浙江大学 文本 Acute negative inotropic effects of homocysteine are mediated via the endothelium_生理学

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Frgp.hed Apr08,204:101L5s2pMe10102203, Acute negative inotropic effects of homocysteine are mediated via the endothelium Richard H.Kennedy,Richard Owings,'Nawal Shekhawat,and Jacoh Joseph2 Deparner of Plarwacewical Scfences -eral Mndicime,o Phcrmccology ad 了ag头0 rsiry of dkaw西or Medicul Selence,L企ka西T225 Subeillol 5 Navrirlx 2003,teerped in firal fun 2 Aptil 2004 Kennedy,Richard H..Richard Owings,Nawal Shekhawat,and In addition to the accumulatingg evidence linking Hhe to Jaroh Juseph.Acule negative inotropic cffocts of homucysicinc are vascular puthology and a prochrombotic state,recent studies mediated via the endothelium.Am Piisiof Mleav?Cie Pinsio 287: have demonstrated that chronic IThe elicits adverse effects on H312-H817,2004.Fest published Apnl 8.2004;10.1152/apheart. 01042 2003-Previous su:ics have shown thet chronic hypertumo the myocardium.Blncher and coworkers (2)examined 75 patients with end-stage renal disease undergoing hemodialysis cysteinemnia is associated with an adverse cardac remodeling ad heart tailure.'This study.whch utilized cocorary-pertused hearts ned nnd showed a positive correlation between echoenrdiogrnphi- superfinod popdary micle.was dexigned lo delrrmine whethe cally measured left ventncular mass index and plasma bomo- hoettocysteine acts acutely to alter cardine contractile function.Left cysteine,even alter adjustment for age.gender.systolic blood veniriclar dkvelup司pw整深著neaaire of syslo pressure,and hemalocrit.In addition,a recent Framinghsm function in the Lagendoeff pertused heart whereas Bometri devel study report (30)indicaled that plasma homocysteine levels are oped tension was used papllary muscle.All prepartions were i independent risk factor for congestive heart failure.Studies hithedl in pysiolgieal huffer an poed elertrically.Initia tllx in or laboralory (11)using rat models showed that 10 wk of showed that homocysteine clicits a relatively rapid oreet (maximum Hhe elicits un adverse cardinc remodeling and diastolic dys- cffoct ohscrvod within 5 min).cncmniraliun-dependenl (10-300 function in normotensive animals.and exacerbates the remod- HM)and moderate negative inctropec action (maxumum decretse in cling nnd dinstol c dystinction ohserved in the hypertersive tension wis -15%of control values)Langendortt-pertused hearts heart (11).Thus.in addation to its well-described vascular hut no in papillary misck.In cunlrxsl,cmuent frum homocyslcine treated hearts decreased contractility in pepillary masck,and all octions.chronie Hhe fets in the absenee of other stimuli to inotropic actions were largely eliminated ween brief Inton X-100 elicit direct adverse effects on cardiac structure and functon. Irealment wi ulilined to iretivate the porceary endnthelium in the suggesting that it is an independent risk factor for cardiac intact henrt.The bomocyseine-induced decrease in contraztile fine. remodeling and dysfunction. liun was n antenmed by M-niln-minine,a rilric atalc syn This study was designed to determne whether homocysteine thase inhibnor,or the cyclooygenase inhibnor indomethacin.Thus also has acute effects an cardiac systolic function,because data suzgest that pothophysiological concentrations of homocyseine such nctions miry complement the prevously deseribed chrome dicil teure negalive inolrpic effed on veninear myoeardaim effects on cardiac structure and contribute to the associaled that is mediated by a oocorary endothelium-derived agent other than cardiac dysfuncton.Previous work by Tvagi et al.(27)in ex nitnc oxide or products of cyclooy genase.Future studies are requred vivo cardiac rings of the rat right vertricle and left ventricle to eloci山de the mechan☒n by which homocysteine acts切elicit the showed that homocysteine erhances diastolic tersion and that relense of the proposed endothelal mediator,the entiy o the this nction is enhanced by removal of the endocardial endo- thelium.In addition,studies in various tissues demonstrated action. neure effects of'homoevsteine that could aftect cardine con- contractlity;Langendorf-perfuted heart popilry musele Tntoa tractile fimction were they to occur in the beart.Foe excample. X-100 Eraimenl:ral homocyateine wis shown to inhbit Na-K-ATPase netivity in the hippocampus (24).decrease intracellular calcum con- iPuuooeYsTENEMIA (IThe)has been lirked to cardiovascular oentration in arteriolar smooth muscle (28)and ihibet the disease in several studies:bowever,most of these reports have transient outwurd current in rat ventricular myocyes(22). focused on its vascular and endothelial actions.For exumple,in MATERIALS AND METHODS 1969.MeCully (16)reponed that severe Hhe resuting from inborn errors of metabolim results in vascular disease.and ats All proced山en色s study were3 ved by the Inst other ivestigators (18)subsequently found that the prevalence ulioredl Animal Crc and U实C=me浦he University of Ark of elevated plasma homocystein increases from an estimated sas for Medical Sciences.Theee-mont-old male Sprague-Duwley rats 5%in the general population to 13-47%in patienes with 13U0-325 g)were purchased fros Harian Sprague Doley (Inanap- symptomatic vauscular disease.Retrospective and prospective alis,IN]and manlainol in or indiiona Diion of Isboralory Animal Medicine on a 12:12-h lighl-dark cycle with free tocess to studies (3.17.21)also showed an nssociation hetween Hhe and chow and waer. atherothrombote dscases,including myccardial in farction and lorii-owrfin beur民were inesthe国a with a volande stroke. incsthctic,and hcarts were remowed and immodiacly porfusod vis the corta with an axygenaned Krebs Henseleit (KH)solution (37C)of the Adlresa for teptint roy33 a odkr c4nxR且家8， Dept.of Phammoceuncal Sciences.Mail Slt 522.Unw.of Arkansas for M5 a Scic,4301W.MGa,1业Rd,A级722n5E4u kenedyriharchgcans.cduk 年ccordance with1手Us.C.S8a01T240ky0 ndicote th fict. 日球12 0161-613504 $5 00 Copyright e 20044 the American Prysialegical Seciety prifuww.ajpheart尼

