congestive heart failure induces profound effects on ventricular dimensions (Fig. 6A). Namely, left ventricular chamber diameters are significantly (P 0.01) greater in diastole and systole in the NTg (5.64 0.24 mm; 5.24 0.29 mm) and eNOS TG (5.60 0.22 mm; 4.95 0.27 mm) hearts compared with sham (3.86 0.09 mm; 2.80 0.09 mm). The differences between the NTg and eNOS TG mice were not statistically significant (P NS). Fractional shortening (Fig. 6B) was also significantly (P 0.01) lower in both heart failure groups compared with sham (27.8 1.2%). More importantly, eNOS TG (12.4 1.6%) hearts exhibited a significantly (P 0.05) greater extent of fractional shortening compared with NTg (7.5 1.6%). Indices of Cardiac Function. At 4 wk of coronary artery occlusion, sham NTg, NTg, and eNOS TG mice were subjected to in vivo left ventricular catheterization for determination of intracyclic ventricular pressures (Fig. 6C). NTg [4,336 623 mmHgsec (1 mmHg 133 Pa)] and eNOS TG (4,112 611 mmHgsec) exhibited significantly (P 0.05) diminished maximum () dPdt compared with sham (6,109 491 mmHgsec). Similarly, minimum () dPdt was significantly attenuated in NTg (4,145 578 mmHgsec) and eNOS TG (4,225 425 mmHgsec) hearts compared with sham (7,055 662 mmHgsec). There were no significant differences between NTg and eNOS TG in maximum or minimum dPdt. By using Doppler flow analysis during echocardiography, cardiac output was measured in all groups of mice after 4 wk of myocardial infarction (Fig. 6D). Cardiac output only in NTg (421 28 lming) mice was significantly (P 0.01) reduced compared with sham (545 30 lming). However, cardiac output was not significantly (P NS) diminished in eNOS TG (500 25 lming) mice compared with sham. Cardiac output in eNOS TG was also significantly (P 0.05) higher than NTg. Pulmonary Edema. Pulmonary edema is associated with depressed cardiac function in the setting of infarct-induced LV failure. Pulmonary edema (Fig. 7) is expressed as the absolute fluid weight in the lungs of sham NTg, NTg, and eNOS TG after 4 wk of myocardial infarction. Sham (102 2 mg) fluid weight was not significantly different from eNOS TG (105 3 mg). However, NTg (112 3 mg) pulmonary fluid accumulation was significantly (P 0.05) higher than sham NTg mice. Discussion We provide compelling evidence for a beneficial role of enhanced eNOS-derived NO in congestive heart failure. Improvement in survival of congestive heart failure in the eNOS TG group is the paramount finding of the present study. In addition to improvements in mortality, mice overexpressing eNOS by 10- to 12-fold exhibited reductions in pulmonary edema and improvement in cardiac function. All of these improvements occurred in the eNOS TG mice without apparent differences in baseline ventricular morphology and function. However, eNOS overexpression did not attenuate the extent of cardiac hypertrophy or improve cardiac contractility. It is likely that the improvements in cardiac function and survival are a result of a decrease in vascular resistance in the eNOS TG mice. These findings support the idea that physiological levels of NO exert beneficial effects in the setting of congestive heart failure. Before the discovery that the endothelial-derived relaxing factor was NO, it was not appreciated that the primary mechanism of action of traditional nitrates was via the release of NO and subsequent vasodilation. Nitrovasodilators have been used since at least the mid-19th century (24) for the treatment of angina pectoris. More recently, nitrovasodilators have been used for the treatment of heart failure (25–27). Nitrovasodilators have also been shown to improve exercise tolerance in patients with severe congestive heart failure (25, 26). It is now known that the nitrovasodilators’ mechanism of action is via the release of NO from the parent compound. Interestingly, administration of inhaled NO to congestive heart failure patients exerts beneficial effects without significantly altering systemic hemodynamics (28, 29). Although systemic hemodynamics were unchanged, the mechanism still appeared to be related to alterations in pulmonary blood flow (i.e., decreased pulmonary vascular resistance). In the present study, it is possible that overexpression of eNOS exerted beneficial regional effects on pulmonary blood flow. Table 1. In vivo left ventricular dimensions of NTg and eNOS TG mice not subjected to any surgery LVFS, % LV chamber LV anterior wall LV posterior wall Diastole, mm Systole, mm Diastole, mm Systole, mm Diastole, mm Systole, mm NTg 25.0 1.1 3.55 0.18 2.68 0.16 0.87 0.03 1.15 0.03 0.95 0.05 1.12 0.05 eNOS TG 26.4 2.1 3.53 0.15 2.63 0.17 0.88 0.03 1.20 0.03 0.89 0.06 1.13 0.06 There were no significant differences between the two groups in any of the parameters shown. LVFS, left ventricular fractional shortening; n 10 per group. Table 2. Systemic hemodynamics, ventricular hemodynamics, and cardiac hypertrophy in sham, NTg, and eNOS TG mice subjected to 1 mo of coronary artery occlusion HR, bpm MABP, mmHg SBP, mmHg DBP, mmHg LVSP, mmHg LVEDP, mmHg HWBW, mgg Sham 393 12 73 4 86 4 60 4 88 3 1 1 4.43 0.15 NTg 403 10 72 3 85 3 61 3 84 4 10 4* 6.25 0.24** eNOS TG 422 12 66 5 78 5 56 4 81 5 7 5* 6.46 0.25** HR, heart rate; MABP, mean arterial blood pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; LVSP, left ventricular systolic pressure; LVEDP, LV end diastolic pressure; HWBW, heart weight to body weight ratio. *P 0.05 vs. sham; **P 0.01 vs. sham; otherwise P NS; n 10–12group. 4894 www.pnas.orgcgidoi10.1073pnas.0837428100 Jones et al.
