JACC Vol. 27, No. 7 June 1996~1777-86

1777

Preconditioning Does Not Prevent Pastischemic Dysfunction in Agirig Heart PASQUALE ABETE, MD, PHD, NICOLA FERRARA, MD, ANGELO CIOPPA, MD, PIETRO FERRARA, MD, SABATINO BIANCO, MD, CLAUDIO CALABRESE, MD, FRANCESCO CACCIATORE, MD, GIANCARLO LONGOBARDI, MD,* FRANC0 RENGO, MD Naples and Lhawento, Italy

Objea-ltiw. This study was performed to investigate tbe e&t of siagle or multiple brief periods of kebemia and the adminktra- tion of exogenous norepiaepbrinc before a more prolonged kcb- emit period aad after reperfusiin in adult and mescent isolated and perhed rat hearts.

fJae&md. Tbe mortality rote fur wronary artery dkease is greater in the elderly. Iscbemic prwonditioniug bas beea pro- posed as an endogenous form of proteetion against kehemia- ~~~~~~~. However, tbe role of precoaditioniag in aging

. .Misds. we compared the protdvv e&et of p!woRditii~

traasient kcbemic aad norepinephine stimuli against 28 I&I of global normotbermic iscbemia and 40 min of regwhsion in isolated pehsed heurts ofadalt (6 months old) aad senescent (24 months old) rats. Norepinephrine release in coronary asent was determined by Mgb perfonaance liquid chromatagrapby.

Itest&. Final recovery of percent developed pressure was improved after single precoaditioniog transient k&emic and aorepinepbrine stiumli in adult huts (87.7 f % and 82.3 f 8.7%) versns uncunditioned control hearts (50.6 + 4.%, p C MI [mean + SD]). The e&t of precondkioni~ 08 developed pres- surerecov~wasaotfwesentin senescent bearts after traasieat

The mortality rate for coronary artery disease increases pro- gressively with age although the specific factors involved in thii phenomenon have not been clearly identified (l-5). Experi- mental studies, however, have show that an episode of ischemia determines a more severe myocardial dysfunction in senesxnt than in adult hearts and that the recovery after a period of myocardial ischemia is less evident in senescent than in adult hearts (6,7). The reason for this age-related increase in severity of coronary artery disease is not clear. One hypothesis

iscbemic stimulus (39.8 + 4.% vs. 41.6 f 5.& p = NS) but vws present after aorepinepbrine stimulw (74.3 f 10.5, p < Q.01). Norepinepbrine release significantly increased after pre- conditiuning ban&at is&emic stJmuias in adolt but nat in sewscent bearts (p C Wl vs. ad&). Transieat ischaic- and mwepinepbrlw-i preamd&Gag was blodud by alpha- adreaer&recephraatagoaistsiabothadaltaadsfwsceat hearts.!Wultipletransieutkcbemicstimuliwereabletoredaee

. . . emit plvmmmq iibdwsau-iaBorrsinephrbw

(/Am cdl olnlial1996$7:1777-86)

could be that some endogeoous mechankms involved in the protection of the heart against ischemia-reperfikon injury may decrease progressively with age.

Recently, several authors have reported that brief periods of ischemia (“preconditioning”) induce intrinsic changes within the myocardium enhancing its resistance to subsequent ischemia-reperfkion injury (8). Several mechanisms have been suggestedtoexpiaintheischemkprewnditioningsuchas reduced energy demand (9). inl&ition of mitwk&al aden- osine triphosphate (ATP) protein (IO), iaductiott of stress protein (11,12X or prosW@n (13) or adewsine release (14), activation of ATP-regulated K’ chanuek (1516). Toombs et al. (17) reported that catecltolamh de.pktion with rewpine totally prevented the card@otective effects of preconditkt- ing in rabbits lherjee et al. (18) ako demonstrated that a transient kchemic stimulus before a more prolonged khernic periudreducedektricaiandmechanicaidysfwXiitithe sympathetic -~norepinephriaehymyoeardia alpha-adrewrgicreceptorsinrat~’

1778 AnmEErAL PRJXONOITiONING AND AGING HEART

Age-related reduction in heart catecholamine release should be the consequence of the lower tissue catecholamine levels found with advancing age and of the partial degenera- tion of sympathetic neurons best descriid in the rat (19-21). We hypothesized that the greater susceptibility to myocardiai ischemia observed in the senescent heart might bc due to a reduced effect of preconditioning, probabiy a result of decreas- ing ability of the transient ischemic stimulus to induce the norepinephrine release responsible for the preconditioning phenomenon.

For these reasons, the aim of the present study is to investigate the effect of single or multiple brief periods of ischemia and the administration of exogenous norepinephrine before a more prolonged ischemic period and after reperfusion in adult and senescent isolated and perfused rat hearts. In addition, we tested the ability of the alpha-adrenergic receptor blockade, phentolamine, to inhibit the effects of precondition- ing transient ischemic and norepinephrine stimuli in both adult and senescent hearts.