Acute negative inotropic effects of homocysteine are mediated via the endothelium Richard H. Kennedy,1,3 Richard Owings,1 Nawal Shekhawat,3 and Jacob Joseph1,2 Departments of 1 Pharmaceutical Sciences, 2 Internal Medicine, and 3 Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Submitted 5 November 2003; accepted in final form 2 April 2004 Kennedy, Richard H., Richard Owings, Nawal Shekhawat, and Jacob Joseph. Acute negative inotropic effects of homocysteine are mediated via the endothelium. Am J Physiol Heart Circ Physiol 287: H812–H817, 2004. First published April 8, 2004; 10.1152/ajpheart. 01042.2003.—Previous studies have shown that chronic hyperhomo￾cysteinemia is associated with an adverse cardiac remodeling and heart failure. This study, which utilized coronary-perfused hearts and superfused papillary muscle, was designed to determine whether homocysteine acts acutely to alter cardiac contractile function. Left ventricular developed pressure was used as a measure of systolic function in the Langendorff-perfused heart, whereas isometric devel￾oped tension was used in papillary muscle. All preparations were bathed in physiological buffer and paced electrically. Initial results showed that homocysteine elicits a relatively rapid onset (maximum effect observed within 5 min), concentration-dependent (10–300
M), and moderate negative inotropic action (maximum decrease in tension was 15% of control values) in Langendorff-perfused hearts but not in papillary muscle. In contrast, effluent from homocysteine￾treated hearts decreased contractility in papillary muscle, and all inotropic actions were largely eliminated when brief Triton X-100 treatment was utilized to inactivate the coronary endothelium in the intact heart. The homocysteine-induced decrease in contractile func￾tion was not antagonized by N-nitro-L-arginine, a nitric oxide syn￾thase inhibitor, or the cyclooxygenase inhibitor indomethacin. Thus data suggest that pathophysiological concentrations of homocysteine elicit an acute negative inotropic effect on ventricular myocardium that is mediated by a coronary endothelium-derived agent other than nitric oxide or products of cyclooxygenase. Future studies are required to elucidate the mechanism by which homocysteine acts to elicit the release of the proposed endothelial mediator, the identity of the proposed paracrine agent, and the mechanism of its negative inotropic action. contractility; Langendorff-perfused heart; papillary muscle; Triton X-100 treatment; rat HYPERHOMOCYSTEINEMIA (Hhe) has been linked to cardiovascular disease in several studies; however, most of these reports have focused on its vascular and endothelial actions. For example, in 1969, McCully (16) reported that severe Hhe resulting from inborn errors of metabolism results in vascular disease, and other investigators (18) subsequently found that the prevalence of elevated plasma homocysteine increases from an estimated 5% in the general population to 13–47% in patients with symptomatic vascular disease. Retrospective and prospective studies (3, 17, 21) also showed an association between Hhe and atherothrombotic diseases, including myocardial infarction and stroke. In addition to the accumulating evidence linking Hhe to vascular pathology and a prothrombotic state, recent studies have demonstrated that chronic Hhe elicits adverse effects on the myocardium. Blacher and coworkers (2) examined 75 patients with end-stage renal disease undergoing hemodialysis and showed a positive correlation between echocardiographi￾cally measured left ventricular mass index and plasma homo￾cysteine, even after adjustment for age, gender, systolic blood pressure, and hematocrit. In addition, a recent Framingham study report (30) indicated that plasma homocysteine levels are an independent risk factor for congestive heart failure. Studies in our laboratory (11) using rat models showed that 10 wk of Hhe elicits an adverse cardiac remodeling and diastolic dys￾function in normotensive animals, and exacerbates the remod￾eling and diastolic dysfunction observed in the hypertensive heart (11). Thus, in addition to its well-described vascular actions, chronic Hhe acts in the absence of other stimuli to elicit direct adverse effects on cardiac structure and function, suggesting that it is an independent risk factor for cardiac remodeling and dysfunction. This study was designed to determine whether homocysteine also has acute effects on cardiac systolic function, because such actions may complement the previously described chronic effects on cardiac structure and contribute to the associated cardiac dysfunction. Previous work by Tyagi et al. (27) in ex vivo cardiac rings of the rat right ventricle and left ventricle showed that homocysteine enhances diastolic tension and that this action is enhanced by removal of the endocardial endo￾thelium. In addition, studies in various tissues demonstrated acute effects of homocysteine that could affect cardiac con￾tractile function were they to occur in the heart. For example, homocysteine was shown to inhibit Na-K-ATPase activity in the hippocampus (24), decrease intracellular calcium con￾centration in arteriolar smooth muscle (28), and inhibit the transient outward current (Ito) in rat ventricular myocytes (22). MATERIALS AND METHODS Animals. All procedures in this study were approved by the Insti￾tutional Animal Care and Use Committee at the University of Arkan￾sas for Medical Sciences. Three-month-old male Sprague-Dawley rats (300–325 g) were purchased from Harlan Sprague Dawley (Indianap￾olis, IN) and maintained in our institutional Division of Laboratory Animal Medicine on a 12:12-h light-dark cycle with free access to chow and water. Langendorff-perfused hearts. Rats were anesthetized with a volatile anesthetic, and hearts were removed and immediately perfused via the aorta with an oxygenated Krebs-Henseleit (KH) solution (37°C) of the Address for reprint requests and other correspondence: R. H. Kennedy, Dept. of Pharmaceutical Sciences, Mail Slot 522, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205 (E-mail: kennedyrichardh@uams.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Am J Physiol Heart Circ Physiol 287: H812–H817, 2004. First published April 8, 2004; 10.1152/ajpheart.01042.2003. H812 0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society http://www.ajpheart.org