A ■动m aNTg Tp 120 4 105 7 100 1415 四1416 L￥▣山 LVESD 95 W 15 1 NTg F.7.Aumo0灯ede0hs函ssed by net fuid weigh飞in11sham,1S NT4.nd16eM05 TG mous纶ns.NTg mice exhibitedsignificant中0P≤G09 mice did not demonstrate any sipnificant ditferences compared with NTp. Endathclial dysfunetin ooors in palicnls with hear failure (3033).Expermental studies (11-14)indicate that a decrease in NO booavailability is associated with heart failure and actually u 1 cxerts dellerous cffcets durin heart failure.cNOS-deficienl mioe NTg have been subjected to coconary artery hgation and beart faihare (34).This study revealed the important rode odf endogenous NO in tenusting the因hKpy司with coeistne hcarl 000 mailure (34)Scherrer-Croshe er al.(34)Sound that end dinstolic 400 diameter and end diastolic volume were increased,whereas frac. 2000 tional shortuning.contractility.and survial were depressed in eN(-deficient mkce compared with wild-type after 4 wk of LAD) d ligation.Furthermoee.LV hypertropby and myucyte width were 2000 simnificantly increasod in the eNOS-defcient mice.Interestingly. 4000 ■8h capillary denry was lower in the post-myocardial intarction eNOS -4000 deficient mice than wild type.implying a rok for NO in post 8000 infarction cipillry preservation.Scherrer-Crosbie er af.(34,35) aso demonstrated that genetic deficency of eNC resuled in D 60 significant palmonary hypertension and right wertricular remodel ing In the present study,eNOS TG mice mary have impruved 600 enary vaselariry eompared with NTg miee during congestive 550 p<ats heart failure.These findings,in conjunction with the present results proide significant suppart for a procective role of eNOS-derived 500 Nin infaret-indlueed pongestive heart failure. Several lines of eNOS TG mice have been developed that 450 overexpres eNOS in endothelial cells under the direetioe of a 400 preproendotbelin promoter (3b)and cardiac myocytes con trolled by an a MHC promoter (37).Although no specific 350 cardise phenotype has yel been reported st baseline in the 8ham NTg Tg endothelial eNOS TG mouse (36),Brunrereral (37]found mild Fig.6.(AI LV camber diameders during diastole (LVEDD]and spstole negative etfects in terms of cardiac-inducible cacium sensitivity. Although eakium sensiliily was not the focus of the prescnt cct uit.NTg and uNO乃TamK起hi也tud aiggiftant(t",P或Q.1)LV study.dobutamine-stress data indirectly indicate no difference belween the cNOS TG and NTg hearts in torms of caleium mie.Thee'e we's eo sigrificanl dilferern inLYciamlun butwean NIg and scmsitivily.Il is likely that this differenlial effeel is relaled to the higher abundance of eNOS protein in the Brunner er af.studhy signitiranty atteruated in hoth groups OTg and eNos TGl nf mkce sjected (37)cmpared with the present findinggx Additiunally,the sile of tn comnry aetery oerludon compared with sham P<0 01).Hoeer. cNOS owruxpression may be of critical importancc. cnmpared with NTg.Left ventricular mac m.m(+)and min mum(-)dP/dt NO has recently been suggested to coetribute to the patho- pencsis of heart failure (38-40).a tenet that is not uniformly nduced sipnifican [P<005 depression of dP/dtin NTg and eNOsTG mice accepted (40)'The current stody does not support the idea of NO compared with sham.However.dP/dt wos dimferent a deleterious mediator ot congestive heart failure.Although NTg and NDS TO M5).1 By uwng tramthoncit Dopplar-anntud not the foous of our study perse,it is possibl that everproduction echocardiog'aphy.output was determined in sham NTg.NTg and ot NO vin iNOS actually canses negathve etfects daring heart eNOs TG mke atter I mo of coronary artery ocduson.Card ac output was failare due to the high capaciry ot iNOS to produce NO).More studies of iNOS and eNOS in congestive heart failure are 4 ham Dwerexpretunn of AND%TGI nesi止dn与nHcanttyIP,nn司Nigher required to fally elucidate this complex physiological system. output compared with NTg mice after the 4wk congesve heer The prescnt data cemoestrate signifcant bencficial effects in fail.re protocol.N.mbers of mice are located within the bars. eNOS TO mice during heart failure.Although the mechanism of 经n酒时 7MA5|AF1520如|aLt0|r@.I|45
