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

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A number of factors, including the temperature of the medium into which the atrial tissue is collected and processed, the slice thickness and the Po, of the in- cubation media may affect the viability of the preparation. In preliminary studies (data not shown), we observed that all these factors play an important role in maintaining the stability of the preparation. To ensure stability, the temperature of the medium for the collection of the specimen should be 4-10 'C; for recovery and processing, the media temperature should be 37 'C and the P02 25-30 kPa, and the thickness of the slices should be no more than 0.5 mm. These findings are supported by Paradise et al. [4] and Prasad and Callaghan [26], who reported that the thickness of the muscle preparation and the P02 of the media are the determinants of the oxygen diffusion rate and the viability of the preparation. The small, not statistically significant decrease in tissue viability seen after the initial 30 min equilibration period, and associated with increased LDH leakage, is possibly the result of mechanical injury sustained during sectioning of the tissue. It is unlikely that ischaernic injury is a contributor to this phenomenon, because the procedure was carried out under hypothermic conditions and the time spent in processing the tissue samples was less than 2 min, which is clearly insufficient time to induce myocardial damage. Furthermore, during this short period of sample processing, the muscles are not preconditioned when subjected to a long period of ischaernia [27]. Studies on ischaernia To the best of our knowledge, the present study is the first to characterize the response of the human myocardium to various degrees of ischaemic insult. We have shown that a period of simulated ischaemia of only 30 min induced significant tissue injury, as measured by MTT reduction and LDH leakage, and that lengthening the ischaemic time to 2h resulted in a loss of more than 75 % viable tissue and substantial LDH leakage. It is worth noting that the decrease in oxygen consumption observed after 30 min of ischaemia was not further affected by 60 or 120 min of ischaemia. This suggests that, in fact, the remaining viable tissue augments oxygen consumption when compared with aerobically perfused tissue or with tissue subjected to shorter periods of ischaernia. This phenomenon of an oxygen-sparing effect after ischaemia has been described for several animal preparations [28-31], suggesting that it may not be a reliable index of tissue damage. It is from evident these studies, not unexpectedly, that, although the atrial myocardial slices aerobically incu- bated for 24 h are still viable, they become more susceptible to ischaemic injury. This observation is of particular relevance when investigating the delayed effects of ischaemic syndromes, such as the second window of ischaemic preconditioning. Myocardial ischaemia/reperfusion in human right attial slices Studies on reperfusion Our findings show that two different pictures can emerge from the injury sustained during reperfusion, depending on whether LDH leakage or MTT reduction are examined. On one hand, the absence of a significant net increase in LDH leakage over the 24 h reperfusion period compared with that seen during the first 2h of reperfusion [compare Figure 9 (upper panel) with Figure 4] suggests that in our preparation tissue injury is limited to the initial 2h reperfusion period. On the other hand, the results of the MTT reduction support the view that reperfusion injury is a progressive process throughout the 24 h reperfusion period. A possible explanation for this apparent discrepancy may be that LDH leakage represents enzyme loss from necrotic tissue, whereas MTT reduction reflects the loss of tissue viability via both necrosis and apoptosis. If this is the case, then one may be tempted to conclude that necrosis is confined to the early reperfusion period (, < 2 h), and that apoptosis is the main mechanism for the loss of tissue viability during the late reperfusion period. Support that apoptosis may play a role in the injury sustained during ischaemia and reperfusion comes from recent reports on different experimental preparations and animal species [32-34]. Certainly, more studies are required to clarify the role played by apoptosis in the ischaemia/reperfusion injury of the human heart. It is worth noting that the presence of blood components may influence the response and the degree of injury sustained during ischaemia/reperfusion. Our studies have shown that neutrophils may exacerbate late reperfusion injury and therefore the presence or absence of blood components should be taken into account when designing a study and interpreting the results. The present model may facilitate the investigation of the underlying mechanisms of injury during early and delayed reperfusion in the human myocardium. This model will also allow us to elucidate the true effects of therapeutical interventions. By looking exclusively at the early reperfusion period, it is possible that many interventions, thought to be beneficial in the past, in fact may have limited therapeutic value if their action is delaying rather than reducing myocardial injury. Comparison with other preparations The use of in vivo or in vitro experimental models each has advantages and disadvantages but both are regarded as necessary and complementary. Thus, for example, vivo models are useful to study the physiological relevance and long-term effects of the processes under investigation; however, they are complex and the influence of factors such as blood elements, the neuro- endocrine system and even animal welfare and seasonal variations cannot be ruled out. By contrast, in vitro models are not exposed to the internal and external 0 200 The in 451
4S2 J. -G. Zhang and othen effects of living animals, but they are limited by their short duration (usually a few hours) and stability. Studies on myocardial ischaernia in the clinical setting are difficult to carry out and to interpret and frequently they are ethically unacceptable. To overcome these difficulties, an alternative is the use of the right atrial preparations, such as the one described in the present study. Certainly, our right atrial preparation is relatively easy to use, the tissue is readily available and inexpensive, since it is regarded as 'surgical waste' in open-heart procedures, and, more importantly, it provides mean- ingful information on the human myocardium. The use of atrial tissue for studies of ischaemia may also offer advantages over isolated and cultured myocytes, because these are more difficult to obtain, they do not retain the normal cell-to-cell contact, ischaernia is more difficult to accomplish and they are viable only for short periods of time [35,36]. A notable benefit derived from the pro- longed stability of our preparation is that the effects of reperfusion can be studied for a period that extends beyond the first few hours, which is usually not possible with in vitro preparations. Limitations of the preparation The present study has several limitations which need to be mentioned. First, the preparation is superfused ('simulated ischaemia') as opposed to being arterially perfused. However, the preclusion of the vasculature as the natural pathway for the provision of substrate may also be advantageous in that the confounding effects of the vascular changes and collateral flow induced by ischaernia and reperfusion are separated. In the present study, atrial tissue was used to characterize the effects of ischaemia and reperfusion in the human myocardium. However, atrial and ventricular myocardium possess characteristics of their own that may influence the susceptibility to ischaemia/reperfusion injury and consequently the results from one may not be applicable to the other. Thus, for example, the reported differences in the distribution of potassium channels [37,38], which contribute to the characteristic differences between atrial and ventricular action potentials, may determine a different response to ischaemia/reperfusion. Although atrial tissue is generally stable and disease free, individual biological variations between patients may result in different susceptibility to ischaemia/ reperfusion injury and this must be accounted for when interpreting results from any human study. Conclusion We have characterized a model of ischaemia and reperfusion in the human myocardiurn using right atrial tissue obtained from patients undergoing cardiac surgery. This is readily available, the preparation is inexpensive and stable for at least 24 h, which permits the, study of the 0 2000 The Biochemical Society and the Medical Research Society early and delayed consequences of ischaemia and reperfusion. In addition, the extended stability of the model may be potentially useful for genetic manipulation to investigate the pathophysiological mechanisms underlying injury sustained during ischaemia and reperfusion, and to develop new therapeutic strategies to combat the undesirable effects. ACKNOWLEDGMENTS This study was funded by grants from Heart Link and Link-up Charities, Glenfield Hospital NHS Trust and the University of Leicester. Our thanks to Mr T. Jefferson for his technical support with the morpho- logical studies, Dr R. T. Smolenski for his analysis of the metabolite studies and Mrjames Brown for his invaluable technical help with the Figures. REFERENCES 1 Ikonomidis, J. S., Turniati, L. C., Weisel, R. D., Mickle, D. A. G. and Li, R. K. (1994) Preconditioning human ventricular cardiomyocytes with brief periods of simulated ischernia. Cardiovasc. Res. 28,1285-1291 2 Piper, H. M., Probst, I., Hutter, J. F. and Spieckermann, P. G. (1982) Culturing of calcium stable adult cardiac myocytes. J. Mol. Cell. Cardiol. 14,397-412 3 Cleveland, Jr., J. C., Wollmering, M. M., Meldrum, D. R., Rowland, R. T., Rehring, T. F., Sheridan, B. C., Harken, A. H. and Banerjee, A. (1996) Ischaernic preconditioning in human and rat ventricle. Am. J. Physiol. 271, H1786-HI794 4 Paradise, N. F., Schmitter, J. L. and Surmitis, J. M. (198 1) Criteria for adequate oxygenation of isometric kitten papillary muscle. Am. J. Physiol. 241, H348-H353 5 Cleveland, Jr., J. C., Meldrum, D. R., Cain, B. S., Banerjee, A. and Harken, A. H. (1997) Oral sulphonylurea hyEoglycaemic agents prevent ischaernic preconditioning in uman myocardium. Two paradoxes revisited. Circulation 96,29-32 6 Rump, L. C., Schwertfeger, E., Schaible, U., Fraedrich, G. and Schollmeyer, P. (1994) Beta 2-adrenergic receptor and angiotensin II receptor modulation of sympathetic neurotransmission in human atria. Circ. Res. 74,434-440 7 Rump, L. C., Berlit, T., Schwertfeger, E., Beyersdorg, F., Schollmeyer, P. and Bohmann, C. (1997) Angiotensin converting enzyme inhibition unmasks the sympathofacilitatory effect of bradykinin in human right atrium. J. Hypertens. 15,1263-1270 8 Walker, D. M., Walker, J. M., Pugsley, W. B., Pattison, C. W. and Yellon, D. M. (1995) Preconditioning in isolated superfused human muscle. J. Mol. Cell. Cardiol. 27, 1349-1357 9 Cohen, G., Shirai, T., Weisel, R. D. et al. (1998) Optimal myocardial preconditioning in a human model of ischaernia and reperfusion. Circulation 98 (19 Suppl. ), 11-184-194; discussion 11-194-196 10 Speechly-Dick, M. E., Grover, G. J. and Yellon, D. M. (1995) Does ischaernic preconditioning in the human involve PKC and the ATP-dependent K' channel? Circ. Res. 77,1020-1035 11 Auchampach, J. A., Grover, G. J. and Gross, G. J. (1992) Blockade of ischaernic preconditioning in dogs by novel ATP-dependent potassium channel antagonist sodium 5-hydroxydecanoate. Cardiovasc. Res. 26,1054-1062 12 Gross, G. J. and Auchampach, J. A. (1992) Blockade of ATP-dependent potassium channels prevents myocardial preconditioning in dogs. Circ. Res. 70,223-233
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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,
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14. Auchampach J, Cavero, I and Gro
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27. Becker LB, Van den Hoek TLV, Sh
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41. Bums PG, Krunkenkamp IB, Calder
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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
Page 203 and 204: 97. Ganote C, Armstrong S and Downe
Page 205 and 206: 110. Grover GJ, D'Alonzo A, Hess T,
Page 207 and 208: 122. Hamilton T and Weston A (1989)
Page 209 and 210: 137. Hu H, Sato T, Seharaseyon J, L
Page 211 and 212: 151. Jennings R, Steenbergen C, Kin
Page 213 and 214: 164. Kelpzig H, Kobert G, Mafter C,
Page 215 and 216: 178. Lamping K, Christensen C, Pelc
Page 217 and 218: 192. Liu GS, Thornton J, Van Winkle
Page 219 and 220: 206. Maulik N, Yoshida T, Das DK. (
Page 221 and 222: 219. Miyawaki H, Zhou X, Ashraf, M.
Page 223 and 224: 233. Okazaki Y, Kodama K, Sato H, K
Page 225 and 226: 248. Pryzlenk K, Li G, Whittaker P
Page 227 and 228: 260. Saito S, Tamura Y, Moriuchi M,
Page 229 and 230: 274. Schulz R, Post H, Valhaus C, e
Page 231 and 232: 288. Stewart JR, Blackwell WH, Crut
Page 233 and 234: 300. Tani M, Suganuma Y, Hasegawa H
Page 235 and 236: 312. Tosaki A, Engelman DT, Engelma
Page 237 and 238: 324. Walker D, Marber M, Walker J a
Page 239 and 240: 337. Yamashita N, Nishida M, Hoshid
Page 241 and 242: 351. Zhang JG, Lindup WE. (1993). R
Page 243 and 244: 444 J. -G. Zhang and others are dif
Page 245 and 246: 446 J. -G. Zhang and others Assessm
Page 247 and 248: 448 J. -G. Zhang and others 0.9 0.8
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Page 253 and 254: ELSEVIER Abstract Cardiovascular Re
Page 255 and 256: 936 S. Ghosh et al. / Cardiovascula
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Page 259 and 260: 940 S. Ghosh et al. [41 Goto M, Liu
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