Methods Experimental procedure. LangendorEperfused isolated

hearts from male Wistar rats aged 6 (adult) and 24 (senescent) months were studied. Mean body weights were 284.1 i 43.44 g at 6 months and 524.4 t 47.5 g at 24 months whereas mean wet ventricular weights were 1.1 2 0.5 g at 6 months and 1.6 ? 0.6 g at 24 months. Animal care was performed according to the “Position of the American Heart Association on Research Anii Use.” The animals were anesthetized with diethyl- ether, and sodium heparin (200 U) was injected intravenously. Hearts were then rapidly excised and attached via the aorta to a moditied LangendorE perfusion apparatus. The basal perfu- sion medium (37T, pH 7.4, perfusion pressure 66 mm Hg) comained iu mM was Nail 117, KCl 4.6, NaHsPO., 0.8, NaHCOs 25, @Cl, 1, CaCl, 2 and glucose 5. The perfusion liquid was oxygenated by a mixture of 5% Co1 and 95% 02. Left ventricular pressure was measured using an intraventric- ular balloon attached to a l-mm diameter hollow cammla and co~ected to a Statham P 23 pressure amplifier and to a h&eve1 preamplifier and direct differentiator (OTB Bio- medica model 2080 pressure meter) to obtain 6rst derivative of left ventricular pressure (+dP/dt). Isovolumetric loading con- ditions were established by setting left ventricular enddiastoiic pressure at approximately 5 mm Hg. Heart electrogram was obtained by an atraumatic epicardial electrode (0.8 mm diam- etcr, silver wire) attached to the free wall of the right ventricle (to avoid a&cting left ventricular function). The electrical signal was obtained from a bioelectric amplifier (OTB Bio- medica model 2077 EC0 amplifier). Pacing wires were tixed to thepulmonaryoutaowtract,andtheheartswerepaeedat6Hz (pacer off during ischemia and drug infusion). Pacing was resumed3minafterthestartofreperh&m,andthebearts were d&r&ted when neccmary. Coronary Row rate was measmed in grahted cyiiuders at intervals of 5 min before, ~~~~~~d~8~.~~~

JACC Vol. 27, No. 7 June 1996177746

flow rate was related to wet ventricular weight (mi/min per g) in both age groups. During the ischemic period, perfusion pressure was 0 mm Hg and the hearts were maintained at 37°C in a thermostatted chamber. Developed pressure (mm Hg), end-diastolic pressure (mm Hg), tdP/dt, -dP/dt, and core- nary flow rate (ml/mm per g) were monitored and recorded on a Harvard oscdlograph at a paper speed of 0.1 mm/s at intervals of 5 min.

Experimental design. After 20 min when electrical and mechanical variables stabilized, the hearts were randomized into 8 groups of 14, each divided into two subgroups of seven (adult vs. senescent): group I = hearts perfused for 80 min to demonstrate the preparation stability;group 2 = hearts treated with ischemic perfusion for 20 min and with reperfusion for 40 min (standard &hernia-reperfusion insult); group 3 = hearts treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (window) and then a standard &hernia-reperfusion insult; group 4 = hearts treated with preconditioning norepinephrine stimulus (10 nmollmin) for 2 min followed by 10 min of reperfusion (window) and then a standard ischemia-reperhtsion insult; group 5 = hearts treated with phentolamine (450 nmol/min) for 7 mm and followed by 5 min of reperfusion (window) and then a standard &hernia-reperfusion insult; group 6 = hearts treated with phentolamine (450 mnoi/min) and transient isch- emit stimulus followed by 10 min of reperfusion (window) and then a standard ischemia-reperfusion insult; phentolamine was infused for 2 min before transient ischemic stimulus and then resumed for another 5 min during perfusion of preconditioning window; group 7 = hearts treated with phentolamine (450 nmobmin) and norpinephrine stimulus followed by 10 min of reperfusion (window) and then a standard ischemia- reperfusion insult; phentolamine was infused for 2 min before norepinephrine infusion and then maintained for another 3 min during perfusion of preconditioning window; group 8 = hearts treated with four cycles of 5 mitt of preconditioning transient ischemic stimulus followed by 5 min of reperfusion (window) and then a standard iscbemia-reperfusion insult.

Adysis of arrhythmias. The incidencea of ventricular fibrillation during reperfusion were determined before pacing (started at 3 ruin of reperfusion) in all groups. Ventricular fibrillation was defined late-fibrillation when it occurred after initial conversion to paced rhythm. Late-ventricular fibrillation was evaluated independently by either the presence or the absence of ventricular fibrillation before pacing hearts. Ac- cording to Lambeth Conventions (22), we stated ventricular fibrillation as a signal in which iudividuai QRS complexes could not be distinguished from one another and for which a rate could not be determined.

Drugs. Norepinephrine and phentolamine were obtained from Siia Chemical Company, St. Louis, Missouri. The drugs were prepared fresh in 5 mmobliter phosphate-buffered saline solution and infused through a port above the aortic root at 0.068 dmin (not circulated).

NoropSmplsriae asays. Norepinephriae !evels were deter- minedaccordingtoHaWsnbethd(23)bycoUectingwronary

JACC Vol. 2l, No. 7 June 19%377X%

effluent after 2 min of transient global ischemia accumulated over the prexxmditioningwindow and related to wet ventricular weight (mI/min per g) in both age groups (pmoI/ml per g). The assay was performed by high performance liquid chromatog- raphy, Beckman; separation and quantitative analysis were detected electrochemicalIy (ESA 51OOA Coulochem System).

StatistIcnI anaIysII Results are expressed as mean + SD. A two-way analysis of variance was performed to compare in each procedure both functional variables at identical time points (developed pressure, end-diastolic pressure, +dP/dt, -dP/dt, coronary Bow rate) and arrhythmias between adult and senescent groups. If the F ratios were signhicant, post hoc Scheffe’s F test was applied to assess signikance. Values less than 0.05 (p < 0.05) were considered sign&ant.

aesatlts E&et of Ischemla and repesfusion In a&It and senescent

hearts. Control adult and senescent hearts simply perfused for 80 min reserved >90% of their originaI function. Twenty minutes of normothermic global ischemia induced a rapid decrease of developed pressure in both adult and senescent hearts and a more gradual increase of end-diastolic pressure that was more pronounced in seneccent hearts (35.5 2 12.0 mm Hg in adult versus 44.8 + 7.6 mm Hg in senescent hearts, p < 0.05). With this kind of injury, after 40 min of reperfusion, adult hearts recovered by -50% and senescent hearts by -30% (Fig. 1, Table 1).

E&et of precomIIt&nIng stlmnIl (transient IsehemIa or nurepinephrise) t3S isebemIa-reperPosion I+wy In adnIt and senescent he&s. Adult hearts preconditioned with a transient ischemia showed a developed-pressure improvement com- pared with &hernia-reperfusion control hearts (87 -C 13 mm Hg vs. 50 + 8 mm Hg, p < 0.01; Table 2) and contractihty recovery was more complete (+dP/dt 2,157 -C 210 vs. 1,240 + 100 mm Hg per s and -dP/dt 1,790 2 140 mm Hg per s vs. 1,028 + 128 mm Hg per s, p < 0.01; TabIe 2). End-diastolic pressure also showed a significantIy greater recovery compared with ischemia-reperfusion control hearts, returning to near baseline values (12.1 2 10 mm Hg vs. 39.6 C 10 mm Hg, p < 0.05; TabIe 2). SurprisingIy, in preconditioned senescent hearts neither developed press-me nor enddiastoIii pressure showed any improvement compared with ixhemia- reperfusion control hearts (Tabk 2). In both adult and senes- cent hear@ 2 min of exogenous infusiin of norepinephrine (10 nmopmin) simulated preconditioning by 2 min of transient ischemia by inducing an improvement of cardiaf function atier ischemia-reperfiisiin injury similar to that observed in prexon- ditioned adult hearts (Table 3).

As shown in Figure 1, recovery of percent developed pressure at 4O-mi0 reperfusioa demOOsh&s that transknt ischemiaimpednsedtaniealfuncti0000lyinadult~ ~~~~~p~~~~p~~

senesu&heatts.TbasedatasIlggesttIleaufial mkoftheadretlergicsystemonule *mm-

ABETEmAL 1779 PRECONDITIONING AND AGING HEART

W -I- t#

Fiil. BargrapbssbowiagreQJvelyofpereentdevelopedpressnre (DP%)at40minofrepemtsiooinadult(~~)andsenacent (did bura) hearts subjeued to isdlealia-reperfnsioa iujly without preconditiining (coatml [ClXL)), suL+ted to preconditioning tran- sient isehemic (Tl) or aorephepbriw (NE) stimuli for 2 mia followed by10rnin(preconditioniogwirldmv);hearts~withphentoIamme subjected to isdemia-reperfusion injty without ~$tionhg (PHE) and treated with ptiemolamine before PrecondNonmg sient isckmic (PHE + TI) or 0orephKphrine (PHE + NE) .5tiZi l p c 0.05 versus senescent tp < O.Oi versus amtrot @I < 0.05 versus pbcntolamine. gp < 0.01 versus transient irdwmia. 3 < o.m versus rlorepinephrine.

enon and the ineffectiveness of transient ischemia in senescent hearts against iscIle.mia--iOn injlny.

ftJn&nal reuwetyafter standad isehemia-m&i00 injury (Table 4, Fii 1). Phentokmine, however, worsened myocar- diaI contmcbhty during p- transient ischemk stimulus (TabIe 5) and signikmtly reduced inotropic effects of norepinephrine in both a&It and senescent hearts (TabIe 6). Phentolamine aboIii the pmkctive effects of premndition- inghansientischemiestimul~in~thearts(Mt-5mmHg vs. 50 + 8 mm Hg, p < 0.01) and seems to exaggerate postischemic dysfundion in senescent hearts (35 C 12 vs. 39 t 13) (Table 5). In the pmsence of pbentotamiw, the protective effects of norepinephrine disappear in both adult and series- cent hearts (Table 6).

Figure 1 ako shows that phenmhunine did not at&t percent developed premure recovery in the absence of precon- ditioningstimuIi.Duringtransientiscbemicstimoh&pbentd- aminebbcksthepmtectiveeffectinduadbypreoo~ inaddtheartsandseemst0exacerbatepdihXOk~-

PitentoIamheinhbitstbepnrieaive uIusinbo4haduh

1780 ALImEETAL JACC Vol. 27. No. 7 PRECGND~ONING AND AGING HEART June 199bdm-86

T&& 1. Hef&ym& V&ables During lschemia (20 min) and Reperftlsion (40 min) in Adult and Senescent Rat Hearts - Add Heam(n = 7) salescent Heans(n = 7)

--.

ihe (alliP&) 0 (A@&)

-dPl& DP LVEDP +dm -dP/dt CFR

W-W (mm Hgls) (srg) (mm Hg) (mm W (mm Hgk) (owl Wh ~8)

a- 9.528 35+15 2,232+150 l,Y54+n, 11.8 2 0.7 101 z 9 4.1 ? 0.6 2,505+14a 2,@77+100 124 2 0.8 10 93 ?8 3.6 + 1.7 2JlO t WI 1,9w+70 11.7 + 0.7 102+8 4.0 + 0.5 2540~140 2.020~1w 125 ? 0.9 24 I-begin 9427 3.6215 2,220’150 1,940?~ 11.7-tO.6 101 + 7 4.2 + OS 2,540 ?r 130 2,040 2 110 123 -+ 0.7 ‘%l-eod 0.0 355 +12* 0.0 0.0 0.0 0.0 448 + 7.6 0.0 0.0 0.0 4.5, R-5 21~79 582212' 520+50* 431 250' 12.7 2 3.1 12211 82.6 + 7.4 297240 246240 13.2 + 21 50, R-10 2929' 4722 10’ 719 lr 70’ 596270 11.5 -t 4.1 17 + 12 68.6 t 5.4 4212 50 394230 11.9 + 20 6q R-20 39%9' 41.6 + 11: 942 r 110’ 781+ 130’ 9.1 2 2.8 24 2 12 56.4 f 10.3 595270 493260 9.4 2 13 m, R-i?d-J 49~8' 405 i: lff 1215 2 loo’ l,OD7 + 110* 8.1~ 2.5 38 2 10 54.6 + 8.4 342250 781'M 8.7 2 2.1 80, R-40 50? 8' 396 2 10’ 1,240 + 1w* 1,028 _f ml* 7.9 k 2.6 39213 53.0+ I1 967+1Dil Fdz+_so 8.6 2 13

‘p c 0.05vermssenewnthearts CPR = cmmaryflcw r&wet hem weight;DP = developed pressure(endsystolic-enddiastolic); tdP/dt = first derivative ofdevelopedpressuq I= isdtemia; LVEDP = leAventricularenddiastolicpressure;R = reperfwion.

Ekts of maltiple cycles of transient pwconditioning stim- ahts aa iscbemia-reperfasioa iajwy in adult and senescent hkts. Table 7 shows that four episodes ot 5 min of ischemia followed by 5 min of rep&&on are able to reduce postisch- emit dysfun&n in adult but not in senescent hearts. After sustained ischeia-xperfusion injury, in fact, the final percent develwd pressure recovery was 80 t 10 mm Hg in adult and 51 -C 11 in Senescent hearts @ < 0.05). When preconditioning transient ischemic stimulus lasted 2 min, the final percent developed PressIre recovery in senescent hearts was ouly 415 12 mm Hg (Table 2), indicating that a more intense precondi- tioning protocol is more efficacious in inducing protection against ischemia-reperfasion injury in senescent hearts. How- ever? the difference on 6nal percent developed pressure recov- ery between senescent hearts subjected to one cycle or multiple cycles of preconditioning stimulus was not statistically different (Fig. 2).

Elfects of preconditioning on iscbemia-reperfosioo- in&cd ventricuhu a&y&m& Although several studies indicate that preconditioning reduced ventricular arrhythmias at tbe beginning of reperfusion (24~26), preconditioning stim- uli in our experiments are able to reduce only ventricular

late-fibrillation after the establishment of paced rhythm. The incidence of repcrfusion-induced ventricular fibrilla!ion, how- ever, was higher in senescent than in adult hearts in all groups (Fig. 3A). The time required to reach paced rhythm was longer in senescent (7.4 t 2.1 min) than in adult (5.1 2 1.8 min) hearts in all groups (p < 0.05). Figure 3B shows that ventric- ular late-fibrillation was significantly reduced by prccondition- ing transient ischemic stimulus in adult hearts (2 of 7 vs. 6 of 7, p < 0.01) and by preconditioning norepinephrine stimulus in both adult (3 of 7 vs. 6 of 7, p < 0.01) and senescent (4 of 7 vs. 7 of 7, p < 0.01) hearts. Phentolamine, however, abolished the antiarrhythmic effect of ischemic preconditioning stimulus in adult hearts and of norepinephrine preconditioning stimulus in both adult and senescent hearts.

Norepioephriae release after traasient ischemia. Results of this study con&m previous results indicating that cardiac adrenergic neurons release norepinephrine in response to ischemic stress (27). The total coronary effluent obtained during the 10 min before preconditioning transient ischemia revealed a norepinephrine background of 0.45 ? 0.09 pmol/ml per g in adult and 0.53 + 0.08 pmol/ml per g in senescent hearts. After 2 min of transient global ischemia, norepi-

AddtHearts(n=?) -t(n=7) --- +dP,# -dPhll @- w t-w) @GfLg) &I%~ fLS (:&I (:&) C&d o,l!a&iae Y7z7 3.7 2 15 QXl5~160 l,Y95ZsO l20+0.8 ll33’-7 39~0.9 2,554+1Xl Zll8i: 100 lU?O.Y lQw 721* 7.62 1.6 164rtW 143 ,+ l(r a0 221 8.2-13 49210 41 i- 10 oil B,W-eod 9326 4.1 2 0.8 uo6+130 1,913 + al 119+0.9 6727 6.1~1Jl 2,044+130 1,78Y?!Xl 125’1.0 :k.? 412 0.0 14' 276 225 2 + 107 wt 1,165 ll0 -c 17tP 956illW 0.0 129 oil + 32 15 Ofl ? 10 41.6 73.6 f C 9.4 8.7 372240 0.0 308fr40 0.0 l3.7+22 0.0

5& R-10 n?ll* l&4 + ll‘t 1,4l3 2 1% 1,172 + 11w l.ZZ2.2 21?11 65.4 + 7.4 520550 431 + 50 128~24 60, R-20 66 + Wt 13.4 + 8’t t,6%+-lwt lJ573n+21W l20 + 21*t 26212 5z29.6 64ir90 534270 98215 X& R-30 86+12ui l24+1oLi ~132~1Ywt lJ6Y+wt 10.7 + l.Tt 42211 9l32zY.9 l,w~lao 6532% 87 + 1.1 84R-40 8lrwt l21awt u57?21W 1,m+14wt 108+1.vt 41212 Ml?89 l.Ol6~laO &131el 89212

‘p<0J5versusseaerea hearlstp~O.Oi~~adultimdsenescenthea.~=~~ W~~,~~~~T~l.

JACC Vol. 27, No. 7 AnmETAL June 1996z1n7-86

1781 PRECONDfTfONMG AND AGING HEART

o,k&e 95?b 3.9 t 1.4 ‘450 z 1% 1,954+40 10, NEsnd 168 + 12’ 27 fr lb’ 5,150 + zv 4,270ZlW XtWend 911t7 3.2 2 0.9 2Jm+1m 1.950~90 441ad 0.0 29626-t 0.0 0.0 45, R-5 49212 24bklOt 1215 + 1M I@7~lal Xl, R-10 54210 187 f lot 1p9trdl 1,110 2 110 60, R-B 62~2Of 15629t 1537 2 l&It 1,275 + ltxtt 33, R-30 832 11t 13.2*9t w589o3t 1,337? lllp @AR-@ R2~13t 13.4 + lot zO33 f 21M 1$86~12#

122t0.8 L3.9 + 1” 118 + on

0.0 126?2n Iz22tzs 12lk 1st 10.4 -+ 1.6t 106 + 1.6t

101 -t 5 4D?rOX &5@Z140 2,cm~W 146210 36 2 1.4 4.415 f 220 3,7loi240 9329 3811 2450~1?0 2.ClB=80

0.0 416 r 8.7t 0.0 0.0 47217 2b.7r11t 1,165 f 150 966t110 52215 213 + lot l,B9Z 140 lJS9ZllB 60t15t 178 + 9s 1,4axz lxt: IM?l-Dt l2c 13i 15.1 r 12i 1,785 f IRO: 1.481 L lalt 14 2 14t 155 t 1Y 1x35 f Isat 152.2 z 1IOt

125ZlD 129 2 08 121 It 1.1

Oil lz.b+28 I21 226 11.92 1% 10.1 2 1% 102118t

*D < 0.05 versus senescent hearts. to < 0.01 wsus hearts subiied to 20 min of &hernia and 40 mio of reperfusion. hF = norepinephriw other abbreviation as iniables 1 and 2.

nephrioe concentrations in coronary effluent that accumulated over the 10 min preconditioning window was 4.1 ? 1.5 pmoUm1 per g in adult and 1.1 i: 0.9 pmol/ml per g in senescent hearts (II = 4 for each group, p < 0.01). These data contirm our hypothesis that norepinephrine plays a crucial role in the preconditioning phenomenon and that in senescent hearts tbese phenomena are less effective probably because of the age-related reduction of norepinephrine release in response to transient ischemia.

Discussion Although the elderly are known to have increased morbii-

ity and mortality rates for myocardial infarction, the specitic factors responsible for thii adverse prognosis are poorly un- derstood (l-5). Even adjustment for age-associated differences covering risk factor profiles has not explained the higher rate of mortality observed in elderly patients (28). The discovery of the phenomenon of ischemic preconditioning has focused attention on the heart’s capacity to protect itself and on the possibihty of an endogenous form of protection. Several

In the present study, the beneticial effects of precondition- ing on electricat and mechanical functions foiknving ischemia- reperfusion injury were 5een in adult but not in senescent hearts. However, preconditioning was achieved by atpha- adrenergic agonist in both adult and senescent hearts, which disappeared after administration of aipha-adrenergic antagc- nist. These data demonstrate the importance of alpha- adrenergic receptor stimulation in the precomhtioning phe- nomenon which is lost in senescent hearts pmbably because of reduced norepinephrine release in response to transient is&- emit stress.

IsdIemia-~iqiargintbeaging~Ischemia- reperfusion injury determines more severe myocardial dys- tiurction in senescent than in adult hearts (6.7). Aged hearts

studies have demonstrated that ischemic preconditioning by means of brief periods of ischemia and reperfusion is able to protect the heart from a longer and sustained ischemic insult (8). This phenomenon occurs also in humans as demonstrated by some studies showing both repetitive coronary oadusion during angioplasty (2930) and preinfarction angina (3132) conferring an early beneficial effect.

1782 ABETFZ ET AL JACC Vol. 27. No. 7 PRECONLXTIONING AND AGING HEART June 1996~3777-86

T& 5. Hemodynamic V&&S hiring &n~nd&ning Transient lsci~tic Stimuhis (2 min), Ischemia (20 min) and Reperfusion (40 min) in Adult and Seinxent Rat Hearts E&B of Alpha-Adrener@c Antagonist (pknt~lamine)

AdultHeartr(n=l) Fenescent Hearts (n = 7)

;i c:pQl 0

tdP/dt -dP/dt CFR LVEDP cm

(mm W) 6-m H%s) (mvmin per g! (miTP@) (mm 4) c2Qb (:$ (mvmin per@

Qbidiw %?8 35 _+ 0.6 2532 + 120 1,6?nf&l IL1 + 0.6 101 z! 5 43203 LMQ?lM 2077+93 .28 + 1.0 10, Tknd 6%2*t 7.4 ? oE*t 270+30-t noIt3o*t 0.0 3 2 I+ 7.7 2 15t 74 t 13t 61,lOt a0 20,w-end %‘+7 4.0 + 0.7 2,456 2 1M m5t60 128 +0.x 85’8 5.9 5 1.4 ~lMtl10 1.748tw 125213 *Iend 0.0 X.4?6t 0.0 0.0 0.0 0.0 30.6 2 9.2 Old 0.0 0.0 45, R-5 2425’ 53 2 s*t 417zw 219,40* 129 2 1.R 12% 11 ?d.8 2 10 29lr30 245220 13.1 + 21 50, R-10 2828’ 45?9v 650+65* 440+65* 120 + 1.0 17flO 68523.4 424560 391*31 12 + 1.7 f,4j R-20 36zrt 40110”t 830 2 1ai*t 689?124Y 11.7 + I.& 24 c 14 56.9 2 9.4 597’70 495260 9.12 1.7 4 R-30 46+8*t 3828-t 1,239 2 1wt 9Xl-ClWf 115 + 1.5t 39211 549 2 8.64 936z60 l&9+50 8.4 t 25 IR-40 50 2 5*t 352 r 8*t 1,2l8 + 105’1 %I 2 115.t 11.0 + 1.6t 35 z 12 53.6 2 12 8672 110 719?a5 8.82 1.6

‘p c 0.05 versus senescent hearts. tp < 0.01 versus adoh and senescent hearts treated with 2 min of transienr ischemic preconditioning, 20 min of &hernia and 40 min of repetion. Phentolamine was infused for 2 min before and resumed for another 5 min during perfusion of preconditioning window Abbreviations as in Tables 1 and 2.

have accumulated more intracellular calcium during ischemia and recovered less completely during reperfusion than adult hearts (7). Reperfusion- and reoxygenation-induced ventricu- lar arrhythmias were more severe in senescent than in adult hearts and the,! increased incidence was associated with a larger development of contracture during reperfusion (33.34). Our data cunfkn these results as both ischemic and postisch- emit myocardial dysfunction were more evident in senescent than in adult hearts, and this greater contractility impairment was associated with a more frequent number of arrhythmias. In view of these results, we consider it extremely important to verify whether a protective endogenous mechanism such as ischemic preconditioning takes place not only in adult but also in senescent hearts.

Precnnditioniag mechanism. We do not know what mech- anism underlies preconditioning. Increased flow during reper- fusion (3537), antioxidant defenses (38), heat stress (39) and synthesis of new proteins (llJ2) seem not to play any impor-

tant role in this phenomenon. Nor does adenosine-A, receptor stimulation, at least in rat experimental model, substitute for preconditioning in the globally ischemic heart (40). Ischemic preconditioning in the rabbit, in fact, is mediated by adenosine AI-receptor stimulation (40). Tsuchida et al., however (41), showed also that alpha-adrenergic agonists precondition the rabbit heart as well suggesting that alpha-adrenergic and A,-receptors activate parallel pathways that can trigger preconditioning-mediated myocardial protection. Norepineph- rine release after transient ischemia has been suggested as a positive process acting by means of the generation of an adaptive signal (42). Depletion of norepinephrine stores by transient ischemic stimulus and therefore reduction of cate- cholamine toxicity, in fact, seems to be not a determinant because chemical sympathectomy did not reduce experimental infarcts in rabbits and because pretreatment with reserpine did not improve postischemic function in rats (l&43). In view of this, in our experiments, minimal release of norepinephrine in

AdultHearls(n=7J SeoeaotHeart(n=7)

(E$) (2&) -dlWl KFR DP LVEDP tdwt -dPldl CFR

(mm W) W~pergi (m&d (mm w (m Hgk) (mm W) (- I= t?!

abgieline %+6 3.82a7 2$7il+180 2135 2 SO 12520.8 loo?5 4.021 z479+110 2fJ56 + 110 1262 1.1 lQT(end 160 + 8’t 2.5 2 O.s*t5,627 + 28O*t 4$593 r 210.t 138 + 0.9 135 +- lot 3.521t 3347+l@t 2776kllBt 129 2 12 20, wend 94irs 4azos 2,LnO~lal 2,163+50 11.8 + 0.9 99210 3.9 +_ 1.4 2455 + IOU 2,O%t1@I 125t1Jl 40,l-end a0 30.8% 10v 00 a0 0.0 0.0 3a6+92t 0.0 0.0 011 45. R-5 24 2 7* 54.1~ll’t xlOz7(r 403270’ 1242 13 14 It 12 fsa c 7.4t 3xJ -c 3 233230 138 + 1.9 so, R-10 4)Z6* 485%9v l5ozw 6&4~tw 1202 1.6 15 2r 12 705*4t 4242% 388238 11.7rtl 60, R-XI 36+e’t 426 f s*t w7 + 11fft a6r1mt 115 ?: 0.g 26 + 18t 573 + lit 581ZBt 4s5~mt 9.4 2 1.6-t Xl, R-30 42+_wt 3959J+vt 1293+1wt 957 i- llcw 11.42 1fJt 3l? lot 55.7 z lit 95o+wt 775z&?t 9.0 2 15t IR-40 49267 33.8~9=$ 1,4nIkwt 95925wt llD-‘lst 39+18t x6+12t 4BD~loo1 789%93 &9 2 1.9t

Ip~0.05~ssenh~~~O.Oiverslaadultandseoesceruhearizaeatedwitb2rninofnaepbKphrinep~~m~~m~~~ and40mh,ofrepemeion.WestdnmineMsio~forZlniabeforeandhenmaintaintdfor~her3miaduringprfuaioncfpremsditionhig~. Abhaia~sinTableslwd2

JACC Vol. 27, No. 7 ABETEETAL June 1996~177745

3783 PRECONDITlONING AND AGING HEART

9925 3.9 2 0.6 i$4O%lal 3~2 25212 802w 221 31kY 53~21

0.0 52 + 10’ 0.0 0.0 48+10* 0.0

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43zO3 37’9 55211 7or15 6s-ct3 lOL?5 39285

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1CHl R-10 8o+w 135 z Kr 2,140 +- ll(r 1,470 + lrn 8.7%24 33% 10 39.7 + 10 1.176 2 1M 9%r&l 93216 l p < 0.05 vetsus senescent heart.s. Trattsieot kcltemia was applied four times for 5 min. and each step was folknwd by 5 mitt of repemsjon (precoaditi

window). Abbreviations as in Tables 1 and 2.

preconditioning and that both transient ischemia and exoge- nous norepinephrine are able to precondition the heart, an effect blocked by alpha-adrenergic receptor antago&t. Lxal vasoconstriction in response to norepioephriw infuskm could be responsible for preconditioning through adenosine release, according to the hypothesis of Liu et al. (14). However, as demonstrated by U and Kioner (40) adenosine seems extra- neous to preconditioning phenomenon, at least in rat experi- mental model. In fact, senescent hearts, which present en- hanced adenosine levels (46). should be more x+nsitkrt: to preconditioning stimuli, on the contrary, our data sbw that the senescent heart is not pieamditionexl by transient isdb em& which is one of the most powerful stimuli of adenosine release. The fact that transient ischemia determined a more severe impairment with a stower recovery in senescent than in adultbeartscouldbe~bybothagreaterJemitivityof aginghearttomyocardialischemia(~7)andaredu&nof norepinephrine release in respomm to kchemic stirnuhts and therefore of the adenosine release that represents one of the endogenous medmnks against is&e*. injury (45). Inotro- picchangesinducedbydilferentpr~stimuhdonot seem responsiiie for the preconditioning medmnkm. In taa, ourdatashowthatinotropicchangesindueedbyoorepiwph- rine infush was greater in adult than in senescent hearts in agreement with several studies showing a reduckm of beta- adrenergic mntractiie respome in aged hcarts (47,48). Never- thelesr,norepinephrilleisaMetomimicprecoaditiin both adult and senescent bearts. Ahho@ aipha-adrenergic stimulation is usdy interpreted in terms of the prominent bemodpamifeffect$me~~e~suchas&tionof protein kinase C should ako be considered. Norepkpkk, in fact,isltnmltoatupletoproteinkinasec~adrene~ stimutationwh@intmqcotddeithermaketbeheartmore resistanttoischemicdeenergirauonorfacilitatereenergizatioo during repemaian (49,!%). Ytrehus et al (51) found that itlkibhOfproreinhiaaseCtSCtjVatiOObbCkCdprrmrdition- ing and. more recently, hMchdl et al (52) have axtkmed

senescent heart, in which preconditioning is inactive, contkms that depletion of catecholamine stores is not the mechanism responsible for preconditioning phenomenon.

Other recent studies show that attenuation of ischemic injury by a form of “chemical shmning” due to a norepineph- tine release after transient ischemic stimulus is not crucial for preconditioning mechanism (44,45). Our data support this hypothesis because the greater impairment of contractile func- tion obtained by transient ischemic stimulus in senescent than in adult hearts does not result in an improvement of premn- ditioning protection as well.

Alpha-adrenergie receptor pathway. Some investigators (17,18) demonstrated that in the isolated heart, adrenergic receptor stimulation seems to control the transduction of

figure 2. Bar graphs showing recovery of Percent developed presmrc (DP) at 4Omin ofreperfnsion in adult (opeo bars) and senescent (wtid bars) hearts subjected to ischemia-reperfusion injuly without precoo- ditioning (GIRL), and subjected to one cyde (1 X 2 min) or to multiple cycles (4 x 5 min) of preconditioning transient ischemic stimulus. *p < 0.05 versus senescent. tp < 0.01 versus control.

1784 ABEmETAL PRESONDITIONMG AND AGING HEART

that protein kinase C is an effectur of preconditioning in the k&ted rat heart via alpha-adrenergic receptor stimulation. Muitiple reuptm couple protein kinase C in the rat inchrdmg thosz for @ha,, angiotensin II, bradychinin and endothelin (5132). A single 2&t period of ischemia is very near the threshold for triggering the protection such that Ioss of nor- epmephrine shdatim in senescent hearts could put the heart behlw the tII&mId for prote&on. In our experiment& mul-

JACC Vol. 27, No. 7 June 1996:1777-86

postischemic recovery (-10%) with respect to single cycle suggesting that a more intense preconditioning protocol should be able to release other agonists in quantity to trigger prorsin kinase C. Nevertheless, the difference in post-ischemic recov- ery between adult and senescent hearts in multiple-cycle preconditioning protocol remains consistent.

The role of endogenous catecholamines in ischemic precon- ditioning, however, is controversial. Weselcouch et al. (53) found that endogenous catecholamines are not necessary for ischemic preconditioning in isolated rat heart, whereas Asi- makis et al. (54) reported that in the same experimental model, exogenous alpha-adrenergic agonists were not cardioprotective and that beta-receptor blockade abolished preconditioning phenomenon. One possible expianation for the differences observed is that the length of the global ischemic period of our experiments was 20 min. which determined a less contractile impairment than the 30 min of global &hernia performed in the experiments of We&couch and Asimakis and Inners- McBride. In addition, it should be pointed out that we used 2-min preconditioning protocol whereas the other investigator, used 4 to 5-min ischemic periods with a 5-min reperfusion interval to reach the cardioprotective &e&s of precondition- ing. As discussed before, a more intense preconditioning protocol should release other af,onirrs, which can trigger protein kinase C even with alpha-adrenergic receptors blocked (5152). Our opinion is that different ,:reconditioning regimens and global ischemia times should jtrst@ the discrepancy ob- served among the groups of preconditioning experiments.

In our experiments, however, abolition of preconditioning by alpha-adrenergic antagonist such as phentolamine is the jump in logic from the basis of our hypothesis. After phentol- amine admiistration, in fact, adult hearts looked like the senescent hearts, and alpha-adrenergic blockade reduced the protective effect of norepinephrine preconditioning stimulus in both adult and senescent hearts.

Catech&minas and aging. It has been suggested that tissue catecholamine levels decrease with advancing age partly because ofIan age-related diminished ability for catecbolamilip synthesis (19). Dawson and Meldrum (20) found that the catecholamine content per organ was reduced by 25% in the ventricle of aged rats, whereas norepinephrine content per gram of tissue weight was significantly decrezised in the whole heart. Yet, McLean et al. (21) demonstrated a sign&cant sympathetic axonal degeneration between 3 and 24 months of age in rat experimental model. Thus, age-related reduced cat&&mine content, especially during stressful conditions, such as myocardial ischemia, could be a functional diidvan- tage if norepiuephrine release is involved in the precondition- mg phenomenon. The reduction of norepinephrine release in senescent hearts in response to transient ischemic stimuli, the reproduction of preconditioning by exogenous norepinephrine infnsii and the ab&tion of protective preconditioning effects of norepin&uine by phentolamine in adult and senescent ~~n~~~en~~rn~~t~~ inantt‘okoftbeadrenergicsysteminthisintriguingphenom- emit.

JACC Vol. 21, No. 7 June 19%:1777-f%

Clinical implkatfans. Numerous studies have suggested that age is a determinant factor in the in-hospital and long term prognosis of acute myocardial infarction (l-5,2@. Age, in fact, has been shown to be a major risk factor in the develop- ment of the various clinical manifestations of coronary artery diiase (l-5,2g). Several recent studies, on the other hand, have demonstrated the protective effect of prodromal angina against ciinical manifestations of myocardial infarction (29- 32). Although the most reasonable mechanism to explain the beneficial role of prodromal angina is “ischemic precondition- ing,” it should be pointed out that a direct evidence of clinical relevance of preconditioning has not been demonstrated. Given the poor prognosis of elderly patients with acute myo- cardial infarction (l-5,2&, however, the hypothetical lack of ischemic preconditioning in these patients should be consid- ered especially when it is possible to induce preconditioning pharmacologicafly (55). Although ethical considerations limit the investigation of preconditioning in humans, studies are needed in elderly patients who have ischemic episodes that occur naturally or are induced by therapeutical manipulation.

Cnnebmhms. We con&k that ischemic preconditioning significantly reduces electromechanical postischemic dysfuoc- tion in adult but not in senescent hearts. Exogenous norepi- nephtine infusion is able to mimic preconditioning in both adult .and senescent hearts, whereas reduction of aorepirqh- tine releaw In XSponSe to ischemic preconditioning is ob- served only in senescent hearts. Abolition of preconditioning by alpha-adrenergic antagonist (phentolamine) seems to con- firm the predominant role of the adrenergic system through alpha-pathway in this phenomenon. These tindii suggest tt ,at in the senescent rat heart, ischemic preconditioning is missing. probably due to the absence of a mediator that triggers thii protective endogenous mechanism against myo- cardial ischemia.

In this regard, it should be pointed out that the precondi- tioning mechanism in humans seems to be mediated by adenosine rather than norepinephrine suggesting that a re- duced norepinephrine release might not have such a profound effect in humans (55,56). However, it might be considered that a role of norepinephrine in the mediation of preconditioning against postischemic dysfunction has been demonstrated not only in rat but also in dog hearts (57). Further studiis are necessary to verify whether norepinepbrine ako mediates the protective effect of ischemic preconditioning in humans.

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ABETEETAL. 1785 PRECONDITIONING AND AGING HFART

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