ACUTE NEGATIVE INOTROPIC EFFECTS OF HOMOCYSTEINE H813 following composition (in mMl:118.0 NaCl 27.1 NaHCOs 3.7 KCl, 1.8 CaCkz,1.2 MaCT.I.0 KH2PO.and 11.1 glucose.The tlow rme A w料s时70ml-ghgl-3-min-1,value8 imiar Ia thed oha:rveal when ow is examined a constant pressure of mm corrary pressure was moetored continooushy by a Statham pressure trans- ducer Both atri were remoned,and the ventrieles were poenl clectrically at 250 beats/min by platinum cuntact doctrodes poetionod on the ingerventricular sectum.A fuid.alled balloon catheter was placed in the lett ventriele to measure intraverencular peessure,ad the hart was cndosol in a humidificd,lempcralur-onirullod chem ber.Contractile function was mompored by measurinz developed pregure (perk svstolic pressure diastolie peessure)at the prelood halloon vlume tha clicied 90-05%uf mrimsl hisal eoetrartil finction.In addition to a polgraph recording.all data were digtized and analyzed with the use of acqurme and analysis sottware (CODAS DeaQ Imenmens,Akmon,H)Afer equiibeation for 60 101525250的前4时 min,the inotropic effocts of humooyseine were examinod hy the Time imn addition of the amino acid to the pertusing solution. B The corenary vaseuar endothelaum wis inoetivated in some hearts hy bricf perfusiun with Triion X-I00.LangndorI-perfuscd prepem 105 tions were set up as descnbed above.and after 30-min equilibration an infusion of Troon X-100 (1:200 diluionl egvalent to 1%of the flow rate was delivered into the KH butter inmedoely above the aocta or I s (14,20)Subsoquently,the hears were perired for 25 min with normal KH buffer hefore the inolropic adicns uf homucyskinc wcre examined as described above.The effectiveness of the Triton treat ment in inactivating the endothelium wis venfed by ns ahility to diminsh ntrie ovae (OI relearse. Papwlory wcle.Papillry muscl (diameter -0.7 mm)was iso laned from the rat heart,suspended in the KH solution described above (7C).and poerl vi cunact clectrodes 30 Hx Itesling teriont 010010202030 (set9)and force of isomctric contraction were moniloeed HomocysleneμM via force-dsplacemert transducers.After a 6U-min equilibeaton pe- riod,biondl contraetiliry wis reconded,id the inoernpie efferts of Fg1.Effooes of hom33sene国developed p尼e (peok systolkg- homucysicine or the cmunt culkcod from Langcndorf-perfusod hearts were examined.The papillary peeparations were exposed to fBsed with an oxygenaled Krebs-Hlerselet solugon (37C)at 7.0 ml.g effluent withi I min of its ooection from the heart. poniticned on the irirrvetricular septumn A fuid-filled balloon calbeier was acrine wne/nitrie The nilralclilrilc corcenrsion,ax mar plaoee in the kef vekick winh wulune beitg as al the valae tal diciaol sured by a modification of the Geeiss technique (19)was used as an 0-4%af esnmace of NO prodoctioe Equal cocentrotioes ot ettfuent ned isolrupic effecb of 10 pM horaoeyarine.Duts ae prexeried an u proorares Gieeiss ment (08%sfnilamnide in 05 N HC0075%N-nphth- ykthykne diamine)were mixed and shaken penthfo15min room 程0 a sanstically signih3I《P G05)6k■teep6 d presstre temperature.Absocbance was then measared at 343 nM.and concen- all arre poists camited herwen 5 and 45 min Auer.mrpresertatnee tengs tratinn was determined with the use of a standand curve obained with 2+115wt44U上1GL.5,UmcG1L山LL中G1气G1L山U2 sodum nitrilo. Smnsncal awivsis.Dota were evaluated by ANOVA with a Stu den-Newmas-Keuls post hoe test or by s-test as approprine with the nocyseine10l9±23 mmH2)and p6 ysteine (I050±69am日g0c0ol se of SigmSial sfeare (SPSS:Chicag,IL).The eriterion for 4 5E Hou signicance was a P value <0.05.Data are reported as means SE. tratian-deperdet deereaoe in drvelped meore thet uns sigreficarth difhe- lP005femu由valaes u ootrr山山wnuf动HM und giraks RESULTS Fuecis of howeoesieine an Langemdod-perfused heart Ini- betwoen 100 and 303 RM.The amino ncid hod no effect on tal experiments excamined the acule eflects of homocysteine on diastolic pressure (data not shown)but elicited a concertration- contractilty in coronar-perfused myocarduum by exmining dependent roduction m coronary pressure (from a control value chamges in left ventricular developed pressure in Lamgendorff- 74.9±6.5o69.7±5.0n65.1±5.6 mmlfg at30nd perlised preparstioes.As shown in Fig.LA.100 uM bomo- 300 uM,respectively,=5)In comtrasl,equivalent concen- cysteine elicited a negative inotropic effect that reached max- trations of cysteine had no effect on contractility or coronary imum levels of --85%of control vnlues within min The pressure response remained stahle for at least 45 min of comtiruous Efects of homoeysteise an isolated popillary muscle.Be. exposurre.and comtractile function returned to near control cause homocysteine has becn reported to clicit rumerous values (5 2%ofcontrol:S)within 15 min of washoun cffects an the vaseulnr endotheiim (9 32.33).subsequen The concentration-response curve (Fig.18)showed that this experiments were desigred to determine whether the acute nepntive inotropic action was detectnble at concentrations be- irotropic actons of homocysteine could be observed in the tween I0and00 uM wit凸山efr战stistically significant absence of curonary perfusicn.Contractility was monitored in effect being ohserved m 30 uM and little change cccurring superfused pupilary muscle before and during expsure to the P.ifar Cire PAgsiol YoL 157.AUGUET 2004.www.tipheanong

following composition (in mM): 118.0 NaCl, 27.1 NaHCO3, 3.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 1.0 KH2PO4, and 11.1 glucose. The flow rate was set at 7.0 ml
g heart1
min1 , a value similar to that observed when flow is examined at a constant pressure of 70 mmHg; coronary pressure was monitored continuously by a Statham pressure trans￾ducer. Both atria were removed, and the ventricles were paced electrically at 250 beats/min by platinum contact electrodes positioned on the interventricular septum. A fluid-filled balloon catheter was placed in the left ventricle to measure intraventricular pressure, and the heart was enclosed in a humidified, temperature-controlled cham￾ber. Contractile function was monitored by measuring developed pressure (peak systolic pressure  diastolic pressure) at the preload balloon volume that elicited 90–95% of maximal basal contractile function. In addition to a polygraph recording, all data were digitized and analyzed with the use of acquisition and analysis software (CODAS; DataQ Instruments; Akron, OH). After equilibration for 60 min, the inotropic effects of homocysteine were examined by the addition of the amino acid to the perfusing solution. The coronary vascular endothelium was inactivated in some hearts by brief perfusion with Triton