ischaemic preconditioning of the human heart. - Leicester Research ...

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Chni(ad Suence 12000) 99.443 4S3 (Pininted in Great Britain) 443 Characterization of an in vitro model for the study of the short and prolonged effects of myocardial ischaernia and reperfusion in man lin-Gang ZHANG', Sudip GHOSH, Colin D. OCKLEFORD* and Manuel GALIRANES Division of (ardiac Surgery, Department of Surgery. University of Leicester. University Hospitals Leicester NHS Trust, Glenfield (ampus, Leicester LE3 9QP, U. K., and *Department of Pre-clini(al Sciences, University of Leicester, University Road, Leicester LEI 7RH. U. K. ABSTRACT The mechanisms underlying myocardial ischaerma and reperfusion-induced injury have been investigated. mainly by using animal experimental preparations in vitro and in vivo. but little is known of the process in human myocardium. The present studies characterize an in vitro model using human myocardium for the study of early and delayed effects of ischaernia and reperfusion. The right atrial appendage was manually sliced and incubated in buffer through which was bubbled 02 C02 (19 ý11v v) for various time periods. Lactate dehydrogenase (LDH) leakage, 3- [4,5-dimethylthiazol-2-yi]-2.5 diphenyl-2H-tetrazolium bromide (MTT) reduction. oxygen con- sumption, nucleotide levels and tissue morphology were all investigated as markers of myocardial injury. The specimens remained stable and viable up to 24 h. but had significantly deteriorated by 48 h. The preparation responded to ischaernia in a time-related manner. Tissue viability was reduced by 25% after 30 min ischaernia, declined to 60% after 60 min ischaernia and to 75'14 after 120 min ischaernia. Interestingly, the tissue was more susceptible when ischaernia was induced after 24 h of aerobic incubation. The effects of the duration of reperfusion were investigated after a fixed 60 min ischaernic insult. The results of LDH leakage suggest that reperfusion injury is mainly sustained within the first 2h of reperfusion. However, the results of MTT reduction show that there is a progressive decrease in tissue viability over the 24 h reperfusion period. possibly reflecting the occurrence of tissue necrosis and apoptosis at different reperfusion times. In conclusion, the data provide evidence that the incubation of human atrial tissue in vitro is stable, and slices are viable for at least 24 h, which permits the study of early and delayed consequences of ischaernia and reperfusion in the human myocardium. INTRODUCTION Isdiacmic licart disease is the singic most common cau%c of mortallt, N In thc Wc%tern world. ()%cr the last two decadcs, .1 grc. it deal ha% becii Icarned about the patho 1111% %iology ot m. % ocardial ischacniia, dic t: on%cqucncc% of repert . uslon and how the ad%crsc ctfccts may bc com- Kcý words: liuman imocardium, i%tAmcnim, rclicitusimi, tight Itfllllll. haticd. Most of out knowledge ha,, bccn gamcd I, \ ii, ing in and in zvoo exilcrinictital animal motick, and flic cxtrapolation of this infoi mation to the human licart li, i% tc%ultcki in the inipIcnictitation ot no\cl diciapctink appi oac lics and 1na piogrcssi\ c dcc i casc in i lit, kicat h ratc attributcd it, catkilac 1"'Alacink C\Clltl,. Studies on cardia,: i%ducinia and rcpcitusion in nian Abbrc%-iations: ANM A, anal. N sis ot %artaiwc, I. D1 1, la,: t au: d ch% d iogctia%cý NVIT, 3 4,5 Lf I 111C111% I III [A/0]-' %I i 2, S diplicm ! 211 ima/Aium biomide; PMN%, pok morphoncutrophils. Tresent address: Department ot PharflIal: CIJIWAI SIAMIA-1, ( ý1111L-gc ot Pliamia,: % , \\ admigion Statc I 99164-(, 51^,, U. S. A. Pullman, \\A Correspondence: hotc-m M. GaIiiianc, (c mad ings', c 2000 The Boochemi(al sooety and the Medi(al Reseaf(h soofty
444 J. -G. Zhang and others are difficult because of the presence and potential influence of a whole host of clinical factors. The utilization of human isolated myocytes [1,2], papillary 60 min muscle [3,4] and atrial myocardium [5-81 has provided a means to investigate directly the effects and mechanisms of ischaernia and reperfusion in man without the need to resort to assumptions made from animal studies, and to safely test interventions intended to be used clinically. Thus the use of human cultured myocytes [1,91 and right atrial tissue [5,10], for example, has served to identify some of the mechanisms involved in ischaemic preconditioning in the human myocardium, which, compared with those found in other animal species [11-15], has opened the door for its clinical application [16]. The right atrial preparation is of particular interest, because the tissue is easily obtainable from patients undergoing open-heart surgery, it is simple to prepare and the procedure is inexpensive. Yet the preparation has not been fully characterized. The aim of the present studies was therefore to investigate the stability of the human right atrium when incubated in a buffered medium, its response to various degrees of ischaemic insult and the short and prolonged effects of reperfusion. METHODS Preparation of atrial slices Specimens of human right atrium appendage were obtained from patients undergoing elective heart surgery. During surgery, the right atrial tissue is routinely removed for venous cannulation and establishment of cardiopulmonary bypass. Samples were quickly im- mersed in cold (4 OC) Krebs/Henseleit/Hepes medium (118 mM NaCl, 4.8 mM KCI, 27.2 mM NaHCO, 1 mM KH2pO4) 1.2 mMMgCl21 1.25 mM CaCl, 10 mM glucose, 20 mM Hepes). The medium had been pre-bubbled with02/CO2 (19: 1, v/v) to attain aP02 of 25-30 kPa and pH 7.4. The atrial appendage was immediately sliced manually with skin-graft blades (Swann-Morton Ltd, Sheffield, U. K. ), each slice to a thickness of 0.5 mm and a weight of 5-10 mg, as originally described for the preparation of rat renal slices [17]. Briefly, the tissue was