Endothelial dysfunction occurs in patients with heart failure (30–33). Experimental studies (11–14) indicate that a decrease in NO bioavailability is associated with heart failure and actually exerts deleterious effects during heart failure. eNOS-deficient mice have been subjected to coronary artery ligation and heart failure (34). This study revealed the important role of endogenous NO in attenuating the pathophysiology associated with congestive heart failure (34). Scherrer-Crosbie et al. (34) found that end diastolic diameter and end diastolic volume were increased, whereas fractional shortening, contractility, and survival were depressed in eNOS-deficient mice compared with wild-type after 4 wk of LAD ligation. Furthermore, LV hypertrophy and myocyte width were significantly increased in the eNOS-deficient mice. Interestingly, capillary density was lower in the post-myocardial infarction eNOS deficient mice than wild type, implying a role for NO in postinfarction capillary preservation. Scherrer-Crosbie et al. (34, 35) also demonstrated that genetic deficiency of eNOS resulted in significant pulmonary hypertension and right ventricular remodeling. In the present study, eNOS TG mice may have improved coronary vascularity compared with NTg mice during congestive heart failure. These findings, in conjunction with the present results, provide significant support for a protective role of eNOS-derived NO in infarct-induced congestive heart failure. Several lines of eNOS TG mice have been developed that overexpress eNOS in endothelial cells under the direction of a preproendothelin promoter (36) and cardiac myocytes controlled by an -MHC promoter (37). Although no specific cardiac phenotype has yet been reported at baseline in the endothelial eNOS TG mouse (36), Brunner et al. (37) found mild negative effects in terms of cardiac-inducible calcium sensitivity. Although calcium sensitivity was not the focus of the present study, dobutamine-stress data indirectly indicate no difference between the eNOS TG and NTg hearts in terms of calcium sensitivity. It is likely that this differential effect is related to the higher abundance of eNOS protein in the Brunner et al. study (37) compared with the present findings. Additionally, the site of eNOS overexpression may be of critical importance. NO has recently been suggested to contribute to the pathogenesis of heart failure (38–40), a tenet that is not uniformly accepted (40). The current study does not support the idea of NO as a deleterious mediator of congestive heart failure. Although not the focus of our study per se, it is possible that overproduction of NO via iNOS actually causes negative effects during heart failure due to the high capacity of iNOS to produce NO. More studies of iNOS and eNOS in congestive heart failure are required to fully elucidate this complex physiological system. The present data demonstrate significant beneficial effects in eNOS TG mice during heart failure. Although the mechanism of Fig. 6. (A) LV chamber diameters during diastole (LVEDD) and systole (LVESD) in sham NTg, NTg, and eNOS TG mice after 4 wk of coronary artery occlusion. NTg and eNOS TG mice exhibited significant (**, P 0.01) LV chamber dilatation in diastole and systole compared with sham-operated NTg mice. There were no significant differences in LV diameters between NTg and eNOS TG after 4 wk of heart failure. (B) Fractional shortening (%) was