X-100. Langendorff-perfused prepara￾tions were set up as described above, and after 30-min equilibration an infusion of Triton X-100 (1:200 dilution) equivalent to 1% of the flow rate was delivered into the KH buffer immediately above the aorta for 1 s (14, 20). Subsequently, the hearts were perfused for 25 min with normal KH buffer before the inotropic actions of homocysteine were examined as described above. The effectiveness of the Triton treat￾ment in inactivating the endothelium was verified by its ability to diminish nitric oxide (NO) release. Papillary muscle. Papillary muscle (diameter 0.7 mm) was iso￾lated from the rat heart, suspended in the KH solution described above (37°C), and paced via contact electrodes at 3.0 Hz. Resting tension (set at 90% Lmax) and force of isometric contraction were monitored via force-displacement transducers. After a 60-min equilibration pe￾riod, basal contractility was recorded, and the inotropic effects of homocysteine or the effluent collected from Langendorff-perfused hearts were examined. The papillary preparations were exposed to effluent within 1 min of its collection from the heart. Measuring nitrate/nitrite. The nitrate/nitrite concentration, as mea￾sured by a modification of the Greiss technique (19), was used as an estimate of NO production. Equal concentrations of effluent and Greiss reagent (0.8% sulfanilamide in 0.5 N HCl-0.075% N-naphth￾ylethylene diamine) were mixed and shaken gently for 15 min at room temperature. Absorbance was then measured at 543 nM, and concen￾tration was determined with the use of a standard curve obtained with sodium nitrite. Statistical analysis. Data were evaluated by ANOVA with a Stu￾dent-Newman-Keuls post hoc test or by t-test as appropriate with the use of SigmaStat software (SPSS; Chicago, IL). The criterion for significance was a P value 0.05. Data are reported as means  SE. RESULTS Effects of homocysteine on Langendorff-perfused heart. Ini￾tial experiments examined the acute effects of homocysteine on contractility in coronary-perfused myocardium by examining changes in left ventricular developed pressure in Langendorff￾perfused preparations. As shown in Fig. 1A, 100
M homo￾cysteine elicited a negative inotropic effect that reached max￾imum levels of 85% of control values within 5 min. The response remained stable for at least 45 min of continuous exposure, and contractile function returned to near control values (98  2% of control; n  5) within 15 min of washout. The concentration-response curve (Fig. 1B) showed that this negative inotropic action was detectable at concentrations be￾tween 10 and 300
M, with the first statistically significant effect being observed at 30
M and little change occurring between 100 and 300
M. The amino acid had no effect on diastolic pressure (data not shown) but elicited a concentration￾dependent reduction in coronary pressure (from a control value of 74.9  6.5 to 69.7  5.0 and 65.1  5.6 mmHg at 30 and 300
M, respectively; n  5). In contrast, equivalent concen￾trations of cysteine had no effect on contractility or coronary pressure. Effects of homocysteine on isolated papillary muscle. Be￾cause homocysteine has been reported to elicit numerous effects on the vascular endothelium (9, 32, 33), subsequent experiments were designed to determine whether the acute inotropic actions of homocysteine could be observed in the absence of coronary perfusion. Contractility was monitored in superfused papillary muscle before and during exposure to the Fig. 1. Effects of homocysteine on developed pressure (peak systolic  diastolic pressure) in Langendorff-perfused rat hearts. Preparations were per￾fused with an oxygenated Krebs-Henseleit solution (37°C) at 7.0 ml
g heart1
min1 and paced electrically at 250 beats/min by contact electrodes positioned on the interventricular septum. A fluid-filled balloon catheter was placed in the left ventricle with volume being set at the value that elicited 90–95% of maximal basal contractile function. A: time-dependent negative inotropic effects of 100
M homocysteine. Data are presented as a percentage of prehomocysteine control values (95.7  7.6 mmHg; n  5). Homocysteine elicited a statistically significant (P  0.05) decrease in developed pressure at all time points examined between 5 and 45 min. Inset, representative tracings that were recorded before exposure to 100
M homocysteine and after a steady-state response was observed. B: concentration-dependent inotropic effects of homocysteine (F; n  5) and cysteine (E; n  5) as examined with the use of cumulative addition. Data are presented as a percentage of preho￾mocysteine (101.9  2.3 mmHg) and precysteine (105.0  6.9 mmHg) control values. Vertical bars represent means  SE. Homocysteine elicited a concen￾tration-dependent decrease in developed pressure that was significantly differ￾ent (P  0.05) from control values at concentrations of 30
M and greater. ACUTE NEGATIVE INOTROPIC EFFECTS OF HOMOCYSTEINE H813 AJP-Heart Circ Physiol • VOL 287 • AUGUST 2004 • www.ajpheart.org

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amino acid. As shown in Fig. 2, homocysteine did not affect contractile function in these preparations. No significant ino￾tropic effect was observed during a 30-min exposure to 100
M homocysteine or with cumulative exposure to concentra￾tions ranging from 10 to 300
M (allowing a 10-min exposure to each concentration). Similarly, homocysteine had no effect on resting tension in isolated papillary muscle (data not shown). Effects of Langendorff effluent on papillary muscle. Contin￾ued experiments examined contractile function in papillary muscle exposed to effluent collected from Langendorff-per￾fused hearts. Figure 3 shows that effluent collected from hearts perfused with control buffer tended to increase developed tension in papillary muscle; however, this effect was not statistically significant. When homocysteine was added to the buffer perfusing the Langendorff preparations, the effluent elicited a decrease in contractility that increased in a dose￾dependent manner with the concentration of homocysteine. Statistically significant differences were first observed using effluent obtained from hearts exposed to 100