placed with the epicardial surface face down on filter paper fixed to a rectangular glass base (5 x 25 cm). A ground-'glass slide (2.5 x 7.5 cm) was then pressed against the tissue and the blade was drawn between the slide and the tissue. The slicing apparatus and the tissue was kept wet at all times with ice-cold medium (4-10 'C). Experimental time course After preparation, the slices (3-5 slices per specimen) were blotted with wet filter paper, loaded into glass 25 ml Erlenmeyer flasks and 5 ml of medium, which was 0 2000 The Biochemical Society and the Medical Research Society 30 min 120 inin Equilibration Aerobic Incubation Figure I Experimental protocol for Study I /Z 240 360 min 12 hr 24 hr 48 hr Tissues from Groups 1-4 were equilibrated for 30 min in aerobic conditions at 37 "C. The right atrial slices (n = 6/group) were further incubated aerobically for the time periods shown. continuously bubbled with O, /CO, (19: 1) to maintain a P0, of 25 kPa and a pH of 7.4, was added. The flasks were then placed in a shaking water bath (100 cycles/min) at 37 'C for 30 min to allow equilibration. The slices were then rinsed with the medium, blotted and placed in clean flasks containing 5 ml of oxygenated medium for various time periods to serve as time-matched aerobic controls. For the induction of simulated ischaemia, the slices were washed with one rinse of medium bubbled with N, /CO, (19: 1) at pH 6.8. In this case, glucose in the medium was replaced with 10 mM 2-deoxy-D-glucoSe (grade II). The slices were transferred to clean flasks containing 5 ml of the same medium which was continuously bubbled with N, /CO, (19: 1) and maintained at 37 'C during the entire ischaemic period. Monitoring of P02 with an oxygen detector electrode (Oxylite"-; Optronix Ltd, Oxford, U. K. ) revealed that the P02 in the medium was 0 kPa. At the end of each ischaemic period, the non-oxygenated medium was removed, the slices were rinsed with oxygenated medium (02/Coll 19: 1) and incubated in 5 ml of oxygenated medium containing 10 mM glucose at 37 OC for a further 120 min. Study groups Three different studies were performed to investigate: the stability of the preparation (Study 1), the effect of the severity of ischaernia (Study 2), and the effect of the duration of reperfusion (Study 3). In Study 1 (Figure 1), atrial slices (n = 6/group) were subjected to various periods of aerobic incubation after an initial 30 min equilibration period. At the end of the experimental time, samples of the incubation medium
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ISCHAEMIC PRECONDITIONING OF THE HU
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ABSTRACT Ischaemic preconditioning
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andomised clinical study with the f
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CHAPTERI Introduction 35 Matefials
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Discussion 151 CHAPTER Vill Conclus
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44 CK leakage and MTT reduction on
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The Wellcome Trust and The British
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Presentations International "The Ef
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Abbreviations An attempt has been m
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1.1 Introduction Ischaernic heart d
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1.2 Myocardial Ischaemia Myocardial
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hours up to several days to complet
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phosphates and its influence on hyd
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1.4 Myocardial Protection This refe
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performed. This process is repeated
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1.6 Possible Mechanisms of Ischaemi
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kilodalton stress proteins. These p
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and these proteins have been sugges
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K vi-1, channels were initially fou
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tissues the K.. %,.,, channels are
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Subsequently, Jaburek et al 1145] i
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venous capacitance vessels. At ther
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preconditioning to be investigated
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In the angioplasty model, Tomai et
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patients were randomized into two g
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CHAPTER 11 THE HUMAN RIGHT ATRIAL T
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inexpensive. Briefly in this model,
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of each ischemic period, slices wer
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30 nini FO linlin L 60 min 120 inin
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In Study 3 (see Figure 2.4), slices
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D. Metabolite analysis Samples were
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leakage is calculated as the net LD
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B. Effect of the severity of ischae
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2.11 DISCUSSION: The present studie
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clearly insufficient to induce myoc
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The present model may facilitate th
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2.12 Conclusiow I have characterise
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3.5 3 V 2.5 3: m Je m Z0 z 2 1.5 0.
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(3 3 2.5 a 1.5 - -r T 0 2 0.5 0 I P
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Q oro . 21 . 0 'a IP 10 '. R -C3 A
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=3 0 h X CD 0 x CD :3 0 , 7, CD 0 0
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CHAPTER III PRECONDITIONING THE HUM
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3.2 MATERIALS AND METHODS-. 3.2.1 E
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A117'reduction- At the end of the e
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-4 ýi r. () .1 a 0 1 tli n F5* - C
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v M. %; n C) .0 ý U ý ý - - - -2
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leakage resulting from precondition
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3.5 DISCUSSION: The present studies
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during cardiac surgery totalling 10
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different. The cellular mechanisms
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cn Je 8 7 6 5 4 1 0 0.8 0.7 0.6 0.5
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8 6 C) 2 0.7 0.6 Q) 0.5 0) E 25 0.4
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6 5 CY) ,u3 CF) m -ýe m 2 1 0 -0.8
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CHAPTERIV THE ROLE OF THE KATPCHANN
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It has been reported that K.. %-I-p
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0 a- I a 3 I cr =r Cl. 2 qQ 0'-ý 0
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Figure 4.7 shows that ischaemia alo
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Figure 4.2 Dosc-responsc experiment
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Figure 4.4 Dose-response experiment
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Figure 4.6 Dosc-response experiment
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1 0.9 -ý 0.8 0.7 E 0.6 0.5 0.4 0.2
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activity, and showed that diazoxide
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Furthermore, Van den Hoek et al [3
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mechanism by which the opening of m
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5.1 INTRODUCTION: Within the enormo
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5.2 MATERIALS AND METHODS: 5.2.1 Ex
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M77'rechiction: At the end of the e
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oc 0 0 cr F; * 10 eb I ý; o I 5 0
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Pages missing original in the
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esulted in a decrease of CK leakage
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are on long-term hypoglycaernics or
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absence of uniformity of expeniment
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5.7 Conclusion Preconditioning is a
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0.9 0.8 07 m 0.6 E -0 0t 4 0.3 02 0
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-0 1.2 1 &8 0.6 04 0.2 o Aerobic Is
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6.1 INTRODUCIFION' Life expectancv
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(MI-T) to blue t'()rmazan product C
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The demonstration that ischaemic in
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Table 61 Patients' demographic and
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Nure 6,1: Creatine Kinase (CK) leak
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CHAPTER VII IN VIVO STUDIES ON T"E
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The apparent discrepancy bet,.,,, e
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In the off-pump group, coronary byp
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eduction as described in section 3.
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7.4.2 In vitro studies Figures 7.3A
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preconditioning stimulus in man is
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myocardium. Several investigators [
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Table 7.2 - Patients haemod-v-namic
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(0 -I 3 CD CD CD 1 3 0 :3 0 0 =r w
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0 co t X 3 co 0 0 Cumulative Releas
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-& a) c) UT 0 =r w CD 3 CD 0 1ý (a
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When ischaernic preconditioning was
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evidence tbr the involvement ot'PKC
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institution o fcardiopu Imo nary by
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Biblioqraphv 1. Abete P, Ferrara N,
Page 191 and 192: 14. Auchampach J, Cavero, I and Gro
Page 193 and 194: 27. Becker LB, Van den Hoek TLV, Sh
Page 195 and 196: 41. Bums PG, Krunkenkamp IB, Calder
Page 197 and 198: 55. Cohen G, Shirai T, Weisel RD, e
Page 199 and 200: 69. Currie RW and White FP (1983).
Page 201 and 202: 83. Downey JM, Liu GS and Thornton
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Page 205 and 206: 110. Grover GJ, D'Alonzo A, Hess T,
Page 207 and 208: 122. Hamilton T and Weston A (1989)
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Page 249 and 250: 450 J. -G. Zhang and others C E 07
Page 251 and 252: 4S2 J. -G. Zhang and othen effects
Page 253 and 254: ELSEVIER Abstract Cardiovascular Re
Page 255 and 256: 936 S. Ghosh et al. / Cardiovascula
Page 257 and 258: 938 S. Ghosh et al. / Cardiovascula
Page 259 and 260: 940 S. Ghosh et al. [41 Goto M, Liu
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Page 265 and 266: 716 chmä et aL Pi unamOso äu In O
Page 267: 718 ehe* et &L ditioning in the hum
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