Glucose Metabolism during Ischemia Due to Excessive Oxygen Demand or Altered Coronary Flow in the Isolated Arterially Perfused Rabbit Septum

Transcription

1 640 Glucse Metablism during Ischemia Due t Excessive Oxygen Demand r Altered Crnary Flw in the Islated Arterially Perfused Rabbit Septum ROBERT C. MARSHALL, WILLIAM W. NASH, KENNETH I. SHINE, MICHAEL E. PHELPS, AND NICHOLAS RICCHIUTI SUMMARY The islated arterially perfused interventricular rabbit septum was adapted fr the study f glucse metablism during ischemia prduced by either increased xygen demand r altered crnary flw. The septum perfused at 1.5 ml/min and stimulated at a rate f 72/min at 37 C was shwn t be a lw wrk preparatin in which glucse utilizatin, lactate prductin, xygen cnsumptin, and develped tensin were stable fr at least 90 minutes. The metablic and functinal respnses f the septum were evaluated during nnnxic increased wrk prduced by intrducing a paired stimulus at 90/min and increasing flw t 3.5 ml/min, during demand-induced ischemia prduced by intrducing a paired stimulus at 90/min and maintaining flw at 1.5 ml/min, and during lw flw ischemia prduced by decreasing flw and maintaining stimulus rate cnstant at 72/min. During nnnxic increased wrk, glucse utilizatin increased by 115 ± 26% (±«KM) ver cntrl, while xygen cnsumptin increased by 100 ± 11%, lactate prductin by 13 ± 9%, and develped tensin by 45 ± 8%. Tigsue glycgen, lactate, and lactate:pyruvate ratis were unchanged cmpared t cntrl. During demand-induced ischemia, glucse utilizatin increased by 116 ± 24%, while xygen cnsumptin increased nly by 29 ± 7%, lactate prductin rse by 106 ± 16%, and develped tensin declined by 20 ± 4%. Tissue glycgen cntent was significantly decreased and tissue lactate and lactaterpyruvate ratis were significantly increased during demand-induced ischemia cmpared t bth cntrl and nnnxic increased wrk. These results are cnsistent with accelerated glucse xidatin during nnnxic increased wrk and accelerated anaerbic glyclysis during demand-induced ischemia. During severe lw flw ischemia, glucse utilizatin declined by 44 ± 8% while develped tensin and xygen cnsumptin decreased by 80 ± 8% and 74 ± 2%, respectively. The results f this study suggest that the dependence f glucse metablism in ischemia n residual perfusin fr washut f metablic end prducts is als bserved when ischemia is prduced by excessive xygen demand. Ore Res 49: , 1981 DURING mycardial ischemia there is a mismatch between xygen supply and demand. Glucse metablism during ischemia is dependent n residual perfusin fr washut f the metablic end prducts, lactate and hydrgen in (Rvett etal., 1975). This finding expains the bservatin that glucse uptake and metablism accelerate if the mycardium is made hypxic by perfusin with xygenfree perfusate at maintained flw rates, but des nt accelerate r even declines if the mycardium is made hypxic by decreasing flw rates (Rvett et al., 1973; Neely et al., 1975). Extraplatin f Frm the Departments f Medicine, Radilgy, and Physilgy and Ls Angeles Cunty Cardivascular Research Labratry, University f Califrnia at Lt Angeles Center fr the Health Sciences, Lt Angeles, Califrnia Supprted by US Public Health Service Grant HL Ls Angeles Cunty Heart Assciatin Grant LACHA 629, and the Castera Fundatin. Address fr repnnu: R C Marshall, Divisin f Cardilgy, Department f Medicine, UCLA Center fr the Health Sciences, Ls Angeles. Califrnia Received January 4, 1980; accepted fr publicatin March 17, these results t the evaluatin f xygen supply and demand during ischemia suggests that glucse uptake and metablism might accelerate if the mycardium were made hypxic by increasing demand withut changing flw, but wuld nt accelerate r wuld even decrease if the mycardium were made hypxic by decreasing flw. Evaluatin f reginal mycardial exgenus glucse utilizatin is nw pssible in humans using 18 F-2-flur-2-dexyglucse (FDG) and psitrn-cmputed tmgraphy (Phelps, 1977; Phelps et al., 1978). If the flw dependence f ischemic glucse metablism culd be shwn t apply t xygen supply-demand imbalance, then FDG culd be used as a physilgical tracer which culd reflect the interplay f xygen supply and demand in the prductin f ischemia if used with an independent indicatr f perfusin. T evaluate this pssibility, the islated arterially perfused interventricular rabbit septum was adapted t study mycardial glucse metablism during ischemia prduced either by increased xygen demand r altered crnary flw.

2 GLUCOSE METABOLISM DURING ISCHEMIA/Marshall et al. 641 Methds Experimental Preparatin The islated arterially perfused interventriculax rabbit septum was btained frm male New Zealand rabbits (1-2 kg). After the administratin f 10 mg f sdium heparin and 180 mg f pentbarbital via an ear vein, the heart was rapidly excised thrugh a median sterntmy and placed in a warm, xygenated perfusate. Fllwing remval f bth atria and the right ventricle, the septal artery was identified by pening the left crnary artery thrugh its stium in the arta. The septal artery was cannulated with a small plyethylene cannula (Clay-Adams). The cannula was secured in place with a 6-O silk suture. An interval f n mre than 5 minutes elapsed frm the time the heart was excised t cannulatin. Flw was maintained at 1.5 ml/min by a Gilsn Minipuls 2 peristaltic pump. Since the final weight f the perfused septum varied between 0.75 and 1.2 g, flw/gm wet weight per min varied between 1.3 and 2.0 ml/min. The perfusate cntained (in HIM): NaCl, 130; Cad*, 2.5; KC1, 6.0; MgCl, 1.0; NaH 2 P0.,, 0.435; NaHCO.,, 12.0). The substrate was dextrse, 5.6 mm. Crystalline insulin, 25 milliunits/ml, was added t ensure adequate availability f glucse t the mycardium. The perfusate was equilibrated cntinuusly with 98% O 2-2% CO 2, yielding a stable ph f Well perfused tissue was identified visually, and a triangular segment was cut ut with the cannula at its base. The perfused mycardium was suspended between tw clamps at its base and tied t a strain gauge-type tensin transducer at its apex using a 5-O silk suture. The tensin transducer was munted n a geared slide, and rest tensin was manipulated by mving the transducer up r dwn n this slide. N rest tensin was applied between the tw clamps. After suspensin f the septum, stimulating electrdes frm a Grass SD-44 stimulatr were attached t the clamps, and 4- t 6-mV, 0.4-msec stimuli were delivered at a rate f 72/min. Under standard experimental cnditins cmmnly emplyed fr the islated arterially perfused rabbit septum, effluent perfusate zes ut ver the septum's surface and is expsed t the atmsphere. T measure effluent xygen cncentratin, a small, sealed Lucite chamber was cnstructed t enclse the septum. Built int the chamber were the cannula, clamps, thermistr, tensin transducer, and effluent sampling access t allw experiments t be cnducted while the septum was enclsed. After searing the septum in the chamber, bth the chamber and the perfusin line were lwered int a cnstant temperature bath. Temperature was maintained at 37 C thrugh all experiments. Cre temperature f the septum was measured using a needle thermistr (Bailey Instruments). An equilibratin tune f 60 minutes preceded any experimental interventin. A preparatin was accepted as suitable fr study if it develped at least 15 g f tensin at a rest tensin f 6-7 g, and if rest and develped tensin were stable ver the equilibratin perid. The dead space f the chamber was 3.2 ml with a 1-g septum in place. T exclude atmspheric cntact with the septum fr effluent xygen cntent measurements, the dead space was filled with effluent perfusate. This maneuver interpses a "mixing chamber" between effluent perfusate cming frm the septum and effluent perfusate cming frm the chamber such that time must be allwed fr washut f the dead space fllwing a change in steady state metablism f the septum. T estimate this time, the chamber with septum in place was laded with 3 H 2 O, and washut curves were cnstructed at flws f 1.6 and 1.0 ml/min. At bth flws, 94-96% f the 3 H 2 O had been washed ut f the chamber in 3x the mean transit time (vlume/ flw). This calculatin was used t estimate the minimum sampling time fr all experiments reprted in this study. Data Cllectin and Analytic Prcedures Rest and develped tensin, maximal rate f tensin develpment, and maximal rate f relaxatin were recrded cntinuusly n a fur-channel Hewlett-Packard 7404A recrder at a paper speed f 5 mm/min. Glucse utilizatin was evaluated by *H 2 O prductin frm J H-2-glucse (Katz and Dunn, 1967). T separate ^O frm :1 H-2-glucse in effluent perfusate, small dispsable clumns cntaining the hydrxide frm f Dwex-AG-1 X8 200 t 400-mesh resin were used. Since previus investigatrs have used the brate frm f this resin (Neely et al., 1972; Rvett et al., 1975), validatin f quantitative separatin was accmplished by placing n the resin knwn activities f 'H^O and! H-2-glucse standards (btained frm New England Nuclear) and eluting with 3 ml f distilled water; 96 and 0.06% f the 1 H 2 O and ; 'H-2 glucse were recvered, respectively. Cntinued validatin f quantitative separatin was perfrmed with each experiment by measuring retentin f a H-2-glucse frm a sample f perfusate. Samples were disslved in Beckmann Ready-Slv HP ccktail and cunted in a Beckmann LS3133P liquid scintillatin cunter. All analyses were perfrmed in duplicate. Effluent perfusate lactate cncentratin was measured by using a standard spectrphtmetric technique (Gutman and Whalefield, 1974). Oxygen cntent was measured in a Lex O? Cn xygen analyzer (Lexingtn Instruments). Oxygen cnsumptin was calculated as the arterial-venus difference in uml/ml multiplied by the flw in ml/ min. Bth lactate cncentratin and xygen cnsumptin were determined in duplicate. Septa used fr tissue glycgen, lactate, and pyruvate analysis were rapidly frzen by clamping them with cpper tngs cled in liquid nitrgen.