M homocys￾teine. Similar results were obtained in papillary preparations pretreated with homocysteine before exposure to effluent. The effluent had no effect on resting tension (data not shown). Effects of Triton X-100 treatment on inotropic actions of homocysteine. To further verify that the inotropic actions of homocysteine were mediated by the vascular endothelium, experiments utilized Langendorff-perfused hearts that were pretreated with Triton X-100 (1:200 dilution introduced as 1% of the buffer flow for 1 s) to inactivate endothelial function (14, 20). The inotropic effects of 100
M homocysteine were examined before and 25 min after the Triton X-100 treatment. The detergent treatment itself had little effect on contractile function; developed pressure before and after Triton treatment was 104.5  4.8 and 109.9  4.2 mmHg, respectively (n  5) with no change in diastolic pressure. However, as shown in Fig. 3B, the nearly 15% reduction in developed pressure elicited by homocysteine before treatment with Triton X-100 Fig. 2. Effects of homocysteine on developed tension in papillary muscle isolated from rat heart. Preparations were bathed in an oxygenated Krebs￾Henseleit solution (37°C) and paced electrically at 180 beats/min by field electrodes. A: time-dependent effects of 100
M homocysteine. Data are presented as a percentage of prehomocysteine control values (0.56  0.07 g; n  5). No significant difference was observed at any time. B: concentration￾dependent effects of homocysteine as examined using cumulative addition. Data are presented as a percentage of prehomocysteine control values (0.58  0.05 g; n  5). Vertical bars represent means  SE. No significant effect was observed at any concentration. Fig. 3. A: negative inotropic effects of effluent collected from Langendorff￾perfused rat heart on rat papillary muscle. Experiments were performed using heart and papillary preparations similar to those described in Figs. 1 and 2. Effluent collected from the coronary-perfused heart preparation was super￾fused onto the papillary muscle before and after cumulative addition of homocysteine to the buffer perfusing the heart. Data for developed tension in the papillary muscle are presented as a percentage of the control values obtained before exposure to effluent (0.68  0.07 g; n  5). The bar on the far right shows developed tension in a different group of papillary preparations that were exposed to effluent obtained from Langendorff hearts that were pretreated with Triton X-100 before exposure to 100
M homocysteine (control value: 0.70  0.06 g; n  5). Significant reductions (P  0.05) in developed tension were observed with effluent collected from hearts exposed to 100 and 300
M homocysteine; this effect was eliminated by Triton X-100 pretreatment of the hearts. B: effects of 100
M homocysteine on developed pressure in hearts before and after treatment with Triton X-100. The Triton treatment consisted of a 1-s exposure to a 1:200 dilution introduced as 1% of the flow rate of the Krebs-Henseleit buffer. Data are presented as a percentage of prehomocysteine control values (104.5  4.8 and 109.9  4.2 mmHg before and after Triton treatment, respectively; n  5). The significant decrease (P  0.05) in developed pressure elicited by 100
M homocysteine was eliminated by Triton X-100 pretreatment. Vertical bars represent means  SE. H814 ACUTE NEGATIVE INOTROPIC EFFECTS OF HOMOCYSTEINE AJP-Heart Circ Physiol • VOL 287 • AUGUST 2004 • www.ajpheart.org

he d80.6±66 14.3±3.1% f the ±6.1o7 d71.D in the NO cine clicits ±5.7 Ah b30 5

was almost eliminated by endothelial inactivation; as in na¨ıve hearts, homocysteine had no effect on diastolic pressure in Triton-treated hearts (data not shown). Time-control studies showed that the response to homocysteine was not significantly different during a second exposure if the hearts were not treated with the detergent after the first exposure (data not shown). In contrast to its effect on the response to homocys￾teine, Triton treatment did not significantly affect the response to a relatively low concentration of the calcium channel blocker nifedipine (0.03
M), which reduced developed pres￾sure by 22.4  4.9% and 28.8  5.5% before and after endothelial activation (n  4). Coronary pressure was in￾creased by the detergent treatment (from 67.9  2.6 to 81.7  4.4 mmHg; n  5), and the endothelial inactivation was supported by the fact that Triton X-100 treatment decreased the nitrate/nitrite concentration (a measure of NO release) of the Langendorff effluent from 0.99  0.06 to 0.20  0.07
M (n  5). In contrast to its effects in untreated hearts, homo￾cysteine had no effect on coronary pressure after Triton X-100 treatment (81.7  4.4 and 80.6  6.6 mmHg before and after homocysteine; n  5). The effects of Langendorff effluent on papillary muscle were also examined after Triton X-100 pretreatment of hearts. As shown in Fig. 3A, the negative inotropic effect of effluent from hearts treated with 100
M homocysteine was eliminated when hearts were treated with Triton X-100 before homocysteine administration. Effects of N
-nitro-L-arginine and indomethacin on inotro￾pic actions of homocysteine. Continued experiments were de￾signed in an attempt to determine the mechanism underlying the observed negative inotropic action of homocysteine. Be￾cause the action was antagonized by inactivation of the endo￾thelium, it seemed possible that the amino acid was acting to release NO, an agent with negative inotropic effects, from the vascular endothelium. Thus experiments compared the concen￾tration-dependent inotropic effects of homocysteine in Lange￾ndorff-perfused hearts in the presence and absence of 0.1 mM N-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor. The inhibitor was added to the buffer solution 30 min before cumulative addition of homocysteine. L-NNA alone increased coronary pressure from 76.7  5.7 to 96.6  8.1 mmHg and decreased developed pressure from 103.6  10.2 to 92.9  9.2 mmHg (n  5) before treatment with homocysteine. As shown in Fig. 4, hearts treated with L-NNA showed an enhanced negative inotropic response to homocysteine that was signifi- cantly greater than that observed in control hearts at concen￾trations of 30
M and above. Coronary pressure was not affected by homocysteine in the presence of L-NNA (e.g., coronary pressures were 96.6  8.1 and 94.9  6.7 mmHg before homocysteine exposure and after treatment with 300
M homocysteine; n  5). Because arachidonic acid derivatives have also been re￾ported to affect cardiac contractile function (5, 13), continued experiments were designed to determine whether the proposed endothelium-derived paracrine agent is a product of the cyclo￾oxygenase pathway. Indomethacin (50
M), a cyclooxygenase inhibitor, was added to the perfusion buffer 20–30 min before the cumulative addition of homocysteine. This concentration of indomethacin was shown in preliminary studies to essentially eliminate the acute negative inotropic action elicited by ara￾chidonic acid in Langendorff-perfused hearts (10
M arachi￾donic acid elicited a 14.3  3.1% decrease in developed pressure under control conditions and only a 1.5  1.1% decrease after pretreatment with 50
M indomethacin). Indo￾methacin alone did not affect contractile function or coronary pressure in the Langendorff-perfused hearts, and had no effect on the negative inotropic response to homocysteine (Fig. 4). As in the absence of indomethacin, coronary pressure was de￾creased by homocysteine in the presence of the cyclooxygen￾ase inhibitor (e.g., from 81.2  6.1 to 73.4  4.4 and 71.0  5.4 mmHg before homocysteine exposure and after treatment with 30 and 300
M homocysteine, respectively; n  5). DISCUSSION Results of this study demonstrate that homocysteine elicits an acute negative inotropic effect on ventricular myocardium and that this effect is observed at concentrations similar to the homocysteine levels observed in individuals with Hhe. The action appears to be specific for homocysteine because it was not observed with identical concentrations of cysteine. The data also suggest that the acute negative inotropic action of homocysteine is not mediated by the release of NO or cyclo￾oxygenase products from the coronary vascular endothelium. The effect of homocysteine was not observed in superfused papillary muscle, but it was observed in papillary muscle exposed to coronary effluent collected from Langendorff-per￾fused hearts. In addition, the decrease in contractility was eliminated when hearts were preperfused with Triton X-100, a method of inactivating endothelial function. The magnitude of the negative inotropic action is somewhat limited, reaching approximately a 15% depression of contractility at 100–300
M homocysteine; however, even minimal acute decreases in contractile function can be clinically relevant over a long period of time and contribute to the recently reported link between Hhe and cardiac dysfunction (11, 12, 30). As mentioned above, Tyagi et al. (27), by using ex vivo cardiac rings of the rat ventricle, showed that homocysteine Fig. 4. Effects of N-nitro-L-arginine (L-NNA) and indomethacin on the negative inotropic actions of homocysteine in Langendorff-perfused rat hearts. The hearts were prepared as described in Fig. 1. L-NNA (0.1 mM; ■; n  5), indomethacin (50
M;
; n  5) or vehicle (F; n  5) was added to the buffer 20–30 min before the cumulative addition of homocysteine. Data are presented as a percentage of values obtained immediately before the addition of the first concentration of homocysteine (see text for effects of L-NNA and indometh￾acin alone). Vertical bars represent means  SE. L-NNA enhanced (P  0.05) the effect of homocysteine at concentrations of 30
M and above. No differences were observed when control and indomethacin-treated hearts were compared. ACUTE NEGATIVE INOTROPIC EFFECTS OF HOMOCYSTEINE H815 AJP-Heart Circ Physiol • VOL 287 • AUGUST 2004 • www.ajpheart.org

int of nlterine be 241. eless,it is pos d in n the I-per

enhances diastolic tension and that this action is enhanced by removal of the endocardial endothelium. Current results showed no significant effect of acute homocysteine exposure on left ventricular diastolic pressure in the electrically stimu￾lated whole heart preparation or on resting tension in electri￾cally paced papillary muscle (data not shown). The cause of this disparity is unknown but is likely caused by the differing preparations and conditions used in the two studies. The absence of direct effects of homocysteine on contractile func￾tion in papillary muscle and Triton-pretreated whole heart suggests that previously reported actions of the amino acid on Na-K-ATPase activity (24), intracellular calcium concen￾tration (28), and Ito (22) were not elicited by concentrations up to 300
M in intact tissue maintained under the chosen experimental conditions. Nonetheless, it is possible that posi￾tive inotropic (inhibition of Na-K-ATPase and Ito) and negative inotropic (decreased intracellular calcium) influences were offsetting, thereby resulting in no detectable change in contractile function. The concentration dependency of the negative inotropic action of homocysteine observed in this study falls within the range observed in individuals diagnosed with Hhe. Plasma protein binding accounts for 70% of circulating homocys￾teine levels in humans with 25% being in free oxidized forms and 3% being in free reduced forms (6). Thus plasma concentrations of total homocysteine in individuals with mod￾erate (30–100
M) and severe ( 100
M) Hhe would result in free concentrations similar to those that elicited the acute effects in current experiments (10–300
M with the first statistically significant effect being observed at 30
M). Con￾tinued experiments are required to determine the relative con￾centrations of the oxidized and reduced forms of homocysteine in physiological buffers, as well as the role of these different forms in observed effects. The mechanism by which homocysteine acts to elicit the release of the proposed endothelial mediator has not been elucidated, nor has the identity of the proposed paracrine agent or its mechanism of action. L-NNA, a NOS inhibitor, did not antagonize the inotropic action of homocysteine, indicating that the endothelial factor is not NO. This is not too surprising in light of data showing that homocysteine acts acutely to inactivate NO via oxidative mechanisms (29) and impair NO synthesis via production of asymmetric dimethylarginine (25). Hhe has been associated with increased levels of NOS, both endothelial NOS and inducible NOS (31), but this requires more prolonged exposure. In addition, indomethacin did not alter the response of Langendorff-perfused heart to homocys￾teine. This suggests that the endothelial factor is not a product of cyclooxygenase such as PGE1, which has been shown to decrease contractile function in the rat heart (13). It is possible that other metabolites of arachidonic acid, such as the negative inotropic leukotrienes (5) or products of cytochrome P-450, are involved. Current experiments were restricted to examining the possible role of cyclooxygenase products because preliminary studies showed that indomethacin nearly eliminated the previ￾ously reported acute negative inotropic response to arachidonic acid (26) under the experimental conditions used in this study. Brief Triton X-100 exposure of the coronary vasculature was utilized to inactivate the endothelium. Previous studies (14, 20) demonstrated the effectiveness of this treatment by showing that it inhibits the response to endothelium-dependent vasodi￾lators without producing morphological changes in the heart. Similarly, current data indicated a significant reduction in endothelial function after Triton X-100 treatment. Coronary pressure rose significantly following treatment with the in￾crease being similar to that elicited by L-NNA. In addition, experiments showed that the Triton X-100 exposure reduced NO release into the effluent by 80%, while having little effect on left ventricular developed pressure (eliciting approx￾imately a 5% increase) suggesting that it did not damage the endothelium to the point of altering barrier function or dam￾aging cardiomyocytes. Current data would seem to suggest that the acute effect of homocysteine on the Langendorff-perfused heart is more po￾tent than the effect of homocysteine-containing effluent on papillary muscle. The first statistically significant effect of homocysteine on heart was observed at 30
M, whereas the first significant change in papillary muscle was observed with effluent containing 100
M homocysteine. Efficacy did not seem to be different as both preparations showed approxi￾mately a 15% decrease in contractile function at 300
M. Although the cause of this slight rightward shift is not under￾stood, it may reflect a simple dilution of the paracrine mediator in the buffer compared with its local concentration in the heart. Alternatively, it is possible that the paracrine mediator is unstable in aqueous solution. Previous studies (20) have demonstrated that even under control conditions the coronary vascular endothelium releases both positive and negative inotropic agents with the relative release depending in part on coronary flow and oxygen tension. A review by Brutsaert (4) suggests that NO, angiotensin II, endothelin, prostanoids, and even polypeptides may be in￾volved. In current experiments, the effluent collected from Langendorff-perfused hearts before exposure to homocysteine tended to increase systolic function in the papillary muscle even though this trend was not statistically significant. Future studies are required to identify the mediators of the endothelial action as well as the mediator released by homocysteine. In addition to its acute negative inotropic effects homocys￾teine altered coronary pressure in Langendorff-perfused hearts. It decreased coronary pressure 13% in control conditions, but had no significant effect after Triton X-100 or L-NNA pretreat￾ment. This would seem to suggest that the coronary dilation elicited by homocysteine was mediated via an increase in NO release or via the acute formation of S-nitroso-homocysteine, a potent vasodilator (15, 23). Somewhat in contrast, other reports suggest that homocysteine acts acutely to inactivate NO (29) and impair its synthesis (25). Future studies are required to clarify this issue. Nonetheless, the more important question for the current study is whether the changes in coronary pressure played a role in observed inotropic actions. Gregg (10) re￾ported in 1963 that changes in coronary perfusion can affect cardiac contractility, and subsequent work suggested that this effect may be caused by changes in oxygen supply (8) or the “garden hose” effect (1) with more recent studies indicating that it is related to capillary perfusion (7). Although current experiments were performed under constant coronary flow, it is possible that changes in arteriolar diameter may have influ￾enced left ventricular systolic function by changing flow dis￾tribution. This, however, seems unlikely as homocysteine ei￾ther decreased or had no effect on coronary pressure while decreasing systolic function. Similarly, it might be proposed H816 ACUTE NEGATIVE INOTROPIC EFFECTS OF HOMOCYSTEINE AJP-Heart Circ Physiol • VOL 287 • AUGUST 2004 • www.ajpheart.org

ACUIE NEGATIVR INOTROPK FFFRCIS OF IVOCYSIFINF H817 that the relatively large increase in cocorary pressure elicited 12 Jeweph J.Wasliaglau A Jeeph L Koclikr 1.Fisk LM.Hauer- by 1-NNA may have contributed via this mechanism to the lemsen M.and Kennedy代H Hyperh3 OYc1nC鱼D达ead小ese concurrent fall in systolic function;however.a similar fall in andi:teaoddilt it hyps leive us.e Pirnw Hearl Cvr Pitmoi 25H2567-H2s74202. cardac cortractility did not occur when Triton X-100 in- 15 karmazyn M,1*ng《KIl.and Dhall山N发og7 candm actions and creased coronary pressure. as厘Iwiw国nearts Ca5 Pirmol Pharocn5 In summary.ths study shows that pathophysiological con- 127-122.194 centrations of homocysteine elicit an acute negative inotropic eflect on ventncular myocardium that is medialed by a coro- ureul viactlar endothdiun国n myocadal perfiemuance in iok可 nary endochelium-derived agent other than NO or cyclooxy. 68-77.1998 I5 Leacalen J.The roci山al由z efr匀neni JCln Ien9g gennse products Firture studies are required to elucklae the 7.19新 mechanism by which homocysteine acts to elicit the relense of 163ClbK线V上upuh3 legy ofhy perbomozysie nenio iriplicatiors the proposod endotheltal mediator.the identity of the proposed fet the devdlugnerl uf alericockxoub.Ao J Pua 56:111-128,1969. paraerine ngent,and the mechanism of its negstive mnotropie 17.Nyzard O.Nordrehauz JE,Refsun H.Lelsad PM.Farstad M,and Yollser SE.Plasma homocyseire levels and mortality n patients wrh action Possible mechanisms of its inotropc eflect include d3720-261 actions on voltage-dependent calcium infux,sarcoplasmic re 1&ygard 0.Yolker SE,民efs用L.Brttstrome L,a同Uelaad P51.To2 ticular calcium release and/or reuptake,calcium efflux (most likely vin the Na/Ca exchanger),andor myofilament calcium 1 sensitivity. nmts at nrrnc and ritmiia in Gl er印a女mmhw可jm ACKNOWLEDGMENTS u小0.4 w BouchEA23l.383-3%.1005. 