Total Ischemia in Dog Hearts, in Vitro

Transcription

1 901 Ttal Ischemia in Dg Hearts, in Vitr 2. High Energy Phsphate Depletin and Assciated Defects in Energy Metablism, Cell Vlume Regulatin, and Sarclemmal Integrity KEITH A. REIMER, ROBERT B. JENNINGS, AND MARY L. HILL SUMMARY This study was dne t assess the capacity f tissue, injured by varying perids f ttal ischemia, t perfrm integrated cellular functins. The principal aim was t learn the nature f the assciatins between the decreasing ATP f the ischemic tissue and the appearance f defective high energy phsphate regeneratin, cell vlume and in regulatin, and membrane permeability. Ttal ischemia in vitr was prduced by incubating papillary muscles frm dg hearts at 37 C. Slices were cut frm cntrl tissue and frm injured tissue after minutes f ttal ischemia. We incubated these slice in xygenated phsphate Krebs-Ringer phsphate buffer cntaining M C-inulin in rder t assess their capacity t resynthesize ATP and CP. t maintain in gradients and water cntent, and t retain membrane impermeability t inulin and creatine. The results demnstrate that there was a clse assciatin between ATP depletin and the failure f the damaged tissue t regenerate high energy phsphates, and t preserve cell vlume and inic regulatin. As lng as the ATP f the tissue was nt depleted belw 6 fiml/g dry weight prir t incubatin, n cellular abnrmalities were detected by subsequent aerbic incubatin f slices f the injured tissue. Hwever, lwer ATP levels were assciated with depressed high energy phsphate resynthesis and failure f cell vlume regulatin. These defects preceded the develpment f vert membrane damage, which ccurred nly after the tissue ATP cntent decreased t less than 2.0 pml/g. When vert membrane damage was present, it was assciated with marked Impairment f ther integrated cellular functins. Althugh the pathgenesis f the membrane damage in ischemia is unknwn, its presence is an bjective sign f lethal injury in this system. Ore Re* 49: , 19S1 THE nset f irreversible mycardial ischemic injury in viv is assciated with marked depletin f ATP and with breaks in the plasmalemma f the sarclemma (Jennings et. al., 1978; Jennings and Reimer, 1979). Incubatin f slices f the damaged tissue in vitr has demnstrated cncmitant lss f cell vlume regulatin, lss f the capacity t maintain nrmal inic gradients, and inability t exclude inulin frm the intracellular fluid (Gante et al., 1976; Jennings et al., 1978). Based n these bservatins, we have hypthesized that marked ATP depletin results in irreparable lss f membrane functin and thereby mediates the nset f irreversible injury (Jennings et al., 1978). In thery, cell vlume and inic regulatin culd fail either because insufficient ATP is available t fuel membrane inic pumps, because the pumps are damaged, r because altered membrane permeability permits mre rapid lss f inic gradients. The present study was designed t assess mre precisely the assciatins between tissue ATP cntent, lss f cell vlume and inic regulatin, and Frm the Department f Pathlgy f the Duke University Medical Center, Durham, Nrth Carlina. This study was supprted in part by Natinal Institutes f Health Grant R01 HL Addrew fr reprinu: Dr. Keith A. Reimer, Department f Pathlgy f the Duke University Medical Center, Durhiun, Nrth Carlina Received Nvember 7, I960; accepted fr publicatin April 16, altered membrane permeability. Because irreversible ischemic cell injury des nt develp simultaneusly thrughut an area f ischemia in viv, an in vitr mdel f ttal ischemia was emplyed t prduce large samples f unifrmly injured mycardium. Methds Ttal Ischemia, in Vitr Seven healthy mngrel dgs f either sex were anesthetized with intravenus sdium pentbarbital (30 mg/kg). The left side f the chest was pened and the heart was quickly excised. Blcks f muscle cntaining the anterir and psterir papillary muscle were cut frm the still beating heart. Part f ne papillary muscle which served as cntrl tissue was remved and drpped int ice-cld (0-l C) istnic KC1. The remaining "ttally ischemic" tissue was placed in a pre-warmed 2-unce jar which was then clsed and placed in a 37 C water bath. The ttal time elapsed frm beginning the chest incisin t beginning tissue incubatin was apprximately 30 secnds. Experiment Design After 60, 75, 90, 120, and 150 minutes f ttal ischemia, the ischemic muscle was briefly remved

2 902 CIRCULATION RESEARCH VOL. 49, N. 4, OCTOBER 1981 frm the water bath and slices were cut frm a prtin f the ischemic tissue fr analysis f metablites, electrlytes, and cell vlume cntrl (Figure 1). Ten thin slices (0.5 mm r less thick, mg) were cut with a hand-held razr blade (Grchwski et al., 1976), frm cntrl mycardium and frm ischemic tissue at each sampling time (Fig. 1). The first tw slices were weighed immediately n a Cahn mdel DTL micrbalance and transferred t icecld 3.6% perchlric acid (PCA) fr metablite assay (see belw). Tw additinal slices were weighed and transferred t rinsed, in-free vials (Cming minivials) fr analysis f ttal tissue water (TTW) and electrlytes (see belw). The remaining six slices were used fr analysis f cell vlume regulatin. They were incubated fr 1 hur in 15 ml f xygenated Krebs-Ringer phsphate (KRP) cntaining 30 mml mannitl, 1% bvine serum albumin (Pentax frm Miles Labratries), and trace quantities f H C-hydrxymethylinulin. The KRP was prepared fresh fr each experiment using analytic, reagent grade chemicals. The final medium was ph and cntained the fllwing ins, in millimles per liter Na +, 151.6; K +, 4.81; Ca 2+, 1.29; Mg^, 1.20; SO 4 2 ", 1.24; PO 4, 15.63, and Cl, The flasks cntaining the incubating slices were cntinuusly equilibrated with humidified 100% xygen and were shaken 