significantly attenuated in both groups (NTg and eNOS TG) of mice subjected to coronary artery occlusion compared with sham (**, P 0.01). However, fractional shortening was significantly (P 0.05) higher in the eNOS TG group compared with NTg. (C) Left ventricular maximum () and minimum () dPdt was assessed by using pressure transduction catheters. Myocardial infarction induced significant (*, P 0.05) depression of dPdt in NTg and eNOS TG mice compared with sham. However, dPdt was not significantly different between NTg and eNOS TG (P NS). (D) By using transthoracic Doppler-assisted echocardiography, cardiac output was determined in sham NTg, NTg, and eNOS TG mice after 1 mo of coronary artery occlusion. Cardiac output was significantly (P 0.01) reduced in the NTg compared with sham NTg. eNOS TG mice did not exhibit a significant decrement in cardiac output compared with sham. Overexpression of eNOS (TG) resulted in a significantly (P 0.05) higher cardiac output compared with NTg mice after the 4-wk congestive heart failure protocol. Numbers of mice are located within the bars. Fig. 7. Pulmonary edema was assessed by net fluid weights in 11 sham, 15 NTg, and 16 eNOS TG mouse lungs. NTg mice exhibited significantly (P 0.05) greater fluid accumulation compared with sham lungs. However, eNOS TG mice did not demonstrate any significant differences compared with NTg. Jones et al. PNAS April 15, 2003 vol. 100 no. 8 4895 PHARMACOLOGY
emhanced suvid pears to be raaded to impemvement in ven- eNO5 overexprssio does not diredtly impenve left ventriculr Iricbr fundtion xnl/or ahngalion of pulmunary cles,Ihe function the setting of cenzestve heart falure.Ultimately. madhanion of impnwell ventricubr fundicn i smdear,One- additional studies wil be regared to elucidate the multtde of sble mochanism is unhancod hlod fow lu the cumpenssting mechanstic posibiities and their relative cnntrbutions to the i surviving myocardium i the eNOS TG mice during heart failure. wivo pathophsiology of congestive heart failure. This efect may occur by blunting the vasoconstrictor response In sumary,overexpression of the eNOS caryme significanthy (angotensn l,endothelin etc.)duringg heart failure.The increased mproves survival and attenuates the extent ot conzestive heart bionnilabuiry ot NO may attenuate the lss ct capellanes that hailure afer svore myocandal infardion in m.Thiee dla occurs during heart flure and may nctually potentiate the forma- pruvidod ckar evidenoe of the bemeficial rulo of enhanoed cNOS- tion o new vesseks.In addition,the enhanced NO production in the derived NO peodaction during coagestive heart tailure.Future eNO5 TG mice:moy serve xs an exkbnt sevenger during beart ludlis xhoukl be:ilirededl foranl ilentifying the medismim ofthis fxilen:thenelry mimimxing the ilelerirs effeiix af arercrcile lictiv:effa In the future,therxpio mighl he davkredl lu amd ather nexctive mygon spcci A very likdy explnation for the prie vascubr cNOS funcio箱落means lo impruve cfn或l significant improwmont in cardiac function and survial in the outcomes in patints with congostive heart failure. cN0sTG■e is relaned to a reduction m systemic viecu▣ resistance.Systemic overexpression of the eNO enzyme withn the ascular erdothelim may simply reduce cardiac afterlood,and thi dimne th解coure of these smude两：1 This wnrk wa trnded5.tinn3l mechantan moy be responsible for the increase in cardiac output of Grants RO1 HL-M159 and POI DK-43785 atenuatian of pulmomary edema,and increied srvivl Thiks (in DJ1) 2uEE,EA,hr5,sK,caE4DB.ctkr.D.Y.,ks4白，P.J. AT-减nH.A&4caL0年C红25%16-lm Igrara L1月上数0awk发21l-24. 3..P.N.&.D.N (Proc.Mat Sct 2548. 1-601 n【212A元h119升1%w2211177-1I14s 4 Melios,R T Igeamm 1.1.0lkneie,F.H.Poateerm F G Hpron A.I. 4月nea.T,1.lTf MR,.PI(8I)d,968 5N日.Cm.1 N.E..I.A.【C10 5.Igtre.L.J.Bugt.G M.Wei L H.Bater.P.H.Wu.G.de Seldvn.P. NEA6.2.87-S2 31).4置A44w,40-4s. tam.L A..Zels.R erdan.E.A.Mon.D.T. 4.KR.A,1ead.1.LA Smth,T.W.10g是年7%-11 1门y5L当1当151，-6 22.上aa3ea,.A￥：nhiran,L.A&n.N.151 LAre.Mind.Araoc,2机 s1t,L.1,XAm,M.生，L3l.M.Leam.II at 44-44 《1万M.上L人345-1u 发.Fte3LRF表zslA人V.(I%影Ne3s线.3T3S% X Nhturrotn,A.Meramra 5.Iinaia.Y-Ao.T.Saghea 5.(ra 4171清Jg月-2L 9.Kau.8.D.Khm.T..2ctalon.G.A.Nouhew.L.Petharlea.P.Krcch.M. 24.T.M.M.D Sahn,T.Gi,Dee,G W..Zapal W.M AWw01.(19W口kw01I年11 Agaolni.1..Crh,S Rucheei,T.,Miacams F.Gub.G fanni 1.. 54nI,I环)m人wdL11-t7. 3 Dhirde.H.Hyro.n.MerodlT Hrmig R.Ieg.H Rrsaer.H.R.&Zelk 1人1一P, 1L.1gCG,1g,G,HT.kr2W国I)hm4n3线17-LT7. 3l.uePM.(1t种2k风Cef Conlal3机.2233-24排. 12.Arirua K.Egutin.K.Nakamea R.W.T.Tsitti.H.Shinkrn.H. 32 Seih CLSer.D..Hxcglet C.Rut B.5.Zhing X.Zoo G_N X.B.Kohuri &1b31A02里上hmaL2乳Hs-, ￥，宁生aL长r.53aw,CkH匹【.且C元an,0-6d 35e.T Hourg.A,刀nG,ahR,FB.下iaX,K4A. 级HL,a白LI,Uaa,o到上A3 aktlt L.Pezl P,LaL Kies G A Hisn.T.mAn人，hd.1-月4g CS.kR1利7kC8C3线.12n 14.Reoctiz F.A.P.L.Be .B.D.Vogel T.R.Xa.X. 3s.Scherer-Croie.M..Lllich.B..Bloch.K.D..H.Nesseri.B 10t,T.1北e易aW- Areu.T..Lindbey.M.L.Vincen.A.C..Haing P.L.Lee.R.T.e.12et1 15.van Haneian,R van Td.A.Vemeakn P lautiiree.M wn Gert,T. Cmaknon 14 Lt-1291. 3在LW,S.hcmr-Conbi:.hK.国LLaF,.L (N.Jeroeth.ar.18.1083-LU. a反Cha4从且&2pLW.L.(人红加x.L AaE1apeE黑，L,d如and M,.n DeLE,ca,且，KtM,anAben 1434-2477. K.Hirtring.J.Groovel.F,ulei,D.1.&kC及20B的人试 .Otbl Y,Krrthra星，1 irata.K.Yarradta,T.hhT,le,N Salrsb T..Kur hora,I.Yaouci Y.Yoloyaria.M.(1999).CM Aas. 122%-. 19和的JAd门i,11华1472 97 Rnmse.F.Alie.P.Wntan.G 7edhaer.R Myrr,R Pfe) Cnn43所-302 1Ene,Y,Ma由d,s,4npoL,从keE,K则以aM 38 Senper.D.B.Celoci W.5.(1966)Cavfd Cin 16.657664 2落4-441 3从DL,L.(15静an22-25 复keLR口hpcn民，JE8Ta民，Vbc,Pwat民 LPlB,a.a点5&Kclk线.A.2I01)Oaan14 435【ww*asee水t为V0.10g月pnan.08中429120 5rw时a碱
enhanced survival appears to be related to improvement in ventricular function andor abrogation of pulmonary edema, the mechanism of improved ventricular function is unclear. One possible mechanism is enhanced blood flow to the compensating, surviving myocardium in the eNOS TG mice during heart failure. This effect may occur by blunting the vasoconstrictor response (angiotensin II, endothelin, etc.) during heart failure. The increased bioavailability of NO may attenuate the loss of capillaries that occurs during heart failure and may actually potentiate the formation of new vessels. In addition, the enhanced NO production in the eNOS TG mice may serve as an oxidant scavenger during heart failure, thereby minimizing the deleterious effects of superoxide and other reactive oxygen species. A very likely explanation for the significant improvement in cardiac function and survival in the eNOS TG mice is related to a reduction in systemic vascular resistance. Systemic overexpression of the eNOS enzyme within the vascular endothelium may simply reduce cardiac afterload, and this mechanism may be responsible for the increase in cardiac output, attenuation of pulmonary edema, and increased survival. Thus, eNOS overexpression does not directly improve left ventricular function in the setting of congestive heart failure. Ultimately, additional studies will be required to elucidate the multitude of mechanistic possibilities and their relative contributions to the in vivo pathophysiology of congestive heart failure. In summary, overexpression of the eNOS enzyme significantly improves survival and attenuates the extent of congestive heart failure after severe myocardial infarction in mice. These data provided clear evidence of the beneficial role of enhanced eNOSderived NO production during congestive heart failure. Future studies should be directed toward identifying the mechanism of this protective effect. 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