20.Ramaciolti C.MeClellan G.Sharkey A.Rose D.Websber A.and The arthors thank Kemey Robeno,Dyom McNece.and Amarda Clnse Winrtrad 5 Cadar cnothdial ol nuddal cortnctiliry of ral heart fre eurellest lechrcal axactnee a o and flow.Bes 72.1044-1064, 1593 GRANTS This uerk wan appored in part hy a (inm-is-Aid fmn fhe Anericn Tru SG.and Hess 0M.Deereased rte of cororery resierosis afer Heel Aouation Horand A面l山e (p R.H Krnslyl,and a Beairmna owerng of hemocyseine levels.N Angl J Ld 1%1595-1t00. Gmart-in-And fmm te Heart.Heorland (to 01 Joscph 22 Sheatz RIl,Xuy P'ard kI,aad Reoanski riJ,Bahhnon oK'cmerrs REE代ENCES 12.175-132001. 1.Arold G.Kosche F.Miessner E.Neitzert A.and Lochaer W,The ad【cat四，Adherwe vaprular女of hanocydeine are moda mpertancs of peerfugion peoure in the commary ameries tor the contre- t3图hegn0om国om0f1ecat.3e29 w-35618 /1wer9川：0明-315,1明3. 24.Sureck EL,Zegau Al.Tarliari B.Surki JF,Wajner M Wamni- 2 Hacher.Demarh K.Ceerin AP.Vad程C,eatti3,Saf每E.d her CMD,ad Wyse ATS 0角ecns■of the inhpitin of 3'K'-A1为3KtW可byh3m0a.r Bev Newvorf2 and cordiaz hypertrophy in endstaze renal dsease.Nepral 12:245- 7-81.20p 255190 3.Boushey CJ.Beresfard SA.Omesn GS.and Meoubky AG.A quarti- Z5.Srihlinger MC.Tsao P ler Jl1,Kimote M.Halint RF,and Caoke alive poxamatt uf plaetu hottueydeite u tiak ficlo fiu vawula: ymnerie dinetylarginine.Cireamon 104:2569-1575,201. 1049-1057.198 4.llrutcert D.Cartac endnthelial-rvoeardal sprolire'its mole in ca A▣ial Med Ger35:1119-1,196. 49-11433交 endocerdial endothdlial funcon.Can PAs Pharncof 77.950-957, 5.Burke J,Levi R.Gue ZG,and Carey EJ.Leukotienes Ca.Da and E: 191 effects on umon and Durea-pc carduc greporanons mn vrre.JPAa- 2&Lugvari Z sad Koller A.H3sex5ehmu线leCF+们 cafa97Ar221:23S-241.192 and consnctoe responses of tsolkted anenoles CovNowsc /amce 5-710 xeine in huna通psab好hi的perfommance liguid chuomotoz4 olry win 29.Upcherch GR.Weleh GN.Fahian Al.Freedman JE,Johnson JL alsaide dtetlion Cremvntyr 4 940 141-151.3002 7.Diglman MA.Heslings JW.Siphema P.and Weserhof N.Perisioo aide by a meckarin imlvin电a山hione peridane了aC2 id过dupo in erdia o由il向depenad en cpellary pfia 1012-117.1w7. 3 Vasan RS Beiwer A,D'Agoatina RB,Lrvy D.Sehuh J.Rawenherg IH. 8.Dowaty J Nhyocirdid comtnctile farction a s fundimn uf aorery and Wibta PWF.Phone hunocykine und tik fi cnolie heul lond fiqu4J为l3r1-6.19% fiuse in eduls wthoat prior myocasdel infurction J 289:1251- 9.Eherhardt RT.Ferginne M.A,Cap A,Leopal JA,Radd M.A,Trellirl 1257.2003. M.Hleydrirk Stark H.kling K Meldlavan I,Yaghoshl M. 31.Waag S.Wright G.Harrah J.Toucbun R.MeCambe W.Geag W. Galdschmidt-Clermont PJ.Farber HW.Cahes R.and Lesealze J. Falrz ME.Abdul-Jall MN.and Wright GL.Shor-emt esposure to lndorhelwl dy stumction in a minne model et mald hy perhemrocyslelne. ia.Ciw Mrer 105 443-491,2000. dent mechantsm.Cae PAyslol Plonocw 78:300-505,1999. 10.Gretr DE.EfTit uf uuotuy peiSDis PoE o twouy fuw ua oxyges usege of the ayocurdium.Cie Res 13:497-500.1963. aon af celuer pltrhicre pemvicase.cu hrmno守室移口 1 Joorpa工，aveph L,5 Shekhawat N5 Devi 5,Wang J.Melcbert RB. h国办shnm.c Now Acod sel L飞w125G-1253k.2101 Hauer-Jctsea M,and Keancdy RH Hyperhomxeysenena letd 线h吧LDim可吧aC,la由ihed A,and Kar女SD.Ffiect nf deem. a鱼dogi:verlricular lypettruphry it memderoive山Aw了pe an on homreydeine-inuced cnothlial dyafinetina in mamal subject Pare 22.F.15-F.18.2000 4r.GP为·14.5g?·a1%到1a4·Wwm列hn吧

that the relatively large increase in coronary pressure elicited by L-NNA may have contributed via this mechanism to the concurrent fall in systolic function; however, a similar fall in cardiac contractility did not occur when Triton X-100 in￾creased coronary pressure. In summary, this study shows that pathophysiological con￾centrations of homocysteine elicit an acute negative inotropic effect on ventricular myocardium that is mediated by a coro￾nary endothelium-derived agent other than NO or cyclooxy￾genase products. Future studies are required to elucidate the mechanism by which homocysteine acts to elicit the release of the proposed endothelial mediator, the identity of the proposed paracrine agent, and the mechanism of its negative inotropic action. Possible mechanisms of its inotropic effect include actions on voltage-dependent calcium influx, sarcoplasmic re￾ticular calcium release and/or reuptake, calcium efflux (most likely via the Na/Ca exchanger), and/or myofilament calcium sensitivity. ACKNOWLEDGMENTS The authors thank Kerrey Roberto, Dawn McNeice, and Amanda Clausen for excellent technical assistance. GRANTS This work was supported in part by a Grant-in-Aid from the American Heart Association, Heartland Affiliate (to R. H. Kennedy), and a Beginning Grant-in-Aid from the American Heart Association, Heartland Affiliate (to J. Joseph). REFERENCES 1. Arnold G, Kosche F, Miessner E, Neitzert A, and Lochner W. The importance of perfusion pressure in the coronary arteries for the contrac￾tility and the oxygen consumption of the heart. Pflu¨gers Arch 299: 339–356, 1968. 2. Blacher J, Demuth K, Guerin AP, Vadez C, Moatti N, Safar ME, and London GM. 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Homocysteine reduces smooth muscle [Ca2]i and constrictor responses of isolated arterioles. J Cardiovasc Pharmacol 37: 705–712, 2001. 29. Upchurch GR, Welch GN, Fabian AJ, Freedman JE, Johnson JL, Keaney JF, and Loscalzo J. Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 272: 17012–17017, 1997. 30. Vasan RS, Beiser A, D’Agostino RB, Levy D, Selhub J, Rosenberg IH, and Wilson PWF. Plasma homocysteine and risk for congestive heart failure in adults without prior myocardial infarction. JAMA 289: 1251– 1257, 2003. 31. Wang S, Wright G, Harrah J, Touchon R, McCumbee W, Geng W, Fultz ME, Abdul-Jalil MN, and Wright GL. Short-term exposure to homocysteine depresses rat aortic contractility by an endothelium-depen￾dent mechanism. Can J Physiol Pharmacol 78: 500–506, 1999. 32. Weiss N, Zhang YY, Heydrick S, Bierl C, and Loscalzo J. Overexpres￾sion of cellular glutathione peroxidase rescues homocysteine-induced endothelial dysfunction. Proc Natl Acad Sci USA 98: 12503–12508, 2001. 33. Zheng H, Dimayuga C, Hudaihed A, and Katz SD. Effect of dexrazox￾ane on homocysteine-induced endothelial dysfunction in normal subjects. Arterioscler Thromb Vasc Biol 22: E15-E18, 2002. ACUTE NEGATIVE INOTROPIC EFFECTS OF HOMOCYSTEINE H817 AJP-Heart Circ Physiol • VOL 287 • AUGUST 2004 • www.ajpheart.org