180 times/min. At the end f incubatin, the slices were remved, dipped int 0.25 M sucrse t remve excess KRP, bltted n Whatman n. 1 filter paper, and weighed. Tw slices were used fr analysis f electrlytes, tw fr metablites, and tw fr the inulin diffusible space (IDS) (Fig. 1). Metablites Fr metablite assays, the slices in PCA were hmgenized with a Tri-R hmgenizer and neu- DURATIONOF TOTAL ISCHEMIA.IN VITRCUMin) Metablites (2 Slices) Metablites (2 Slices) One Hur Incubatin (6 Slices) I OS (2 Slices) Electrlytes (2 Slices) Electrlytes (2 Slices) FIGURE 1 The experimental design is diagrammed. See Methds fr explanatin. tralized t ph with KzCO, and KOH. The extracts were centrifuged t remve KCIO4 and the supernatant was frzen at 70 C. Samples were subsequently assayed by enzymatic techniques fr lactate (Lwry and Passneau, 1972), adensine triphsphate (ATP) (Lamprecht and Trautschld, 1974), adensine diphsphate (ADP) (Jawuk et al., 1974), adensine mnphsphate (AMP) (Jawuk et al., 1974), creatine phsphate (CP) (Lamprecht and Trautschld, 1974), creatine (Bemt et al., 1974), and glucse-6-phsphate (G6P) (Lamprecht and Trautschld, 1974). Ttal adenine nucletides (XAd) were calculated as ATP + ADP + AMP, ttal creatine (2Cr) was CP + creatine, ttal high energy phsphates (HEP) were CP + ATP, and the adenylate charge was (ATP + V4ADP)/ SAd (Atkinsn, 1968). [Nte that this definitin f HEP differs frm that used in the first paper f this series in which the ttal number f high energy phsphate bnds was = CP + (2 X ATP) + ADP (Jennings et al., 1981).] Slices used fr analysis f electrlytes r IDS were dried fr 6-24 hurs at 105 C. The dry slices were reweighed and the TTW calculated as ml H2O/ 100 g dry tissue. Electrlytes Electrlytes were extracted frm each dried slice in 5 ml f 0.75 N HNO3 accrding t techniques described previusly (Jennings et al., 1957; Jennings et al., 1970). Briefly, Mg 2 *, Na +, and K + were determined in a 1 t 100 dilutin cntaining 5000 ppm f RbCl. Ca 2+ was determined in a 1 t 10 dilutin cntaining 10,000 ppm La 3+. These dilutins were dne in in-free glassware, verified prir t use t cntain n detectable sdium. All fur electrlytes were measured in an IL 351 atmic absrptin spectrphtmeter interfaced with a Tektrnix mdel 31 cmputer calculatr. Standard curves were prepared fr all fur ins, and tw water standards equivalent t 100 mg f mycardium and tw reagent blanks were analyzed with every series f unknwns. IDS The dry slices used t measure the IDS were rehydrated by the additin f ne r tw drps f deinized water, and were slubilized in Sluene 350 (Packard). U C activity was cunted in a Packard liquid scintillatin cunter. The IDS was calculated as ml H2O/IOO g dry tissue (Grchwski et al., 1976). Statistics All data are given as means ± the standard errr f the mean. Statistical cmparisns were made using a tw-tailed paired t-test between cntrl and ischemic samples frm each heart. The average f results frm duplicate slices were U3ed as single data pints.

3 HEP, ION GRADIENTS, AND MEMBRANE DAMAGE IN ISCHEMIA/Reimer et al. 903 Results Effect f Ischemia n High Energy Phsphate and Other Metablites Rapid degradatin f high energy phsphates ccurred even during the brief perid required t cut and cl cntrl samples. Cntrl mycardium cntained 29.2 ± 0.8 /unl/g dry weight f high energy phsphates (HEP) f which 24.2 ± 0.7 was ATP and 5.0 ± 0.3 was CP (Table 1). This cntrl ATP is similar t ATP levels btained by rapidfreezing techniques (Braasch et al., 1968), but the CP levels are much less, Presumably, the relative preservatin f ATP during the initial sampling perid was achieved by the cnversin f CP t ATP via the creatine kinase reactin. During ttal ischemia, there was further prgressive depletin f HEP s that by 90 minutes, nly 7.5% f the cntrl HEP remained (Table 1). Creatine prduced frm degradatin f CP was nt further metablized; thus, ttal creatine remained cnstant thrughut 150 minutes f ischemia (Table 1). In cntrast, the degradatin f ATP was assciated with a cncmitant degradatin f ttal adenine nucletides (Table 1; Fig. 2). Initially, ADP decreased slwly and AMP was relatively cnstant, indicating that bth cmpunds were being utilized r further degraded as rapidly as they were prduced. Hwever, after 90 minutes f ischemia, when the rate f ATP and SAd degradatin was slwing, the AMP f the tissue increased 2.7 times ver that fund at 60 minutes. By 2 hurs f ischemia, virtually n ATP (1.6%) remained and ADP was 43% f cntrl. At this time, the rate f 2Ad destructin had slwed greatly and AMP had becme the nucletide in highest cncentratin in the tissue. Changes in High Energy Phsphates and Other Metablites in Incubated Slices Preparatin and incubatin f slices f either cntrl r ischemic mycardium in xygenated me- TABLE 1 Effects f Ttal Ischemia n High Energy Phsphates and Metablites Duratin f ttal ischemia (mini Cntrl HEP ± :1 ± * ± * ± } ± } ±0.12 CP ATP ADP AMP Adenylate charge ZAd 4.97 ± ± ± ± ± ± } ± $ ± f ± ± t ± ± t ± }: ± ± ± ± ± } ± $ ± } ± * ± } ± ± } ± } ± } ± } ± } ± ± } ± } ± } ± } ± } Creatine Cr 91.0 ± ± ± ±2.9 Abbreviatins are as fllws: CP creatine phsphate; HEP ttal high energy phsphate CP 4- ATP, - ttal adenine nucletide* - ATP + ADP + AMP; adenylate charge = (ATP + 05 ADP)/ AD; CR - ttal creatine - CP + creatine. All data are expressed m junl/g dry weight ± SEM. Data frm each heart were based n duplicate measurements btained frm the ttally ischemic tissue f six f the seven dgs in the study Tissue frm ne dg was lst during prcessing. The number f hearts studied at each time is shwn in parentheses. Statistical cmparisns were made between cntrl and ischemic tissue frm the same heart* using a paired (-test. P<0OS;fP<001;4:P< 0001.

4 904 CIRCULATION RESEARCH VOL. 49, N. 4, OCTOBER 1981 hi en E JJ i _?^ N XAd s ATP ' v ADP ^ 'k ' ^ ~ ~ *" ~ * AMP, Jr'^ 1 "Y * Minutes f Ttal Ischemia - a 150 FIGURE 2 77ie prgressive degradatin f ttal adenine nucletides (SLAd) in parallel with the depletin f ATP during ttal ischemia is illustrated. Adensine diphsphate (ADP) cntent decreased slwly thrughut 150 minutes f ttal ischemia. Adensine mnphsphate (AMP) cntent was unchanged at 60 minutes but increased between 60 and 120 minutes, suggesting either that 5'-nucletidase was becming inhibited r that AMP was being sequestered in a cmpartment inaccessible t this enzyme. dia resulted in the lss f a prtin f the ttal creatine and adenine nucletides present befre incubatin, presumably because f washut frm damaged cells n the edges f the slices (Tables 1 and 2; Fig. 3). Adenine nucletide lss was disprprtinately greater frm cntrl than frm ischemic slices. The cause f this disprprtinate lss is unknwn. Previus studies have shwn that this lss ccurs during the first 5 minutes f incubatin (Jennings et al., unpublished bservatins). It may be due t accelerated prductin f AMP and adensine (ADO), with lss f the ADO by diffusin frm the cld slice int the medium during the initial equilibratin and warmup t 37 C. The ttal nucletide cntent f incubated slices, either cntrl r injured, was never greater than that present prir t incubatin. Thus, n detectable de nv synthesis f adenine nucletides ccurred during aerbic slice incubatin. Nevertheless, resumptin f aerbic metablism was assciated with restratin f the adenylate charge and with resynthesis f significant quantities f CP in cntrl slices r in slices ischemic fr nly 60 minutes (Tables 1 and 2). Hwever, with prgressively lnger perids f ischemic injury, slices were nt able t fully re-energize even the small quantities f ADP and AMP remaining. Thus, the adenylate charge was 0.6 r less after 75 r mre minutes f ischemia (Table 2) and was assciated with a great reductin in the capacity f the heart t resynthesize CP (Table 2; Fig. 4). The failure f slices t re-synthesize creatine phsphate during aerbic incubatin was related clsely t the ATP cntent f the ischemic tissue prir t incubatin (Fig. 5). As lng as the tissue ATP was >5.0 ^ml/g dry, CP synthesis was equivalent t that f cntrl shces. Cnversely, an initial ATP cntent f 2.5 r less invariably was assciated with depressed CP resynthesis. The lw energy charge and depressed creatine phsphate synthesis is indicative f failure f glyclytic r mitchndrial energy prductin and/r transprt. The high slice cntents f G6P and lactate suggest that anaerbic glyclysis was still ccurring under aerbic cnditins and that mitchndrial functin was partially r cmpletely inhibited especially in slices f tissue injured by 90 r mre minutes f ttal ischemia. Exhaustin f supplies f substrate seems an unlikely explanatin fr the apparent mitchndrial failure. It previusly has been shwn that glycgen is still present in ttally ischemic tissue at these times. Als lactate, lng chain acyl-c A (Neely and Feuvray, 1981), fatty acids, and pyruvate all accumulate in ischemic tissue and are available as endgenus mitchndrial substrates. Finally, the fact that the lactate cntent f the slices was increased after 60, 75, 90, and 120 minutes f ischemia suggests that the mitchndria were unable t metablize lactate and/ r pyruvate. The calcium cntent f all slices was increased cmpared t the intact tissue, presumably because f calcium entry int damaged cells n the slice surface (Table 3). The average slice calcium cntent was higher in slices with an adenylate charge f <0.75 than in shces with higher adenylate charge. Hwever, the pint scatter was t great t either substantiate r rule ut calcium verlad as a cause f mitchndrial failure. Changes in Cellular Membrane Permeability in Incubated Slices Inulin with a mlecular weight f 5000 is excluded frm nrmal cells. Thus, the inulin diffusable space (IDS) f incubated slices is a measure f the extracellular space plus the intracellular space f damaged cells which have permitted the entry f inulin mlecules. Cntrl mycardium had an IDS f 94 ± 4 ml/100 g which was 30% f the ttal tissue water. The IDS was unchanged after 60 minutes f ischemic injury but prgressively increased with lnger perids f injury, reaching 220 ml/100 g at 150 minutes (Table 3). The latter IDS was 54% f the TTW. Because electrn micrscpy f such slices has nt demnstrated interstitial edema (Gante et al., 1976; Jennings et al., 1978), this increased IDS indicated that inulin entered pr-

5 HEP, ION GRADIENTS, AND MEMBRANE DAMAGE IN ISCHEMIA/Reimer et al. 905 TABLE 2 Effect f Ttal Ischemia and Subsequent Slice Incubatin n High Energy Phsphates and Metablites f Seven Dgs Duratin f ttal ischemia (mm) prir t incubatin f tifleue slices HEP ± * ± * ± $ ± $ ± $ ±0.42 CP 30.9 ± ± ± $: ± $ ± $ ±0.41 ATP ± $ ± $ ± $ ± $ ± $ ±0.19 ADP 283 ± f ± * ± $ ± ) 2.02$ ± * ±0.33 AMP 0.44 ± ± ± ± ± ±0.08 Adenylate charge 0.89 ± :): ± * ± $ ± $ ± $ ±0.05 Ad ± : ± f ± $ ± $ ± $ ±0.49 Creatine ZCr Lactate G6P 18.6 ± ± ± ± $ ± t ± ± t ± $ ± * ± f ± * ± * ± ± $ ± ' ± $ ±3,1 21.4t ± $ ± $ ±1.6 Abbreviatins are thse used in Table 1 G6P glucse-6-phtwphate Data frm each heart were the mean f measurements frm tw slices at each time The number f hearts studied at each time is shwn in parentheses. Statistical cmparisns were made between cntrl and iachemic slices frm the same heartk ufiing a paired -test P < 0.05, } P< 0.01; $ P < gressively mre cells as the duratin f injury was increased. Althugh the exclusin f inulin frm 46% f the TTW after 150 minutes f ischemia culd have been due t the preservatin f many mycytes r lack f injury t interstitial cells and capillaries, it seems much mre likely that the IDS underestimated the number f cells with plasmalemmal defects. The inulin space reaches equilibrium after minutes f incubatin in slices f nrmal tissue (Grchwski et al., 1976). Hwever, it seems likely that it will take lnger t equilibrate with the intracellular water thrugh fci f plasrnalernmal damage, such that equilibratin might still be incmplete at the end f the 60-minute incubatin perid. Als, the intracellular inulin may be excluded frm mitchndria, which ccupy abut 30% f the vlume f the dg mycyte, and/ r nuclei. Such cmpartmentatin als wuld explain why the IDS did nt apprach the TTW. Creatine lss frm slices t the incubatin medium prvided a secnd measure f altered membrane permeability. Cntrl slices and slices f mycardium injured by 60 minutes f ischemia retained mre than 50% f the ttal creatine present in the tissue prir t slice incubatin. With lnger perids f injury, mre creatine was lst frm the slices t the medium and, by 150 minutes, slices retained nly 12% f their riginal ttal creatine (Tables 1 and 2). Creatine lss and inulin influx bth were detected simultaneusly after 90 minutes

6 906 CIRCULATION RESEARCH VOL. 49, N. 4, OCTOBER 1981 = * 20- B 15 Cntrl Isctinnic Ttal Adenine Nucletide Cnlent f Whle Tissue (ju.ml/q dry weight) FIGURE 3 The ttal adenine nucletide cntents f mycardium are cmpared befre incubatin vs. after slice incubatin. The dashed line is the unity line. Data are pltted irrespective f the duratin f ttal ischemia. Sme lss f adenine nucletides ccurred during slice incubatin. This lss was disprprtinately greater in cntrl slices than in slices f tissue injured by varius perids f ttal ischemia. The adenine nucletide lss frm cntrl slices ccurs during the first 5 minutes f incubatin and is mst likely due t catablism f AMP with washut f adensine during the warm-up f the cld-preserved tissue. In additin, adenine nucletides undubtedly were lst frm cut cells n the surfaces f all slices. f ischemia and, within individual slices, there was a clse inverse assciatin between the tw events (Fig. 6). In tw f seven experiments, creatine lss may have preceded inulin entry, but the tw markers f increased membrane permeability were detected cncurrently in the ther five experiments. Thus, increased membrane permeability t the small 135 ml. wt. creatine mlecule was, at mst, a brief transitinal state which was fllwed by the apparently abrupt develpment f defects large enugh t admit the large (5000 ml. wt) inulin mlecule. Bth indices f membrane permeability became abnrmal in later samples and at lwer tissue ATP levels, cmpared t the develpment f abnrmal indices f energy prductin. Increased membrane permeability t these markers was nt bserved in any slice with mre than 2.5 juml/g f ATP, and this evidence f membrane damage was nt invariably detected, even at ATP levels f less than 1.0 /iml/g (Fig. 7). All slices with increased membrane permeability had markedly depressed CP synthesis. Cnversely, membrane impermeability t creatine and inulin still was present in a few slices with a markedly reduced capacity t synthesize CP (Fig. 8). Changes in Cell Vlume Regulatin in Incubated Slices Slices f cntrl mycardium maintained relatively nrmal water and electrlyte cntents (Table 3). There was a mdest gain f sdium and calcium and a mdest lss f ptassium and magnesium which was expected because f damaged r cut cells n the surfaces f the slices. Slices f mycardium injured by up t 75 minutes f ischemia were able t maintain electrlyte cntents similar t thse f cntrl slices. Hwever, after 90 r mre minutes f ischemia, prgressive lss f cell vlume and inic regulatin ccurred, manifested by a prgressive influx f Na +, Ca 2+, and water, and by lss f K + and Mg* + (Table 3). By 150 minutes, K + was reduced by 73% and Ca 2+ was increased by 56% cmpared t cntrl slices. The sdium cntent f these slices indicated nearly cmplete equilibratin with the sdium cncentratin f the medium (0.152 mml Na + /ml medium X 405 ml H 2 O/100 g dry weight 63 mml Na + /100 g dry weight expected fr cmplete equilibratin). Either vert plasmalemmal damage r insufficient HEP prductin t run the membrane pumps (e.g., Na + /K + ATPase) culd cause defective cell vlume regulatin. Since electrlytes f the media shuld have ready access t the sarcplasm f cells with membrane defects, we expected t find and fund that every slice with an increased IDS als demnstrated an increased Na + and water cntent ADENYLATE CHARGE VS CP IN INCUBATED SLICES I ) 5 10 i Creatine Phsphate iiml/g dry Height 1 Cntrl licktmic 1 i FIGURE 4 The capacity f cntrl r injured slices t resume HEP prductin during incubatin was indicated by creatine phsphate resynthesis and by the adenylate charge rati. The dashed lines mark tw standard deviatins belw the cntrl means f these indices, and the data are pltted irrespective f the duratin f ttal ischemia. The adenylate charge in this cntext represents the capacity f the adenine nucletide pl remaining in the tissue t resynthesize ATP. If synthesis were cmplete, the adenylate charge wuld be 1.0, whereas slices cntaining nly AMP wuld have a charge fo. Synthesis f CP frm creatine indicates that net ATP is available, that creatine kinase is functinal, and that all substrates and cfactrs fr the reactin, including Mg 2 *, ADP, and creatine, are present. Nte that an adenylate charge rati f less than 0.75 always was assciated with a reduced capacity t resynthesize CP.

7 HEP, ION GRADIENTS, AND MEMBRANE DAMAGE IN ISCHEMIA/Reimer et al Ccfllrl hchemic 20- c 200 i -Cntrl - Ischemic : i i i ATP ji ml/q dry FIGURE 5 The capacity f cntrl r injured slices t regenerate high energy phsphates during incubatin, as measured by the slice CP cntent, is cmpared t the tissue ATP cntent prir t incubatin. The dashed lines mark tw standard deviatins belw the mean cntrl CP cntent. The data are pltted irrespective f the duratin f ttal ischemia. Regeneratin f high energy phsphates t cntrl r near cntrl levels ccurred in all slices frm tissue with an initial ATP f at least 5.0 liml/g dry weight, whereas energy metablism was defective when tissue ATP was <5.0 unl/g dry. Hwever, sme slices with a relatively nrmal IDS shwed a disprprtinately high sdium cntent (Fig. 9). These bservatins are the principal evidence that defective cell vlume regulatin preceded the develpment f vert membrane damage. 150 ( Ttal Crelme fi mles/g dry weight FIGURE 6 Membrane damage was indicated by increased permeability t inulin and creatine. The dashed lines mark tw standard deviatins abve r belw the cntrl mean IDS r ttal creatine cntent, respectively. The data are pltted irrespective f the duratin f ttal ischemia. There was a clse inverse assciatin betu>een an increased inulin space and creatine lss as tw indices f increased cell membrane permeability. Als, there was a clse assciatin between increased slice sdium cntent and decreased adenylate charge rati (Fig. 9), which indicates that failure t preserve cell vlume and inic gradients always was assciated with failure t restre HEP prductin. Lss f vlume cntrl always ccurred TABLE 3 Effect f Ischemia and Subsequent Slice Incubatin n Ttal Tissue Water, Inulin Diffusable Space, and Electrlytes TTW IDS Na + K + Mg 2 * Cntrl uruncu bated tissue 351 ± ± ± ± ±4 94 ± ± ± ±0.11 Duratin f ttal ischemia (minii pnr t incubatin f tiaeue slices $ ±7 91 ± ± ± ± $ ±6 102 ± ± ± ± f ±8 125* ± * ± * ± * ± $ ±6 ia3t ±15 53.lt ± $ ± $ ± $ ±9 220$ ± $ ± $ ± $ ±0.14 Ca ± ± ± ± ± * ± * ±0.13 Effect f ischemia and subsequent slice incubatin n ttal tissue water, inulin diffusible space, and electrlytes. The ttal tissue water (TTW) and inulin diffusible space (IDS) are expressed in ml/100 g dry tissue and electrlytes in mml/100 g dry useue plue r minus the standard errr f the mean. Data frm each heart were based n measurements frm tw slicesfrmeach time except fr the TTW, which was measured in fur slices at each time (the tw used fr the IDS determinatin and the tw used fr electrlyte determinatin). The number f hearts studied at each time is shwn in parentheses. Statistical cmparisns were made between incubated cntrl (0 time) and iflchemic slices frm the same hearts using a paired (-test. * P < 0.05; $ P < 0.01; P <

8 908 C I R C U L A T I O N R E S E A R C H V O L. 4 9, N. 4, O C T O B E R w h e n H E P p r d u c t i n h a d f a i l e d, w h e t h e r r n t v e r t p l a s m a l e m m a l d a m a g e w a s d e m n s t r a b l e. H w e v e r, l s s f i n i c g r a d i e n t s w a s m s t s e v e r e w h e n p l a s m a l e m m a l d a m a g e a l s w a s p r e s e n t. Discussin The results shw that dg cardiac mycytes injured by ttal ischemia in vitr develp virtually identical signs f injury t thse previusly bserved in severe ischemia in viv (Gante et al., 1976; Jennings et al., 1978). This evidence f severe injury includes defective cntrl f cell vlume and in gradients, signs f cell membrane damage, destructin f the 2Ad pl, and cessatin f energy prductin by anaerbic glyclysis. The marked depletin f ATP and cell membrane damage have been identified as signs f lethal injury in in viv ischemia (Jennings et al., 1978). Their appearance in the same sequence during ttal ischemia in vitr (Jennings et al., 1981) indicates that this is a satisfactry mdel in which t study the bichemical, functinal, and structural changes ccurring as cells becme lethally injured. In the studies presented in this paper, ur aim was t assess the relatinships amng the depletin t-200 k - f t as^ 150 "5 " i F _ * m 10 * * 1 i * -Cntrl -IsCriermc V 1 1 I ATP u. ml/fj dry FIGURE 7 The capacity f cntrl r injured slices t exclude inulin r retain creatine is cmpared t the A TP cntent f the tirftue prir t incubatin. The dashed lines mark tw standard deviatins abve and belw the cntrl mean IDS and ttal creatine cntent, respectively. The data are pltted irrespective f the duratin f ttal ischemia. Overt membrane damage, as indicated by increased permeability t these tw markers, did nt develp until the tissue ATP cntent was less than 2.0 jxnwl/g dry weight. Thus vert membrane damage was manifest later than the defective regeneratin f HEP (cmpare with Fig. 8) Creatine Phsphate p. mles /q dry weight FIGURE 8 The capacity f cntrl r injured slices t regenerate CP is cmpared t the tw indices f membrane damage, i.e., the IDS and ttal creatine cntent. The dashed lines mark tw standard deviatins frm the cntrl means f the three parameters and the data are pltted irrespective f the duratin f ischemia. The capacity f slices t regenerate CP was reduced in seven slices in which membrane permeability was nt yet increased. f ATP, the capacity t resynthesize HEP, cell vlume regulatin, and cell membrane damage. The relatinships have prved t be cmplex. The varius facets will be discussed in the next few sectins- Changes in Mitchndrial Functin, Cell Vlume, and In Regulatin in Incubated Slices Slices f mycardium, injured by 90 minutes r mre f ttal ischemia and then incubated in an aerbic medium, were nt able t resynthesize HEP cmpunds r t regulate cell vlume r inic gradients, and, subsequently, the cell membrane became permeable t inulin and creatine. The failure t resynthesize HEP r regulate inic gradients ccurred 15 t 30 minutes earlier and at higher levels f ATP cmpared t the develpment f membrane permeability alteratins. The failure f slices t restre the adenylate charge rati abve 0.75 indicates that the slices cannt cnvert the available adenylate pl t ATP despite the resumptin f aerbic metablism and represents indirect evidence f failure f mitchndrial ATP prductin, translcatin, r bth. Further supprt fr this cncept is prvided by the

9 HEP, ION GRADIENTS, AND MEMBRANE DAMAGE IN ISCHEMIA/Reimer et al tin f slices f tissue damaged by 90 r mre minutes f ttal ischemia is mst likely a reflectin f the reduced adenylate charge in the tissue and, as such, wuld be a secndary reflectin f mitchndrial failure. High energy phsphate bnds are transferred between CP and ATP via the creatine kinase (CK) reactin: _ 8 -AUwyltt Chrge <0.75. Charge ^0-75 i JDS ml/loog FIGURE 9 The capacity f cntrl r injured slices t exclude sdium is cmpared with the capacity t exclude inulin. The dashed lines mark tw standard deviatins abve the cntrl means f the parameter a. The data are pltted irrespective f the duratin f ttal ischemia. Sdium cntent was increased in sme slices that still had nrmal mulin diffusible spaces, indicating that defective sdium transprt r increased permeability t sdium, preceded the develpment f vert membrane damage. The relatinship between the capacity t resynthesize HEP as manifested by an adenylate charge >0.75 and a nrmal Na* cntent is clearly demnstrated. bservatin that anaerbic glyclysis alne cannt prvide enugh ATP t maintain the adenylate charge and t synthesize CP in anxic slices f cntrl left ventricle (Jennings et al., unpublished bservatins). The cause f the depressed mitchndrial functin was nt established in the present study but culd be due t ne r mre f several general pssibilities: (1) the lss f substrate(s) r cfactr(s) essential fr aerbic xidative phsphrylatin, (2) inhibitin r destructin f mitchndrial creatine kinase and adenine nucletide translcase respnsible fr translcatin f HEP between mitchndria and sarcplasm, (3) damage t the structural integrity f the mitchndria, r the presence f excess cytplasmic calcium and cnsequent mitchndrial transprt f Ca 2+ in lieu f ATP prductin. Althugh substrates were nt added t the incubating medium in this study, the high lactate cntent f ischemic slices shuld have been metablizable by nrmal mitchndria. Mrever, the fact that slice lactate cntent remained increased after an hur f incubatin indicates that glyclysis was ccurring and that aerbic utilizatin f this substrate was depressed. Althugh calcium cntent was increased t 1.3 mml/100 g r mre in all mycardial slices with a depressed adenylate charge rati, the pint scatter was t great t either supprt r disprve a causal relatinship between the tw parameters. The lss f CP synthesis during aerbic incuba- dry CP + ADP CK Creatine + ATP. During ischemia, ADP, prduced by the breakdwn f ATP, is available and the equilibrium f the CK reactin is t the right. Thus, CP is cnverted rapidly t ATP. Hwever, with the resumptin f nrmal aerbic metablism, much f the ADP is rapidly recharged t ATP and the synthesis f CP is favred. The bserved failure t resynthesize CP reflects either lss f CK activity, insufficient ATP r creatine, r a relatively high rati f ADP t ATP in ne r mre cmpartments f the mycyte. Since CP synthesis failed in sme slices in which membrane permeability t inulin r creatine was nt yet increased, i.e., befre washut f creatine had ccurred, the initial failure f CP resynthesis evidently was nt due t creatine lss. CK lss was nt measured in the present studies, but it seems unlikely that it wuld precede the develpment f increased permeability t the much smaller creatine mlecule. There are at least three pssible mechanisms fr the lss f regulatin f cell vlume and inic gradients fllwing ischemic injury. (1) Increased permeability t ins may have caused excessive backleaks f ins such that the membrane pumps were verwhelmed. (2) There may have been insufficient HEP available t supprt the required rate f in transprt. (3) There may have been damage t the membrane bund in pumps, per se. Slice electrlyte cntents always were deranged mst severely when there were als marked increases in membrane permeability t inulin and creatine. In the presence f this evidence f abnrmal membrane functin, severe derangements in cell vlume and in regulatin were expected. Hwever, abnrmal slice electrlyte cntent always became detectable minutes befre vert membrane damage was detectable by changes in inulin r creatine. This suggests that cntrl f in gradients was altered prir t the appearance f structural damage t the sarclemma. Thus, increased permeability t electrlytes may have preceded the develpment f increased permeability t inulin and cntributed t the failure f cell vlume regulatin. The appearance f marked alteratins in cell vlume regulatin cincided precisely with the nset f the failure f the slices t resynthesize high energy phsphates. The clse linkage between these tw events has at least tw pssible explanatins: (1) Inic transprt ccurs via pumps, including-the sarclemmal Na + /K + ATPase, which require ATP

10 910 CIRCULATION RESEARCH VOL. 49, N. 4, OCTOBER 1981 t functin (Sku, 1965; Hkin, 1976). The inability f the cell t resynthesize ATP bviusly wuld impair cell vlume and inic regulatin because f the absence f a surce f energy t drive the pump r pumps. (2) Cnversely, lss f inic gradients and, specifically, the presence f excess Ca 2+ in the cell culd uncuple xidative phsphrylatin and cause subsequent mitchndrial dysfunctin (Lehninger, 1974, Nayler, 1981). In ther wrds, mitchndrial dysfunctin culd cause lss f cell in transprt and/r failure f in transprt culd result in mitchndrial dysfunctin. Which ne f these events precedes and causes the ther cannt be determined frm the present study. Once either mechanism appeared, a vicius cycle wuld ensue in which inadequate ATP prductin wuld cause failure t remve intracellular Ca 2+, and in turn, this wuld further depress ATP prductin. Lss f cell vlume regulatin als culd result frm damage t the membrane pumps, per se. Hwever, ther studies have shwn that at least the sarclemmal Na + /K + ATPase is a stable enzyme cmplex which is relatively resistent t ischemic injury (Schwartz et al., 1973; Beller et al., 1976; Willersn et al., 1977). Membrane Permeability Changes in Incubated Slices The develpment f vert membrane damage, as evidenced by permeability t creatine r inulin, ccurred relatively late in these studies. This membrane damage never was bserved until the ATP f the tissue had fallen belw 2.0 jiml/g dry weight and was nt invariably present at tissue ATP levels f 1.0 /nml/g. Increased membrane permeability always was assciated with severe defects in mitchndrial functin and cell vlume and inic regulatin, but the latter defects ccasinally were present in slices that still demnstrated intact membranes. Thus, increased sarclemmal permeability invariably was assciated with signs f lethal injury (Jennings et al., 1978) but the metablic failure f lethal injury was nt invariably assciated with increased membrane permeability. This bservatin suggests that a metablic deficit must be present fr a perid f time befre sarclemmal disruptin is detectable. The pathgenesis f the sarclemmal defects is unknwn. Small breaks in the plasmalemma f the sarclemma are detectable by electrn micrscpy (Jennings et al., 1978) in severely ischemic cells in viv, and in ttal ischemia in vitr when the EDS is markedly elevated (Jennings and Hawkins, 1980). Thus, the increased membrane permeability t inulin ifi apparently caused by these structural changes. We have previusly pstulated that membrane damage might develp when ATP levels becme inadequate t phsphrylate membrane prteins, with resultant activatin f membrane phsphlipases (Jennings and Reimer, 1981). Hwever, the present results shw that membrane breaks d nt necessarily ccur quickly fllwing severe ATP depletin. If the defects are related t ATP depletin, the reasn fr the variable perid f time during which the ATP must be markedly depressed befre defects develp is nt knwn. In additin t the ptential effects f ATP depletin n phsphlipase activatin, ther mechanisms f membrane damage are pssible in severe r ttal ischemia. These include (1) the effect f cell swelling n the sarclemma due t lss f cell vlume regulatin, (2) release f phsphlipases frm lyssmes, and (3) lipperxidatin. In cnclusin, the results f these studies demnstrate that the events assciated with the nset f irreversible injury in viv als develp in ttal ischemia in vitr. The slwer time curse f injury and the larger amunt f unifrmly injured tissue prduced by ttal ischemia have allwed a mre precise cmparisn f the duratin f ischemic injury vs. severity f ATP depletin, failure f cell vlume and inic regulatin, develpment f vert membrane damage, and the develpment f mitchndrial dysfunctin. A decreased capacity t regenerate high energy phsphates develps at variable times, depending n the rate f HEP depletin. This metablic failure ccurs relatively early, when ATP levels in the tissue are as high as 5.0 juml/g dry weight. Cell vlume and inic regulatin fail simultaneusly. Each f these defects prbably cntributes t the severity f the ther. It is significant that vert membrane damage has been shwn t be a later event in ischemic injury, the pathgenesis f which remains unknwn. It may be mediated either by the marked lss f ATP, per se, the lss f cell vlume regulatin, r ther events such as activatin f endgenus phsphlipases f the mycyte. Acknwledgments Technical assistance was prvided by Nancy J. Kramer and Sherry L. Mntgmery. References Atkinsn DE (1968) The energy charge f the adenylate pl as a regulatry parameter: Interactin with feedback mdifiers. Bichemistry 7: Beller GA, Cnry J, Smith TW (1976) Ischemia-induced alteratins in mycardial (Na* + K + )-ATPas«and cardiac glycside binding. J Clin Invest 57: Bemt E, Bergmeyer HU, Mllering H (1974) Creatine. In Methds f Enzymatic Analysis, edited by HU Bergmeyer. New Yrk, Academic Press, pp Braasch W, Gudbjarnasn S, Puri PS, Ravens KG, Bing RJ (1968) Early changes in energy metablism in the mycardium fllwing acute crnary artery cclusin in anesthetized dgs. Circ Res 23: Gante CE, Jennings RB, Hill ML, Grchwski EC (1976) Experimental mycardia] ischemic injury. II. Effect f in viv ischemia n dg heart slice functin in vitr. J Ml Cell Cardil 8: Grchwski EC, Gante CE, HOI ML, Jennings RB (1976) Experimental mycardial ischemic injury. L A cmparisn f Stadie-Riggs and free-hand slicing techniques n tiisue ultra-

11 HEP, ION GRADIENTS, AND MEMBRANE DAMAGE IN ISCHEMIA/Reimer et al. 911 structure, water and electrlytes during in vitr incubatin. J Ml Cell Cardil 8: Hkin LE (1976) The mlecular machine fr driving the cupled transprts f Na + and K + is an (Na + + K + )-activated ATPase. Trend* Bichem Sci 1: Jawuk D, Gruber W, Bergmeyer HU (1974) Adeneine-5'-diphsphate and adensine-5'-mnphsphate. In Methds f Enzymatic Analysis, edited by HU Bergmeyer. New Yrk, Academic Press, pp Jennings, RB, Hawkins HK (1980) Ultrastructural changes f acute mycardial ischemia. In Degradative Prcesses in Heart and Skeletal Muscle, edited by K WUdenthal. Amsterdam, Elsevier/Nrth Hlland, pp Jennings RB, Reimer KA (1979) Bilgy f experimental, acute mycardial ischemia and infarctin. In Enzymes in Cardilgy: Diagnsis and Research, edited by DJ Hearse, J DeLeiris. New Yrk, Jhn Wiley & Sns, pp Jennings RB, Reimer KA (1981) Lethal mycardial ischerruc injury. Am J Pathl 102: Jennings RB, Crut JR, Smetters GW (1957) Studies n distributin and lcalizatin f ptassium in early mycardial ischemic injury. Arch Pathl 83: Jennings RB, Mre CB, Shen AC, Herdsn PB (1970) Electrlytes f damaged mycardial mitchndria. Prc Sc Exp Bil Med 135: Jennings RB, Hawkins HK, Lwe JE, Hill ML, Kltman S, Reimer KA (1978) Relatin between high energy phsphate and lethal injury in mycardial ischemia in the dg. Am J Pathl 92: Jennings RB, Reimer KA, Hill ML, Mayer SE (1981) Ttal ischemia in dg hearts in vitr 1. Cmparisn f high energy phsphate prductin, utilizatin, and depletin f adenine nucletide catablism in ttal ischemia in vitr vs. severe ischemia in viv. Circ Res 49: Lamprecht W, Trautschld I (1974) Determinatin f ATP with hexkinase and glucse-6-phsphate dehydrgenase. In Methds f Enzymatic Analysis, edited by HU Bergmeyer- New Yrk, Academic Press, pp Lehninger AL (1974) Ca 2+ transprt by mitchndria and its pssible rle in the cardiac cntractin-relaxatin cycle. Circ Res 34/36 (suppl in): Lwry OH, Passnneau JR (1972) Measurement f enzyme activities with pyridine nucletides. In A Flexible System f Enzymatic Analysis. New Yrk, Academic Press, pp Nayler WG (1981) The rle f calcium in the lschemic mycardium. Am J Pathl 102: Neely JR, Feuvray D (1981) Metablic prducts and mycardial ischemia. Am J Pathl 102: Schwartz A, Wd JM, Allen JC, Barnet EP, Entman ML, Gldstein MA, Srdahl LA, Suzulri M (1973) Bichemical and mrphlgic crrelates f cardiac ischemia. I. Membrane systems. Am J Cardil 32: Sku JC (1965) Enzymatic basis fr active transprt f Na + and K + acrss cell membrane. Physil Rev 45; Wdlersn J, Scalee F, Mukherjee. A, Platt M, Templetn G, Fink G, Buja M (1977> Abnrmal mycardial fluid retentin as an early manifestatin f ischcmic injury. Am J Pathl 87: