CLINICAL STUDIES
Renal ischemia/reperfusion remotely improves myocardial energy metabolism during myocardial ischemia via adenosine receptors in rabbits: effects of "remote preconditioning"
Atsushi Takaoka, MDa,
Ichiro Nakae, MD, PhDa,
Kenichi Mitsunami, MD, PhD*,
Takahiro Yabe, MD, PhDa,
Shigehiro Morikawa, MD, PhD ,
Toshiro Inubushi, PhD and
Masahiko Kinoshita, MD, PhDa
a First Department of Internal Medicine, Shiga University of Medical Science, Seta, Otsu 520-2192, Japan
* Department of General Medicine, Medical Coordination Center, Shiga University of Medical Science, Seta, Otsu 520-2192, Japan
Molecular Neurobiology Research Center, Shiga University of Medical Science, Seta, Otsu 520-2192, Japan
Manuscript received May 19, 1998;
revised manuscript received September 1, 1998,
accepted October 2, 1998.
Reprint requests and correspondence: Dr. Atsushi Takaoka, First Department of Internal Medicine, Shiga University of Medical Science, Seta, Otsu 520-2192, Japan
taka5836{at}belle.shiga-med.ac.jp
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Abstract
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Objectives
This study examined the changes in myocardial energy metabolism during myocardial ischemia after "remote preconditioning" and investigated the involvement of adenosine receptors in the mechanisms of this effect.
Background
Recent studies have indicated that a brief period of ischemia and reperfusion (ischemic preconditioning, PC) in a remote organ reduces myocardial infarct size (IS) protecting against subsequent sustained myocardial ischemia. However, the mechanisms of "remote PC" remain unclear. We assessed myocardial energy metabolism during sustained myocardial ischemia and reperfusion after renal PC (RPC), in comparison with that after myocardial PC (MPC) in open-chest rabbits. It has been established that adenosine receptors are involved in the mechanisms of MPC.
Methods
Rabbits that had been anesthetized with halothane were divided into six groups. The control (CNT) group underwent 40-min coronary occlusion followed by 120 min reperfusion. Before the procedure, the MPC group underwent an additional protocol of 5 min coronary artery occlusion and 20 min reperfusion, and the RPC group received a 10 min episode of renal artery occlusion and 20 min reperfusion. In additional experimental groups, 8 sulfophenyltheophylline (SPT, 10 mg/kg), an adenosine receptor inhibitor, was intravenously injected before the 40 min myocardial ischemia (SPT, MPC + SPT and RPC + SPT groups, respectively). Myocardial levels of phosphocreatine (PCr), ATP and intracellular pH (pHi) were measured by 31P-NMR spectroscopy.
Results
RPC and MPC delayed the decreases in ATP levels, preserved pHi during 40-min myocardial ischemia and resulted in better recovery of ATP and PCr during 120 min reperfusion compared with the controls. SPT abolished the improvement in myocardial energy metabolism and the reduction in myocardial IS caused by MPC or RPC. Myocardial IS in the CNT (n = 8), MPC (n = 9), RPC (n = 9), SPT (n = 6), MPC + SPT (n = 8) and RPC + SPT (n = 8) groups averaged 42.8 ± 3.5%, 18.2 ± 1.8%*, 19.6 ± 1.3%*, 44.9 ± 5.0%, 35.6 ± 2.7% and 34.8 ± 3.6% of the area at risk (*p < 0.05 vs. CNT), respectively.
Conclusions
PC in a remote organ, similar to MPC, improved myocardial energy metabolism during ischemia and reperfusion and reduced IS in vivo by an adenosine-dependent mechanism in rabbits.
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Abbreviations and Acronyms
| | AAR | = area at risk | | CNT | = control | | 2,3-DPG | = 2, 3-diphosphoglyceric acid | | IS | = infarct size | | MPC | = myocardial ischemic preconditioning | | PC | = ischemic preconditioning | | PCr | = phosphocreatine | | pHi | = intracellular pH | | RPC | = renal ischemic preconditioning | | SPT | = 8-sulfophenyltheophylline |
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Recent studies have demonstrated that brief episodes of myocardial ischemia render the heart resistant to myocardial injury during subsequent prolonged ischemic episodes (1,2). This phenomenon, termed "ischemic preconditioning (PC)," has been demonstrated in a variety of organs, not only in the heart (3). Recently, it was also reported that an ischemic episode in one organ, such as a kidney, can augment ischemic tolerance in another organ, such as the heart; this is known as "remote PC" (4,5). Since PC is currently the most effective means available of protecting ischemic myocardium, this phenomenon has attracted a great deal of clinical interest. Thus, we examined myocardial metabolic changes following remote PC as well as myocardial PC (MPC).
Biochemical studies have revealed that MPC slows the rate of ATP degradation during subsequent sustained ischemic insult (2). The onset of irreversible myocardial ischemic injury in vivo is associated with marked depletion of ATP and with breaks in the sarcolemma. Myocardial ischemic injury is also related to the myocardial level of phosphocreatine (PCr) and intracellular pH (pHi). 31P-NMR spectroscopy can directly evaluate the levels of high-energy phosphate present in the myocardium (6–11).
In the present study, we investigated the changes in ATP, PCr and pHi during sustained myocardial ischemia and reperfusion after renal PC (RPC) or MPC in rabbits. Because this model has no significant coronary collateral circulation, we do not have to consider the influence of collateral circulation, as in dog models (12,13). Adenosine is considered to be a common initiation trigger for the MPC response in many species, including rabbits (14,15). It has also been shown that infusion of adenosine protects against the attenuation of myocardial function after coronary occlusion (9,11,16) or reduces myocardial infarct size (IS) (17). Thus, we also investigated whether the adenosine receptor inhibitor 8-sulfophenyltheophylline (SPT) affects the changes in myocardial energy metabolism caused by RPC.
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Methods
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Surgical preparation.
Animals were treated according to the guidelines for animal experimentation prepared by the Japanese Association for Laboratory Animal Science.
Male New Zealand White rabbits weighing 2.1 to 3.5 kg were anesthetized by intravenous injection of sodium pentobarbital (25 mg/kg). The animals were intubated by tracheostomy and mechanically ventilated with a Harvard respirator (Harvard Apparatus, South Natric, Massachusetts) using room air mixed with oxygen. The respiratory rate was fixed to 60 per min and tidal volume was initially adjusted to 13 ml. Anesthesia was maintained with halothane (1.0 to 2.0%). The tidal volume and oxygen supplement were adjusted to maintain arterial blood gas values within the physiological range (pH 7.35 to 7.45, PCO2 35 to 45 mm Hg, and PO2 100 to 150 mm Hg). The internal jugular vein was cannulated for maintenance of intravenous infusion. A catheter was inserted into the carotid artery and connected to a pressure transducer to monitor aortic blood pressure. Heart rate was measured with an AT-600G HR counter (Nihon Kohden, Tokyo, Japan) triggered by arterial pressure pulse. Aortic pressure and heart rate were recorded continuously with a polygraph and recorder (model RCM 3000, Nihon Kohden, Tokyo, Japan). The animals underwent median thoracotomy, the sternum was removed, and the pericardium was gently excised exposing the surface of the left ventricle. A reversible snare occluder consisting of 6-0 sutures threaded through an elastic tube was placed around a marginal branch of the left coronary artery. For RPC, laparotomy was performed and the left renal artery was dissected free. A reversible snare occluder consisting of 6-0 sutures was placed around the renal artery. The snare was tightened around the artery during occlusion, and was loosened during reperfusion.
31P-NMR spectroscopy.
In vivo 31P-NMR spectra were obtained using a 2.0 T CSI Omega System (Brucker, Fremont, California). The animals were fixed in a cylindrical plastic case and maintained at a rectal temperature of 37.0 to 37.5° C with a heating pad. A copper wire four-turn surface coil (10 mm in diameter) tuned to both 1H-(85.6 MHz) and 31P-(34.6 MHz) frequencies was placed on the epicardial surface over the region of the myocardium to be rendered ischemic, thereby minimizing contamination by nonischemic myocardial signals. The rabbit was then moved into the magnet and allowed to sit for  30 min to reach a steady state. After shimming with water 1H-signals, 31P-spectra were collected with a repetition time of 2 s and 288 acquisitions using respiratory and cardiac gating. The NMR data were analyzed by an automatic curve-fitting process using the Simplex procedure and were broken down into 9 components of Lorentzian lines: phosphomonoester, inorganic phosphate (Pi) in the myocardium, 2, 3-diphosphoglyceric acid (2,3-DPG) in the blood, phosphodiester, PCr,  -ATP,  -ATP, ß-ATP and broad baseline (18). The ATP level was assigned by the ß-ATP peak area, since ß-ATP is the most uncontaminated ATP resonance. Tissue levels of PCr and ß-ATP were estimated as a percentage of the total phosphorus signal excluding the broad baseline.
Experimental protocol.
Rabbits that had been anesthetized with halothane were divided into six groups: CNT (n = 8), MPC (n = 9), RPC (n = 9), SPT (n = 6), MPC + SPT (n = 8) and RPC + SPT (n = 8). In the CNT group, the rabbits underwent 40 min coronary artery occlusion followed by 120 min reperfusion; in the MPC group, the rabbits underwent MPC consisting of 5 min coronary artery occlusion and 20 min reperfusion before the 40 min coronary artery occlusion; in the RPC group, the rabbits underwent RPC consisting of 10 min renal artery occlusion and 20 min reperfusion before the 40 min coronary artery occlusion; in additional experimental groups, 8-sulfophenyltheophylline (SPT, 10 mg/kg), an inhibitor of adenosine receptors, was intravenously injected for 2 min before 40-min myocardial ischemia (SPT, MPC+SPT and RPC+SPT groups, respectively) (17). SPT (Sigma) powder was dissolved in saline to 5 mg/ml immediately before use.
Determination of infarct size.
After 120 min of reperfusion, the coronary artery was reoccluded, and blue dye was injected into the venous catheter to stain the normally perfused region of the heart. With the rabbit under deep pentobarbital anesthesia, cardiac arrest was produced by injection of 15% KCl through the arterial catheter. The heart was rapidly excised. After the atria, right ventricle and great vessels were removed from the heart, the left ventricle was sectioned into 2-mm slices from the apex to the base using a tissue slicer. Myocardial infarct size was assessed by triphenyl tetrazolium chloride (TTC, Sigma) staining (19). Each slice was rinsed in saline and incubated in a 1% solution of TTC buffered in 0.2 M phosphate buffer to pH 7.4 at 37° C for 10 min. Following staining, the basal surface of each slice was photographed. The areas of the infarct and risk zone were determined by planimetry, converted to volumes and expressed as cm3.
Pilot study.
To examine the influence of laparotomy in the animals that received both thoracotomy and laparotomy in the absence of PC (sham), only myocardial IS was assessed. Myocardial IS after the 40 min coronary occlusion and 120 min reperfusion (%IS, 48.0 ± 8.9%, n = 4) did not differ from that in the CNT group (received thoracotomy). Thus, 31P-NMR spectra were studied in the six groups.
Data analysis.
All data are expressed as mean values ± SEM. The pHi was calculated from the difference between the chemical shifts ( , ppm) of PCr and Pi resonances using the following equation (6): pHi = 6.90 – log [( – 5.805)/(3.290 – )]. The rate-pressure product was calculated as the systolic aortic blood pressure multiplied by the heart rate. The chi square test was used to analyze mortality differences. Statistical significance was determined by analysis of variance (ANOVA) for multiple comparisons. When multiple comparisons were made, Scheffe’s post hoc test was used to determine the significance of differences. Differences in regression lines were tested by analysis of covariance (ANCOVA). A p value of less than 0.05 was considered significant.
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Results
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Mortality of rabbits.
Fifty-three rabbits entered protocol. None of the rabbits died during PC. During the 40 min occlusion, one rabbit in each of the CNT, MPC, RPC, SPT and MPC+SPT groups died due to ventricular fibrillation. These animals were excluded from analysis. In the RPC+SPT group, no rabbit died. The mortality rates were not significantly different among the six experimental groups.
Hemodynamic changes.
Heart rate, mean blood pressure and rate-pressure product were comparable throughout the experiment among these six groups (Table 1).
31P-NMR spectroscopy.
No significant changes were observed in PCr, ATP or pHi during the 30 min of baseline measurement. The time courses of changes in PCr and ATP were thus expressed as percentages of the baseline values obtained for 10 min before the first ischemia. In the SPT group, the changes in these parameters were expressed as percentages of the baseline values obtained for 10 min before the 30 min of first ischemia for a time corresponding to the PC procedure. Representative examples of NMR spectra are shown in Figure 1.
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Figure 1 Representative 31P-NMR spectra from CNT, MPC, RPC, SPT, MPC+SPT and RPC+SPT groups at baseline (BASE), after ischemic preconditioning (PC), before coronary occlusion (Pre-oc), after 40 min of coronary occlusion (Occ) and after 120 min of reperfusion (Rep). CNT = control; MPC = myocardial ischemic preconditioning; MPC+SPT = myocardial ischemic preconditioning plus 8-sulfophenyltheophylline-treatment; PCr = phosphocreatine; Pi = inorganic phosphate; RPC = renal ischemic preconditioning; RPC+SPT = renal ischemic preconditioning plus 8-sulfophenyltheophylline-treatment.
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In the CNT group, PCr decreased to 4 ± 1% after 40 min of sustained ischemia, and returned to 29 ± 3% after 120 min of reperfusion (Fig. 2). In the MPC group, PCr decreased transiently during PC, but recovered after reperfusion. After sustained ischemia, PCr decreased to 13 ± 4%, and then returned to 69 ± 5% after reperfusion. At 10 and 20 min of sustained ischemia, PCr values tended to be higher in the MPC than CNT group, but these differences were not significant (Fig. 2). During reperfusion, except for the first 10 min, PCr values were significantly higher in the MPC than CNT group. In the RPC group, PCr did not change during PC. PCr decreased to 12 ± 2% after sustained myocardial ischemia, and returned to 64 ± 5% after reperfusion. During reperfusion, PCr in the RPC group had recovered and was similar to that in the MPC group.
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Figure 2 Time courses of changes in phosphocreatine (PCr) as percentages of the baseline value in CNT (open squares), MPC (open triangles), RPC (open circles), SPT (solid squares), MPC+SPT (solid triangles) and RPC+SPT (solid circles) groups. *p < 0.05 versus CNT, #p < 0.05 versus MPC+SPT, p < 0.05 versus RPC+SPT. Data points represent the means ± SEM. Abbreviations as in Figure 1.
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In the CNT group, ATP decreased to 34 ± 3% after sustained ischemia, and only reached 36 ± 5% after reperfusion (Fig. 3). In the MPC group, the ATP level decreased transiently during PC, but returned to 93 ± 4% after reperfusion. ATP decreased to 53 ± 3% during sustained ischemia, but returned to 79 ± 5% after reperfusion. Thus, the ATP level was significantly higher in the MPC than CNT group during sustained ischemia, except for the first 10 min, and throughout reperfusion. In the RPC group, the ATP level did not significantly change during PC. ATP decreased to 51 ± 4% after sustained myocardial ischemia, and returned to 68 ± 4% after reperfusion. Thus, during sustained ischemia, the ATP level was preserved to an extent similar to that in the MPC group, and during reperfusion, ATP in the RPC group recovered to a similar degree to that in the MPC group.
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Figure 3 Time courses of changes in ATP as percentages of the baseline value in CNT (open squares), MPC (open triangles), RPC (open circles), SPT (solid squares), MPC+SPT (solid triangles) and RPC+SPT (solid circles) groups. *p < 0.05 versus CNT, #p < 0.05 versus MPC + SPT, p < 0.05 versus RPC+SPT. Data points represent the means ± SEM. Abbreviations as in Figure 1.
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There were no significant differences among baseline pHi values in the CNT, MPC and RPC groups (Fig. 4). In the CNT group, pHi decreased to 5.93 ± 0.07 after 40 min of sustained ischemia. In the MPC group, pHi decreased transiently during PC, and recovered before sustained ischemia. After 40 min of ischemia, pHi values were 6.46 ± 0.09. In the RPC group, pHi did not change during PC. After sustained ischemia, pHi decreased to 6.38 ± 0.11. In these three groups, pHi returned to the baseline values after 40 min of reperfusion. Thus, during sustained myocardial ischemia, pHi was preserved similarly in the RPC and MPC groups at higher levels than in the CNT group.
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Figure 4 Time courses of changes in intracellular pH in CNT (open squares), MPC (open triangles), RPC (open circles), SPT (solid squares), MPC+SPT (solid triangles) and RPC+SPT (solid circles) groups. *p < 0.05 versus CNT, #p < 0.05 versus MPC+SPT, p < 0.05 versus RPC+SPT. Data points represent the means ± SEM. Abbreviations as in Figure 1.
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Throughout reperfusion, except for the first 10 min, PCr values were significantly higher in the MPC than MPC+SPT group, and also higher in the RPC than RPC+SPT group (Fig. 2). Throughout 120 min reperfusion, ATP values were significantly higher in the MPC group than in the MPC+SPT group and also higher in the RPC than RPC+SPT group (Fig. 3). At 30 and 40 min of sustained ischemia, pHi values were significantly higher in the MPC group than in the MPC+SPT group, and at 20, 30 and 40 min of sustained ischemia, significantly higher in the RPC than RPC+SPT group (Fig. 4). There were no significant differences in myocardial values of PCr, ATP and pHi during sustained ischemia and reperfusion among CNT, SPT, MPC+SPT and RPC+SPT groups (Figs. 2, 3 and 4). Thus, SPT abolished the improvement of PCr, ATP and pHi caused by MPC and RPC.
Infarct size study.
All of the experimental groups were comparable with regard to body weight, heart weight and size of area at risk (AAR) (Table 2). Percentage of infarct size (IS as a percentage of AAR) was significantly smaller in the MPC and RPC groups than in the CNT group. There was no significant difference in %IS between the MPC and RPC groups. Figure 5 shows the changes in the relationships between IS and AAR size as a percentage of total left ventricular mass. The regression lines in both MPC and RPC groups were significantly (p = 0.0001) less steep than the CNT regression line. There was no significant difference between the slopes of regression lines in MPC and RPC groups. There were no significant differences in %IS among the CNT, SPT, MPC+SPT and RPC+SPT groups (Table 2). The slope of the regression line in the MPC group was significantly (p = 0.0007) less than that in the MPC+SPT group (Fig. 5). The slope of the regression line in the RPC group was significantly (p = 0.0001) less than that in the RPC+SPT group. There were no significant differences among the slope of regression lines in the CNT, SPT, MPC+SPT and RPC+SPT groups.
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Figure 5 Scatterplots of relations between infarct size (IS) and area at risk (AAR) as percentages of total left ventricular (LV) mass in CNT (open squares), MPC (open triangles), RPC (open circles), SPT (solid squares), MPC+SPT (solid triangles) and RPC+SPT (solid circles) groups. The regression line of the MPC group (y = 0.20 x –0.01, r2 = 0.42) was significantly (p = 0.0001 by ANCOVA) less steep than that of the CNT group (y = 0.53 x –0.03, r2 = 0.43). The slope of the regression line in the RPC group (y = 0.22 x –0.01, r2 = 0.67) was significantly (p = 0.0001) less than that in the CNT group. There was no significant difference between the slopes of regression lines in MPC and RPC groups. The slope of the regression line in the MPC group was significantly (p = 0.0007) less than that in the MPC+SPT group (y = 0.51 x –0.05, r2 = 0.89). The slope of the regression line in the RPC group was significantly (p = 0.0001) less than that in the RPC+SPT group (y = 0.45 x –0.03, r2 = 0.78). There were no significant differences among the slope of regression lines in the CNT, SPT (y = 0.70 x –0.06, r2 = 0.94), MPC+SPT and RPC+SPT groups.
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Discussion
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The major findings of this study were: (a) remote RPC, similar to MPC, delayed the decreases in the ATP levels and preserved higher levels of pHi during sustained myocardial ischemia and improved recovery of the ATP and PCr levels during reperfusion relative to control, resulting in a reduction of IS; and (b) an inhibitor of adenosine receptors, SPT, abolished the improvement in myocardial energy metabolism with the reduction in IS caused by both MPC and RPC.
Usefulness and limitations of 31P-NMR spectroscopy.
In the present study using 31P-NMR spectroscopy, the myocardial level of ATP decreased to 35% after 40 min of coronary occlusion. Compared to the myocardial ATP level measured by biochemical methods, the degree of ATP decrease in this study was somewhat small. Murry et al. showed that subendocardial ATP level decreases to 11% after 40 min of ischemia in canine hearts (2). Their samples were subendocardial, whereas ours were transmural and also somewhat weighted toward the epicardium because of the sensitivity profile of the surface coil. The ischemic zone progresses from the subendocardium to the subepicardium. It was reported (20) that in rabbits this progression began at 5 min after coronary artery occlusion and that subendocardial nontransmural infarction was produced after 15 min. After 30 min of occlusion, both nontransmural and transmural infarctions were observed. Thus, the slower depletion of ATP may have been due to differences in tissue sampling methods.
Using 31P-NMR spectroscopy, Kida et al. reported earlier depletion of PCr and ATP during sustained ischemia in pig hearts in vivo (8). Myocardial PCr disappeared early after more than 10 min of ischemia, and ATP decreased to 20% after 40 min of ischemia. They used a surface coil 17 mm in diameter to collect signals in pigs, whereas the surface coil in the present study was 10 mm in diameter. The measurements represent an average of the signals from all myocardium proximal to the coil. Considering heart size and coil size, the sampling of a relatively larger area in rabbit heart than in pig heart may account for this difference.
In rabbit hearts in vivo, Wroblewski et al. reported that PCr and ATP decreased to 53% and 31% after 40 min of ischemia, respectively, using 31P-NMR spectroscopy and a 13-mm coil (7). In our study, PCr and ATP decreased to 4% and 34% after 40 min of ischemia, respectively. Our data for ATP were similar to the values obtained by Wroblewski et al. Our PCr data could more sensitively reflect myocardial ischemia. We carefully set the surface coil in the center of the ischemic area, and believe that contamination by nonischemic regions was minimal. However, under basal conditions, contamination by chamber blood seemed to have somewhat elevated the Pi resonance due to signals from 2,3-DPG in the blood. Thus, Pi values were not given in the text because of their unreliability. During ischemia, however, the peak was split into myocardial Pi and 2,3-DPG peaks (11). Thus, the pHi values during coronary occlusion can be accurately calculated by the chemical shift of Pi (6). Although it has some limitations, 31P-NMR spectroscopy can be used to monitor pHi, ATP and PCr serially and noninvasively in studies of myocardial energy metabolism.
Myocardial ischemic preconditioning.
Using 31P-NMR spectroscopy in pigs in vivo, Kida et al. showed both preserved high-energy phosphate levels and pHi in MPC hearts during sustained ischemia and suggested that the preservation of pHi is more important because it was more prolonged (8). However, they did not observe myocardial metabolic parameters during reperfusion. Recently, de Albuquerque et al. suggested that the protective effect of MPC is closely related to preservation of pHi during a prolonged ischemia, independent of preservation of high-energy phosphate stores, using 31P-NMR spectroscopy in isolated rat hearts (21). However, isolated heart preparations may metabolically behave differently from intact myocardium. We examined the metabolic changes during ischemia and reperfusion using intact animals. In our study, PCr tended to slowly decrease during ischemia in the presence of MPC, but this was not significant. With higher levels of pHi during ischemia in the PC group, we observed better recovery of PCr and ATP during reperfusion, resulting in a reduction in IS. Consistent with the results of their studies, the protective effect of MPC seemed to depend more on reduced acidosis of the myocardium rather than on the preservation of myocardial energy stores during ischemia.
Kida et al. reported an increase in PCr immediately after PC (overshoot phenomenon [22], which suggests increased energy production) in pigs (8), but we did not clearly observe this phenomenon in rabbits. Kida et al. performed four episodes of 5 min ischemia as PC, whereas we performed only one such episode. They observed little overshoot of PCr at the first and second 5 min coronary occlusions. Thus, this difference may have been due to the number of repetitions of the PC procedure. Li et al. reported that the protective effects of PC are not necessarily enhanced by multiple, repetitive ischemic periods (23).
In the present study, the improvement of myocardial energy metabolism and the reduction in IS in the MPC groups were blocked by SPT. This result indicated that the MPC effect is closely related to adenosine receptors (14,15). In addition, this observation indicated that the higher levels of PCr and ATP in the MPC groups as measured by 31P-NMR spectroscopy were not due to changes in the position between the heart and surface coil, which may have been caused by bulging, stunning etc., because SPT did not affect any hemodynamics. As described above, our data included blood contamination from the left ventricular cavity. Although this may have affected the results of the quantitative analysis of Pi signals, it did not affect measurements of PCr because PCr is not present in the blood. In the present study, the PCr level was higher in the MPC group (MPC 69% vs. CNT 29% after reperfusion). Measurements of ATP may include some blood ATP (24). However, the improvement in ATP level (MPC 79% vs. CNT 36% after reperfusion) was similar in magnitude to that in PCr level in the MPC group. Therefore, we believe that the changes in PCr and ATP analyzed in this study reliably reflected the changes in myocardial concentrations of PCr and ATP. Our results were compatible with reports that adenosine infusion mimicked the improvement of myocardial energy metabolism during prolonged ischemia by MPC using 31P-NMR spectroscopy in rabbits (9) and pigs (11).
Remote ischemic preconditioning.
Conventional PC means that brief periods of ischemia protect the organ, usually the heart, against subsequent episodes of prolonged ischemia in the same area. However, Przyklenk et al. demonstrated that brief episodes of ischemia in the circumflex branch protect myocardium of the left anterior descending coronary artery region from subsequent sustained ischemia in dog hearts (4). Their term "remote" was used to indicate arteries in another artery region in the same organ. Recently, Gho et al. reported that brief ischemia in the mesenteric artery or renal artery region protected myocardium against infarction as effectively as MPC in rats (5). Thus, it has also been shown that brief ischemia in "remote" organs reduces myocardial IS caused by subsequent sustained coronary occlusion.
The mechanisms of conventional MPC remain obscure despite intensive study (25,26). Activation of adenosine receptors is believed to play an important role in mediating the protective effects of MPC in rabbits (14), dogs (27) and pigs (11). Adenosine activates a pertussis toxin-sensitive inhibitory G protein, which can facilitate the activation of ATP-sensitive K+ channels. Stimulation of -adrenoceptors by endogenous catecholamines can also exert PC effects through the activation of protein kinase C via a G protein (25). A G protein is involved in MPC in rabbits (28), and the opening of ATP-sensitive K+ channels has been implicated in MPC in dogs (27). However, in rat hearts neither adenosine (29) nor ATP-sensitive K+ channels (30) mediate the effects of MPC. In the present study, our results suggested that activation of adenosine receptors is involved in the mechanisms of RPC as well as MPC in rabbits.
The mechanisms of remote PC remain unclear. In rats, Gho et al. reported that the mechanism of remote PC caused by mesenteric artery occlusion may differ from that of MPC since hexamethonium abolished the protective effect of remote PC but did not abolish that of MPC (5). This indicated that myocardial protection caused by remote PC acts via ganglia in rats. Permanent occlusion of the renal or mesenteric artery failed to reduce myocardial IS in rats (5). In addition, RPC in rabbits did not reduce myocardial IS when the 40 min coronary artery occlusion was started just before reperfusion of RPC (unpublished data). These findings suggest that cardioprotection by remote PC is mediated through a pathway activated by some factor(s) released during reperfusion (31). In our preliminary study, plasma concentrations of adenosine in the carotid artery were more elevated after RPC than after MPC (plasma adenosine ratio [RPC/MPC] after PC, >10-fold, data not shown). Thus, these effects may be triggered by elevation of plasma adenosine levels.
Gho et al. (5) reported that the effects of remote PC were dependent on body temperature in rats; the cardioprotective effects of remote PC were observed under hypothermic (30 to 31° C) but not normothermic conditions (36.5 to 37.5° C). However, in the present study, cardioprotection by remote PC was observed under normothermic conditions. This cardioprotection was not primarily caused by lower cardiac temperature due to the surgical procedure, because (a) rectal temperature was maintained between 37.0° C and 37.5° C with a heating pad and cardiac temperature was close to rectal temperature under these conditions; and (b) the effect of remote PC was abolished by SPT under similar conditions of body temperature. In addition, a pilot experiment showed that in the animals that received both thoracotomy and laparotomy, myocardial IS after ischemia/reperfusion did not differ from that in the CNT group. Considering that the physiologic temperature of rabbits is 39° C, however, further studies at higher body temperature would also be needed.
Previous studies in rabbits (32) and dogs (33) suggested that volatile anesthetic agent itself mimics PC. Indeed, myocardial IS was smaller using halothane than intravenous anesthetic agents. In previous studies using rabbits under pentobarbital or ketamine/xylazine, myocardial IS averaged 3855% of the AAR after 30 min ischemia and reperfusion (34–36). Thus, in this study, we chose the longer period of 40 min ischemia under halothane so the average of IS would be 40% of the AAR. Although we cannot rule out the possibility that halothane affected our results, the remote PC effect observed in our study cannot be explained by this alone, because we compared 31P-NMR spectra or IS in the RPC group with that in the CNT group under similar conditions. If the influence of PC effect induced by halothane is large, it is expected that blockade of adenosine receptors or ATP-sensitive K+ channels increases myocardial IS following ischemia/reperfusion under halothane. However, neither SPT (this study) nor 5-hydroxydecanoate (unpublished data) increased IS in the absence of PC. Thus, we believe that the influence of PC effect by halothane was very small under our experimental conditions and did not impair our results.
In the present study, remote PC slowed the decreases in ATP and pHi during sustained ischemia and produced better recovery of ATP and PCr during reperfusion. The preservation of pHi during prolonged ischemia appeared to contribute to reducing IS by remote PC, similarly to MPC (8,21). This may lead to the reduced stimulation of Na+-H+ (36) and Na+-Ca2+ exchange (26). Intracellular acidosis is considered to be a major cause of myocardial injury and to be one of the main mechanisms responsible for Ca2+ accumulation in myocardial tissue during ischemia/reperfusion. SPT effectively abolished both reduced acidosis and reduced IS by RPC and MPC. These findings suggest that the mechanism of cardioprotection by remote PC involves, at least in part, a pathway in common with that by MPC. However, further studies are required before a detailed mechanism of remote PC can be proposed.
Clinical implications.
Our results suggest that brief, repeated episodes of ischemia in one organ can confer a protective effect against injury due to a subsequent prolonged period of ischemia in other organs. For instance, clinical events such as transient cerebral ischemic attack, abdominal angina, intermittent claudication (37), renal ischemia by renovascular hypertension, balloon inflation during angioplasty and aortic cross-clamping may have cardioprotective effects against subsequent acute myocardial infarction.
Conclusions.
RPC, similar to MPC, improves myocardial energy metabolism during myocardial ischemia and reperfusion and reduces myocardial IS through a mechanism involving adenosine receptors in rabbits. In addition, the effect of RPC may be related to reduced acidosis during prolonged ischemia.
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Acknowledgments
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We thank Akihiro Sekine, Fujirebio Inc., Pharmaceuticals Research Laboratories, Hachioji, Tokyo, Japan, for valuable suggestions and Ikuko Sakaguchi for technical assistance.
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References
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