3 642 CIRCULATION RESEARCH VOL. 49, N. 3, SEPTEMBER 1981 The frzen tissue was pulverized and extracted with 6% perchric acid. Lactate and pyruvate were assayed by the lactate dehydrgenase prcedure and glycgen by the hexkinase glucse-6-p-dehydrgenase prcedure as described in Bergmeyer (1974). Data are expressed as the mean ± SEM. The data illustrated in Figures 1, 3,4, and 5 and Table 1 were subjected t a repeated measures analysis (Dixn and Brwn, 1979). When a single treatment parameter was varied, rthgnal decmpsitin f the trial effect was perfrmed t determine the nature f the treatment effect n the measured variable. The data f Table 2 were subjected t a tw-way analysis f variance and significance determined using the apprpriate./^-statistics. Results Figure 1 illustrates the functinal and metablic stability f the islated arterially perfused rabbit septum under cntrl cnditins (72 stimuli/min si 8 3 E 50 I I d 75 I J- -? * 5 i i t 2O 3O Time in M mutes FIGURE 1 Evaluatin f the mechanical and metablic stability f the septum perfused at 1.5 ml/min per septum with a single stimulus rate f 72/min. Develped tensin (panel A) and xygen cnsumptin (panel B) were measured at 30-mmute intervals and lactate prductin (panel C) and exgenus glucse utilizatin (panel D) at 10-minute intervals fr 90 minutes. Results represent the mean ± SEM fr fur septa. Althugh the data demnstrated a small but statistically significant variatin with time, n simple trends culd be demnstrated fr any f the measured parameters. and 1.5 ml/min flw) fr 90 minutes. Develped tensin at time 0 averaged 20 ± 1.4 g, did nt change ver the first hur, and declined 11% t 17.8 ± 1.4 g at 90 minutes. Initial xygen cnsumptin averaged 0.94 ± 0.04 /iml/min and increased slightly t 0.99 ± 0.04 after 90 minutes. Average initial glucse utilizatin and lactate prductin were 0.33 ± 0.02 and 0.51 ± 0.05 /unl/min, respectively. By 90 minutes, glucse utilizatin had increased by 12% t 0.37 ± 0.3 juml/min, while lactate prductin increased by 8% t 0.55 ± 0.06 /unl/min. Subtracting lactate prductin frm glucse uptake demnstrates that xidatin f exgenus glucse accunted fr mre than 44% f the xygen cnsumed; the rest presumably was accunted fr by xidatin f endgenus triglycerides. Lactate prductin accunted fr 78% f exgenus glucse utilizatin. T evaluate the pssibility that the high cntrl lactate utput f the septum was secndary t cellular hypxia, lactate prductin and develped tensin were evaluated during graded increasas in flw frm 1.5 t 4.2 ml/min. The results are detailed in Table 1. There was a slight but statistically significant (P < 0.05) decrease in lactate prductin and increase in develped tensin as flw was increased, althugh these changes were small cmpared t the 290% increase in xygen delivery. At least part f the small increase in develped tensin culd be accunted fr by a 20% increase in rest tensin (data nt shwn) secndary t the increased perfusin pressure assciated with the higher flws. These data suggest that mycardial hypxia cntributes minimally t the prminent lactate prductin seen in the islated arterially perfused rabbit septum under cntrl cnditins. The high cntrl lactate utput prbably reflects decreased pyruvate xidatin and diffusin ut f the cell in the absence f lactate in the perfusate. Tensin develpment and xygen cnsumptin in the islated arterially perfused rabbit septum are reduced cmpared t ther islated r in situ experimental preparatins. Since the ability t prduce changes in xygen demand is critical t the prductin f demand-induced ischemia, the effect n xygen cnsumptin f increasing the tensintime index by increasing the single stimulus rate and intrducing an intrpic stimulus was evaluated under nnischemic cnditins (Fig. 2). The value fr basal xygen cnsumptin in the septum, 0.5 ± 0.01 juml/g per min, is in the range f previusly reprted values ( /iinl/g wet weight per min) (McKeever et al., 1960; Klcke et al, 1965; Klcke et al., 1966). Oxygen cnsumptin increased linearly as the tensin-time index was increased by increasing the single stimulus rate. As expected, the intrductin f an intrpic stimulus increased xygen cnsumptin ut f prprtin t the increase in the tensin-time index. These results indicate that, despite the reduced tensin develpment and xygen cnsumptin encuntered in the septum, it is pssible t change xygen cnsumptin almst 3-

4 GLUCOSE METABOLISM DURING ISCHEMIA/Marshall et al 643 TABLE 1 Effect f Flw n Lactate Prductin and Develped Tensin Lactate (^ml/min) Develped tensin (g) ± ± 2.7 Flw ± ± 28 ± ± ± ±3.0 Phe effect n lactate prductin and develped tensin f increasing flw frm 1.5 t 2.5, 3.5, and 4.2 ml/nun, maintaining stimulus rate cnstant at 72/min, is shwn. Each septum served as its wn cntrl with subse<njent measurements taken 15 minutes after each increase in flw t ensure the presence f a steady state. Results are the mean ± SEM f SU preparatins Fr discussin, see text. fld by intrducing stimuli knwn t change xygen demand. Glucse Metablism and Mechanical Perfrmance during Ischemia Exgenus glucse utilizatin, xygen cnsumptin, lactate prductin, and mechanical perfrmance were evaluated during demand-induced ischemia and cmpared t nrmxic increased wrk. Nrmxic increased wrk was prduced by intrducing a paired stimulus at 90/min (180 ttal stimuli/min) and increasing perfusin frm 1.5 t 3.5 ml/min in each septum. Demand-induced ischemia was prduced by intrducing a paired stimulus at 90/min but maintaining flw cnstant at 1.5 ml/ min per septum. The results, illustrated in Figure 3, indicate that the increase in exgenus glucse utilizatin is cmparable fr demand-induced ischemia and nrmxic increased wrk. Hwever, during nrmxic increased wrk, lactate prductin did nt change significantly frm cntrl, whereas xygen Ob PEAK TENSION x STIMULUS RATE (gm x bets/min) x IO" 3 FIGURE 2 The effect n xygen cnsumptin f increasing cardiac wrk. Cardiac wrk was increased by changing the stimulus rate frm 0 t 70, 140, and 180/min and by intrducing a paired stimulus at 20 and 70/min with 15 minutes allwed t elapse at each stimulus rate befre xygen cnsumptin measurements were taken. Each septum was perfused at a cnstant rate f 2.5 ml/min. Perfusate cntaining 10 mm KCl was used t btain ttal quiescence nly during the perid in which n stimuli were delivered. Fr the rest f the experiment, standard perfusate cntaining 6 mm KCl was used. Results represent the mean ± SEM f five septa. Fr discussin, see text. and glucse cnsumptin increased prprtinately, cnsistent with previus reprts demnstrating that glucse xidatin increases during increased cardiac wrk in the absence f alternative substrates (Neely et al., 1972; Neely et al., 1976). In cntrast, during demand-induced ischemia, lactate prductin dubled whereas glucse cnsumptin increased ut f prprtin t the increase in xygen cnsumptin, indicating an accelerated rate f anaerbic metablism f glucse. These findings demnstrate that glucse cnsumptin is accelerated during demand-induced ischemia, and that this increase is due primarily t acceleratin f anaerbic glyclysis. Tissue levels f lactate, pyruvate, and glycgen were btained during nrmxic increased wrk and demand-induced ischemia t substantiate further the presence f ischemia when xygen demand is increased withut a cncmitant increase in xygen supply in additin t evaluating the use f endgenus stres f glucse frm glycgen fr glyclysis (Table 2). Cmpared t cntrl, the tissue cntent f lactate, pyruvate, and glycgen and the lactate:pyruvate rati were nt significantly different during nrmxic high wrk ver the 80-minute perid evaluated. In cntrast, during demand-induced ischemia, tissue lactate and the lactate:pyruvate rati increased significantly fr the entire 80 minutes, and glycgen stres were significantly reduced 20 minutes fllwing intrductin f the paired stimulus when cmpared t bth cntrl and nrmxic high wrk. During the first 20 minutes f demand-induced ischemia, glycgenlysis was rapid and verall glyclytic rate was apprximately twice that bserved at 40 and 80 minutes. The large increase in tissue lactate during the first 20 minutes f ischemia reflects the faster glyclytic rate during this time perid. These results prvide further evidence fr the presence f ischemia during xygen supply-demand imbalance with maintained basal perfusin. A cmparisn f exgenus glucse utilizatin during demand-induced and flw-reduced ischemia is illustrated in Figure 4. Glucse utilizatin, xygen cnsumptin, and develped tensin were evaluated during prgressive increases in the paired stimulus rate maintaining flw cnstant at 1.5 ml/min per septum and cmpared t the results btained during prgressive reductins in flw maintaining the single stimulus rate cnstant at 72/min. Due t

5 644 CIRCULATION RESEARCH VOL. 49, N. 3, SEPTEMBER 1981 CJ» X tn O) ~ O 1. Cl 1 8 ATE PF ACI _i O Q5 -? ~ O 1 Q 8 UJ _J O I Glucse Oxygen CONTROL :XX31 />< g H40 H09 a HIGH WORK IMBALANCE S FIGURE 3 Cmparisn f exgenus glucse utilizatin, lactate prductin, xygen cnsumptin, and develped tensin during cntrl, nrmxic increased wrk, and demand-induced ischemia During the cntrl perid, each septum was perfused at 1.5 ml/min with a single stimulus rate f 72/min. Nrmxic increased wrk was prduced by intrducing a paired stimulus at 90/min and increasing flw t 3.5 ml/min per septum, while demand-induced ischemia was prduced by intrducing a paired stimulus at 90/min and maintaining flw cnstant at 1.5 ml/mm per septum. Each septum served as its wn. cntrl, and measurements were made 15 minutes after the intrductin f each interventin. Results are the mean fr fur hearts. Fr discussin, see text ^ LJ 10,>, the very lng transit times thrugh the chamber at flws f less than 0.8 ml/min, the chamber was emptied f effluent perfusate, and xygen cnsumptin was estimated knwing the amunt f xygen delivered and using the lwest xygen cntent bserved frm the preceding measurements t estimate effluent xygen cntent. In thse preparatins in which demand was prgressively increased withut changing flw, develped tensin initially increased while xygen and glucse cnsumptin decreased (secndary t the decline in the ttal integral f tensin; data nt shwn), suggesting that the septum was nt ischemic at the lwest paired stimulus rate. Hwever, as the paired stimulus frequency was prgressively increased, the develped tensin fell while xygen cnsumptin increased slightly ver cntrl and glucse cnsumptin prgressively increased, changes similar t the results TABLE 2 Values fr Tissue Glycgen, Lactate, Pyruvate, and Lactate.Pyruvate Rati Cntrl High wrk Supply-demand imbalance Glycgen 4/iml/g dry wt) 274 ± ± ± ± ± ± ± ± ± ± ± 15* 179± 9'* 154 ± 13*-** LacLate (/iml/g dry wt) 16 ± ± ± ± ± ± ± ± ± 4.0* 45 ± 7 6'" 60 ± 8.5"* 26 ± 4 0*" 25 ± 3.5'- * Pyruvfite (pml/g dry wt) 0.45 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.09 Lac Late, pyruvate 39 ± 8.36 ± 6 23 ± 5 31 ± 8 33 ± ± 4 29 ± 8 32 ± ± 28* 192 ± 49* 140 ± 37* 108 ± 39* 101 ± 23** At time 0, a paired stimulus at 90/min was intrduced, and flw was either increased t 3.75 ml/septa per min (high wrk) r nt changed frm cntrl flw rates (supply-demand imbalance) Septa were smash -frzen at the times indicated fllwing paired stimulatin. At lime 0. perfusate calcium cncentratin was increased frm 2 5 t 3.5 rrim in high wrk and supply-demand imbalance experiments. Data are the mean ± SKM fr five t eight septa. During high wrk, there were n significant differences fr any f Lhe measured metablites cmpared t cntrl values at cmparable time intervals. In cntrast, during supply-demand imbalance, a significant increase in bth lactate and lactate-pyruvate ratis were nted when cmpared t bth cntrl and nrmxic high wrk Als glycgen was significantly depleted during supply demand imblance 20 minutfl fllwing the intrductin f a paired stimulus. (When asterisks are separated by a cmma, lhe firet represents cmparisn t cntrl and lhe secnd t high wrk.) * P <0.05; "P<0 026, "*/*< 0 01.

6 GLUCOSE METABOLISM DURING ISCHEMIA/Marshall et al. 645 ^ 20-1 E ^ ZD CO z E E a CO O v 0.45 J O C Q e I 8.H B s.-----^ ) J,--"" v ^~~~t-^^^^ [ ^Glucse Oxygen Tensin O PAIRED STIMULUS RATE/m.n "I 140 * Glucse r30. I Q FLOW (cc/tgm/wet wl/min) btained in the previus experiments. In thse preparatins in which flw was prgressively reduced, develped tensin decreased in prprtin t the flw reductin, and xygen cnsumptin als decreased belw flws f 1.3 ml/min per septum. Glucse cnsumptin increased slightly ver cntrl during mdest decreases in flw, and then fell when flw was decreased belw 0.7 ml/min per septum. These data demnstrate that the mycardial metablic rate fr exgenus glucse prgressively accelerates during demand-induced ischemia and fails t accelerate r even declines during prgressive flw-reduced ischemia. When the dead space f the chamber is filled with effluent perfusate, a rapid change in glucse cnsumptin culd nt be detected due t the time required fr washut f metablites reflecting the preceding metablic rate fr glucse and xygen. Accurate evaluatin f glucse and xygen cnsumptin in the presence f this "mixing chamber" depends n the maintenance f a "steady state" metablic rate at least fr the perid f time crrespnding t the lngest transit time thrugh the -20 CO UJ Q CL _J LJ FIGURE 4. The effect n exgenus glucse utilizatin, xygen cnsumptin, and develped tensin f prgressively increasing the paired stimulus rate while maintaining flw cnstant at 1.5 ml/nun per septum (panel A), and prgressively decreasing flw maintaining the stimulus rate cnstant at 72/min (panel B). Each septum served as its wn cntrl, and measurements during ischemia were perfrmed 15 minutes after each change in either flw r paired stimulus rate. Results fr bth types f i.schemia are the mean f fur septa. Statistical analysis revealed that all f the measured parameters changed significantly (P < 0.01) as either flwreduced r demand-induced ischemia was prgressively increased, althugh nly glucse cnsumptin during demand-induced ischemia and xygen cnsumptin and develped tensin during flw-reduced ischemia were linearly related t the treatment effect. Data are the mean ± SEM fr fur septa. system (Zierler, 1961). Since previus investigatrs have reprted a transient suppressin f glucse uptake in the first 15 minutes f ischemia due t metablism f endgenus glycgen (Neely et al., 1975), evaluatin f exgenus glucse utilizatin as a functin f time in the septum during demandinduced ischemia was perfrmed with the chamber empty (Fig. 5). In cntrast t the results btained with the Langendrf preparatin during ischemia caused by reduced flw, exgenus glucse utilizatin increased t 80% f its maximum value in 2 minutes. Since there was nly a small increase in glucse utilizatin ver the first 20 minutes, these results suggest a relative steady state fr exgenus glucse utilizatin during demand-induced ischemia fr at least 40 minutes. Discussin In this study, the islated arterially perfused rabbit septum was adapted t evaluate mycardial glucse metablism during ischemia prduced by excessive xygen demand and reduced xygen supply. The septum pssesses several features which

7 646 CIRCULATION RESEARCH VOL. 49, N. 3, SEPTEMBER 1981 * O p O 3 E =; i CD i I ^ * } 1 i I I L - 1 I T T t T T 1 i i MINUTES CONTRO. "" "" CONTROL FIGURE 5 Glucse utilizatin during demand-induced ischemia as a functin f time. At time 0, a paired stimulus at 90/min was intrduced, maintaining flw cnstant at 1.5 ml/min per septum. Each septum served as its wn cntrl. Glucse utilizatin accelerated 80% ver cntrl at 2 min, and slwly increased by anther 19% ver the first 20 minutes. The increase in glucse cnsumptin with paired stimulatin was statistically significant (P < 0.01). Data are the mean ± SEM fr five hearts. make it particularly well suited fr investigatin f ischemic glucse metablism. First, ischemia in the septum is glbal, independent f whether it is secndary t increased demand r reduced xygen supply. Althugh reginal ischemia mre clsely apprximates the clinical situatin, reducing flw thrugh a single crnary artery prduces a hetergenus ppulatin f cells with sme having near nrmal flw and thers virtually nne. Crrelatin f the severity f ischemia and metablic changes is therefre impssible. Secnd, cntrl flw rates in the septum are nly slightly higher than basal flws in humans. Since glucse metablism during ischemia is flw-dependent, the ability t prduce ischemia at flws cmparable t that which prduce ischemia in humans is a prerequisite fr extraplatin f these results t the clinical situatin. Finally, direct perfusin f the septum thrugh the septal artery allws independent cntrl f changes in wrklad and flw, allwing independent evaluatin f the effect n metablism f changes in xygen supply and demand. The metablic and functinal stability f the septum at 37 C with a flw rate f 1.5 ml/min was demnstrated in this study by shwing that exgenus glucse utilizatin, lactate prductin, xygen cnsumptin, and develped tensin were 3table fr at least 90 minutes. Althugh the flw rates emplyed here are similar t thse in previus reprts utilizing the septum, prir investigatins were perfrmed either at 28 C using red bld cell-free perfusate (Langer and Serena, 1970) r, if at 37 C, using perfusate cntaining red bld cells (Shine and Duglas, 1974). Recently, evaluatin f the energetic state f the septum at 37 C perfused withut red bld cells was perfrmed during an investigatin f the effect f perfusate bicarbnate cncentratin n high energy phsphate cntent, ptassium cntent, and develped tensin (D. Angel, unpublished bservatin). Of relevance t this reprt were the findings that high energy phsphate and ptassium cntents, alng with develped tensin, were nt significantly different frm thse which had been reprted previusly fr the septum evaluated at either 28 C C r 37 C and perfused with red bld cells. These data, alng with the stability f the septum demnstrated here, are evidence fr the metablic and functinal integrity f the septum under the experimental cnditns emplyed in this study. The lw tensin develpment (apprximately g) in the islated arterially perfused rabbit septum prbably accunts fr the stability f the preparatin perfused at 37 C withut red bld cells. The lw level f tensin develpment is nt due t xygen deprivatin since develped tensin is nt significantly different when red bld cells are included in the perfusate (Shine and Duglas, 1974). Als, a 3-fld increase in perfusin rate did nt change tensin develpment substantially. The lw tensin develpment can be understd by cmparing the spatial distributin f develped tensin in the septum t the intact heart. The wall f the left ventricle cntains muscle fibers which are bth curved and varyingly riented and in which tensin develpment is multidirectinal (Streeter et al., 1970). In the intact heart, tensin (r stress) develpment usually is reslved int radial, circumferential, lngitudinal, and shear cmpnents. Since the septum is a segment f the left ventricular wall, it als cntains curved, varyingly riented muscle fibers, and therefre the ptential fr multidirectinal tensin develpment exists. Hwever, unlike the intact heart, the septum is secured nly in the plane frmed by the tensin transducer and the tw clamps. Since it is impssible t have unppsed tensin develpment, the septum can develp tensin nly in this single plane. In additin, since there is n stretch applied acrss the septum between the tw clamps, tensin develpment in this plane is restricted t vectrs detected by the tensin transducer. Since the vast majrity f fibers at the base f the heart are riented circumferentially (Streeter et al., 1969), tensin develpment in the islated arterially perfused rabbit septum prbably represents a vectr f the shear cmpnent in the intact heart. Glucse Metablism and Functin during Ischemia Evaluatin f exgenus glucse utilizatin during ischemia induced by increasing xygen demand has nt been reprted previusly. This is smewhat surprising since clinical ischemic heart disease frequently manifests as demand-induced ischemia in patients with exertinal angina. Dcumentatin f

8 GLUCOSE METABOLISM DURING ISCHEMIA/Marshall et al. 647 the prductin f ischemia by increasing xygen demand is perfrmed daily by bserving ECG changes during exercise n a treadmill and by the presence f left ventricular dysfunctin and lactate prductin during artificial pacing studies perfrmed in the catheterizatin labratry. The bservatin that abruptly discntinuing prpranll in patients with ischemic heart disease can ccasinally have fatal cnsequences (Miller et al., 1975) suggests that excessive xygen demand might, in certain instances, play a primary rle in the pathgenesis f acute mycardial infarctin. In this study, we believe we have simulated the clinical situatin f demand-induced ischemia in the islated arterially perfused rabbit septum. Evidence t supprt this cntentin was btained by cmparing lactate prductin, xygen cnsumptin, mechanical perfrmance, and tissue levels f lactate, pyruvate, and glycgen during demand-induced ischemia t cntrl and nrmxic increased wrk. The increased tissue lactate, lactate:pyruvate rati, and lactate prductin and the reduced glycgen stres in additin t the failure t imprve mechanical perfrmance in respnse t an intrpic stimulus and the inability t apprpriately increase xygen cnsumptin are all cnsistent with the presence f demand-induced ischemia. Cntrl xygen cnsumptin in the islated arterially perfused rabbit septum is nly abut 25% f that in resting humans and 10% f that fund in the Langendrf preparatin (Klcke et al., 1968; Neely et al., 1976). The evidence presented in this reprt suggests that the reduced xygen supply and flw rates encuntered in the septum are secndary t reduced xygen demand and that the septum is nt deprived f xygen under cntrl cnditins. In additin t the lw level f tensin develpment discussed abve, the slwer stimulus rate applied t the septum cmpared t the Langendrf preparatin are the prbable explanatins fr the lw metablic rate f the septum. The lw utilizatin rate fr xygen means that all metablic prcesses (glucse uptake and metablism, ATP hydrlysis and synthesis, NAD + reductin and subsequent xidatin, etc.) ccur at a much slwer rate than in viv r with ther experimental preparatins. If the metablic rate fr the septum were higher, it is pssible that the higher rate f lactate and hydrgen in prductin might inhibit glyclysis during demandinduced ischemia. Evidence against this pssibility is the presence f a well dcumented Pasteur effect during anxic perfusin in the high wrk Langendrf preparatin. Since each f the physilgical (cntrl and nrmxic increased wrk) and pathphysilgical states (demand-induced and flw-reduced ischemia) studied has a prprtinately reduced metablic rate, it is ur belief that the lw metablic rate shuld nt alter the directinal changes bserved in this study. Hwever, quantitative extraplatin f these results t the clinical situatin is nt warranted, and additinal studies in islated muscle preparatins perfrming mre wrk are currently being undertaken t further evaluate the effect f basal metablic rate n glyclysis during demand-induced and flw-reduced ischemia. In cnclusin, the results f this study suggest that the dependence f glucse metablism in ischemia n residual perfusin fr washut f metablic end prducts is als bserved when ischemia is prduced by excessive xygen demand in the islated arterially perfused rabbit septum perfused with glucse and insulin. These results will encurage investigatin int the use f FDG and psitrncmputed tmgraphy t evaluate the interplay f xygen supply and demand in the prductin f acute mycardial ischemia and infarctin in humans (Marshall et al., 1981). Acknwledgments We acknwledge the secretarial assistance f Victria Brden and the technical assistance f Ivan Whitehrn. References Bergmeyer H (1974) Methds f Enzymatic Analysis. New Yrk, Academic Press Dixn WJ, Brwn MB (eels) (1979) BMDP Bimedical Cmputer Prgrams P-Senes, University f Califrnia Press Gutman I, Whalefield AW (1974) In Methds f Enzymatic Analysis, edited by H Bergmeyer. New Yrk, Academic Press, p 1464 Katz J, Dunn A (1967) Glucse-2-t as a tracer f glucse metablism. Bichemistry 6: 1-5 Klcke FJ, Kiaser GA, Rss J Jr, Braunwald E (1965) Mechanism f increase f mycardial xygen uptake prduced by catechlamines. Am J Physil 209: Klcke FJ, Braunwald E, Rss J Jr (1966) Oxygen cst f electrical activatin f the heart. Circ Res 18: Klcke FJ, Kberstein RG, Pittman DE, Bunnell IL, Green DG, Rsig, DR (1968) Effects f hetergeneus mycardial perfusin n crnary venus Ha desaturatin curves. J Clin Invest 47: Langer GA, Serena SD (1970) Effects f strphanthidin upn cntractin and inic exchange in rabbit ventricular mycardium. J Ml Cell Cardil 1: Marshall RC, Schelhert HR, Phelps ME. 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9 648 CIRCULATION RESEARCH VOL. 49, N. 3, SEPTEMBER 1981 effects f anxia and whle heart ischemia n carbhydrate utilizatin. Circ Res 32: Rvett MJ, Lambertn WF, Neely JR (1975) Mechanisms f glyclytic inhibitin in ischemie rat hearts. Circ Res 37: Shine KI, Duglas AM (1974) Magnesium effects n rabbit ventricle. Am J Physil 227: Streeter D Jr, Sptnitz H, Patel D, Rss J Jr, Snnenbhck E (1969) Fiber rientatin in the canine left ventricle during diastle and systle. Circ Res 24: Streeter D Jr, Vasnar RN, Patel D, Sptnitz HM, Rss J Jr, Snnenblick EH (1970) Stress distributin in the canine left ventricle during diastle and systle. Biphys J 10: Zierler KL (1961) Thery f the use f arterial-venus cncentratin differences fr measuring metablism in stead> and nn-steady-states. J Clin Invest 40:

10 Glucse metablism during ischemia due t excessive xygen demand r altered crnary flw in the islated arterially perfused rabbit septum. R C Marshall, W W Nash, K I Shine, M E Phelps and N Ricchiuti Circ Res. 1981;49: di: /01.RES Circulatin Research is published by the American Heart Assciatin, 7272 Greenville Avenue, Dallas, TX Cpyright 1981 American Heart Assciatin, Inc. All rights reserved. Print ISSN: Online ISSN: The nline versin f this article, alng with updated infrmatin and services, is lcated n the Wrld Wide Web at: Permissins: Requests fr permissins t reprduce figures, tables, r prtins f articles riginally published in Circulatin Research can be btained via RightsLink, a service f the Cpyright Clearance Center, nt the Editrial Office. Once the nline versin f the published article fr which permissin is being requested is lcated, click Request Permissins in the middle clumn f the Web page under Services. Further infrmatin abut this prcess is available in the Permissins and Rights Questin and Answer dcument. Reprints: Infrmatin abut reprints can be fund nline at: Subscriptins: Infrmatin abut subscribing t Circulatin Research is nline at: