Myocardial Protection by Brief Ischemia in Noncardiac Tissue
Background Brief coronary artery occlusions (CAOs) protect both the artery's own perfusion territory (“myocardial preconditioning”) and adjacent “virgin” myocardium. Whether ischemia in remote organs protects myocardium is unknown. We examined whether brief occlusion of the anterior mesenteric artery (MAO) or left renal artery (RAO) protects against myocardial infarction.
Methods and Results Area at risk (AR) and infarcted area (IA) were determined in anesthetized rats after 180 minutes of reperfusion following a 60-minute CAO. At normothermia (body temperature, 36.5°C to 37.5°C), IA/AR was 68±2% (mean±SEM, n=11) in control rats and 50±3% (n=9, P<.001) in rats preconditioned by 15-minute CAO 10 minutes before 60-minute CAO. A 15-minute MAO was equally protective (IA/AR=50±3%, n=10, P<.001), whereas 15-minute RAO failed to limit IA/AR (72±5%, n=8). Hypothermia (body temperature, 30°C to 31°C) did not affect IA/AR (67±3%, n=11) in control animals but enhanced protection by 15-minute CAO (IA/AR=22±3%, n=8), whereas protection by 15-minute MAO (IA/AR=44±5%, n=11, P<.001) was minimally enhanced. Hypothermia unmasked protection by 15-minute RAO (IA/AR=46±6%, n=9, P<.01). Hexamethonium (20 mg/kg IV) did not alter protection by 15-minute CAO, but it abolished protection by 15-minute MAO. When MAO was sustained throughout the study, cardioprotection was absent.
Conclusions Brief ischemia in “remote” organs protects myocardium against infarction as effectively as myocardial preconditioning. The mechanism of protection by MAO differs from that of CAO, because ganglion blockade abolished protection by MAO but not by CAO. The neurogenic pathway is activated during reperfusion after 15-minute MAO, because sustained MAO failed to produce cardioprotection.
Ischemic preconditioning has been described not only for the myocardium1 but also for the kidney,2 skeletal muscle,3 brain,4 and liver.5 Furthermore, Przyklenk et al6 reported that a brief CAO preconditioned the myocardium not only within but also outside its perfusion territory (“remote” but intracardiac ischemic preconditioning). It is unknown, however, whether remote organ ischemia can protect the myocardium against infarction.
Therefore, we first examined whether brief remote organ ischemia before a 60-minute CAO limited myocardial infarct size. For this purpose, we produced transient ischemia in the small intestine or left kidney by MAO or RAO in rats and examined its effect on myocardial infarct size. Since body temperature may influence infarct size7 8 and cardioprotection by the adenosine deaminase inhibitor pentostatin was observed only in the presence of mild hypothermia,9 studies were performed at two temperatures. Because results indicated that brief MAO provided cardioprotection at both temperatures, MAO was selected to examine the mechanism of protection by remote organ ischemia. To investigate whether a neurogenic pathway was involved, we repeated the studies after ganglion blockade with hexamethonium. To determine whether activation of the neurogenic pathway occurred during remote organ ischemia or the subsequent 10 minutes of reperfusion, we also determined infarct size after 60-minute CAO in the presence of permanent MAO.
Experiments in male Wistar rats (weight, ≈300 g) fed ad libitum were performed in accordance with the Guiding Principles in the Care and Use of Animals as approved by the Council of the American Physiological Society and under the regulations of the Animal Care Committee of the Erasmus University Rotterdam.
Effect of 15-Minute MAO or 15-Minute RAO on Infarct Size Produced by 60-Minute CAO (Protocol 1)
Nine groups were studied in this protocol (Fig 1⇓). Eight groups underwent a 60-minute CAO followed by 180 minutes of reperfusion at normothermia (body core temperature, 36.5°C to 37.5°C) (groups 1 through 4) or hypothermia (body core temperature, 30°C to 31°C) (groups 6 through 9). Control groups 1 and 6 underwent a 25-minute sham period before the 60-minute CAO. Groups 2 and 7 underwent a 15-minute CAO, groups 3 and 8 a 15-minute MAO, and groups 4 and 9 a 15-minute RAO, each followed by reperfusion starting 10 minutes before the 60-minute CAO. One group of normothermic rats (group 5) underwent only a 15-minute CAO to determine whether the classic ischemic preconditioning stimulus produced irreversible myocardial damage.
Involvement of Neurogenic Pathway in Cardioprotection by 15-Minute MAO (Protocol 2)
Six groups of rats were studied after pretreatment with the ganglion blocker hexamethonium (20 mg/kg IV) 15 minutes before the ischemic stimulus was applied (Fig 2⇓). Groups 10 through 12 were studied during normothermia and groups 13 through 15 during hypothermia. In groups 10 and 13, the effect of ganglion blockade on infarct size produced by 60-minute CAO was studied. The effect of ganglion blockade on cardioprotection by 15-minute CAO was studied in groups 11 and 14 and that by 15-minute MAO in groups 12 and 15.
Importance of Reperfusion for Cardioprotection by MAO (Protocol 3)
To investigate whether activation of the neurogenic pathway occurred during occlusion or reperfusion of the anterior mesenteric artery, we determined infarct size produced by 60-minute CAO in the presence of a permanent MAO (Fig 3⇓).
Surgical and Experimental Procedures
Rats were anesthetized with pentobarbital (60 mg/kg IP) and intubated for positive-pressure ventilation (Harvard) with room air. A PE-10 catheter was positioned in the thoracic aorta for measurement of arterial blood pressure and heart rate (Baxter Diagnostic Inc). A PE-50 catheter was positioned in the inferior caval vein for infusion of a polygeline (35 mg/mL) plus electrolytes (in mmol/L: Na+ 145, K+ 5.1, Ca2+ 6.25, and Cl− 145, pH 7.3±0.3) (Haemacel, Behringwerke AG). After intercostal thoracotomy, the pericardium was opened and a silk 6-0 suture was looped under the coronary artery for later production of CAO.10 11 After laparotomy, a catheter was positioned in the abdominal cavity to allow intraperitoneal infusions of pentobarbital for maintenance of anesthesia. Then, the anterior mesenteric artery or the left renal artery was dissected free, and a suture was placed around the artery to facilitate later MAO or RAO with an atraumatic clamp. After the ischemic stimulus was applied, the abdomen was closed. The control and the classic ischemic preconditioning groups underwent the same procedure but without dissection of the renal or mesenteric artery.
Body core temperatures were continuously measured rectally with an electronic thermometer (Electromedics Inc) and were maintained in the designated range by either heating pads or ice-filled packages. Except during application of the CAOs and reperfusions, the thoracotomy site was covered with aluminum foil to prevent heat loss from the thoracic cavity. The adequacy of this procedure was verified in five rats in which simultaneous measurements of rectal and intrathoracic temperature showed no differences at baseline (37.2±0.2°C and 36.9±0.02°C, respectively) or at the end of 60-minute CAO (36.9±0.2°C and 36.8±0.2°C, respectively). Rats that fibrillated during ischemia or reperfusion were allowed to complete the protocol when conversion to normal sinus rhythm occurred spontaneously within 1 minute or when resuscitation by gentle thumping on the thorax was successful within 2 minutes after onset of fibrillation. Occlusion and reperfusion were visually verified by appearance and disappearance of myocardial, small intestinal, or renal cyanosis.
Measurement of AR and IA
At the end of the experiment, the heart was quickly excised and cooled in ice-cold saline before it was mounted on a modified Langendorff apparatus and perfused retrogradely via the aorta with 10 mL ice-cold saline to wash out blood.10 11 After the coronary ligature was retied, the heart was perfused with 3 mL trypan blue (0.4%, Sigma Chemical Co) to stain the normally perfused myocardium dark blue and delineate the nonstained AR. The heart was then frozen at −80°C for 10 minutes and cut into slices of 1 mm from apex to base. From each slice, the right ventricle was removed and the LV was divided into the AR and the remaining LV. The AR was then incubated for 10 minutes in 37°C nitro blue tetrazolium (Sigma Chemical Co; 1 mg per 1 mL So¨rensen buffer, pH 7.4), which stains vital tissue purple but leaves infarcted tissue unstained. After the IA was isolated from the noninfarcted area, the different areas of the LV were dried and weighed separately.
Data Analysis and Presentation
Infarct area (% total LV mass) was analyzed by ANCOVA with IA as dependent variable, experimental groups as independent factor, and AR (% total LV mass), the heart rate–systolic arterial blood pressure product, and temperature as covariates. Infarct size (IA/AR, in percent) was analyzed by one-way ANOVA followed by an unpaired t test with modified Bonferroni correction.12 Hemodynamic variables were compared by two-way ANOVA for repeated measures followed by the paired or unpaired t test with modified Bonferroni correction. Data are presented as mean±SEM.
Eight of the 89 rats that entered protocol 1 (1 rat in each of the groups 1, 2, 3, 8, and 9 and 3 rats in group 4) and 7 of the 49 rats that entered protocol 2 (1 rat in each of the groups 12 and 15, 2 rats in group 13, and 3 rats in group 14) were excluded because of sustained ventricular fibrillation. In group 17 (protocol 3), 1 of the 9 rats had to be excluded.
ARs in the Three Study Protocols
There were no significant differences between ARs of the experimental groups (Table 1⇓).
Effect of 15-Minute MAO or 15-Minute RAO on Infarct Size Produced by 60-Minute CAO (Protocol 1)
There was a strong linear relationship between IA and AR of control rats that underwent the 60-minute CAO (IA=0.76[±0.04]AR−1.93[±1.20]; r2=.98, P<.001). A single 15-minute CAO limited IA/AR produced by 60-minute CAO to 50±3% versus 68±2% in the control groups (P<.001). The 15-minute CAO itself resulted in negligible necrosis (IA/AR=3±1%, n=4). A 15-minute MAO was equally protective (IA/AR=50±3%, P<.001) as 15-minute CAO, whereas 15-minute RAO failed to protect the myocardium (IA/AR=72±5%).
IA/AR of the hypothermic control groups (67±3%) was similar to IA/AR of the normothermic control group (IA=0.74 AR−1.80, r2=.90, P<.001). In contrast, protection by 15-minute CAO was greater during hypothermia (IA/AR=22±3%, P<.001) than during normothermia (P<.01). The limitation of IA/AR to 44±5% (P<.005) by 15-minute MAO was not different from that produced by 15-minute MAO during normothermia. The 15-minute RAO, ineffective during normothermia, limited IA/AR to 46±6% during hypothermia (P<.01 versus hypothermic control).
Involvement of Neurogenic Pathway in Cardioprotection by 15-Minute MAO (Protocol 2)
Fig 5⇓ and Table 1⇑ illustrate that during normothermia as well as hypothermia, ganglion blockade had no effect on infarct size produced by 60-minute CAO (IA/AR=68±3% and 67±3%, respectively) or infarct size limitation by 15-minute CAO (IA/AR=54±3%, P<.001, and IA/AR=18±4%, P<.001, respectively). In contrast, cardioprotection by 15-minute MAO was completely abolished by ganglion blockade during both normothermia (IA/AR=74±2%) and hypothermia (IA/AR=69±3%).
Importance of Mesenteric Artery Reperfusion for Cardioprotection by MAO (Protocol 3)
Fig 6⇓ and Table 1⇑ show that when MAO was sustained throughout the experimental protocol, myocardial infarct size was not different from that of the control animals (IA/AR=70±3% and 63±3% at normothermia and hypothermia, respectively).
Heart Rate and Mean Arterial Blood Pressure
Under normothermic baseline conditions, heart rate (351±5 bpm), mean arterial blood pressure (86±3 mm Hg), and the product of heart rate and systolic arterial blood pressure (34 500±1300 bpm·mm Hg) were not different in groups 1 through 4 (Table 2⇓). Hypothermia did not affect mean arterial blood pressure (84±3 mm Hg), but it decreased heart rate (285±8 bpm, P<.001) and the rate-pressure product (28 410±1400 bpm·mm Hg, P<.001). There were also no differences in baseline values of heart rate and arterial blood pressure between groups 6 through 9.
None of the ischemic stimuli had an effect on heart rate during either normothermia or hypothermia. A 15-minute CAO resulted in a 12±3 mm Hg (P<.05) decrease in mean arterial blood pressure, which did not recover during the 10 minutes of reperfusion. In contrast, 15-minute MAO and 15-minute RAO produced increases in mean arterial blood pressure of 11±3 mm Hg (P<.05) and 6±3 mm Hg (P<.05), respectively. In the MAO groups, arterial blood pressure decreased to below baseline values during the 10 minutes of reperfusion but was maintained at 11±3 mm Hg (P<.05) above baseline in the RAO groups.
Administration of hexamethonium caused decreases in mean arterial blood pressure of 24±3 mm Hg (P<.05) and 20±6 mm Hg (P<.05) during normothermia and hypothermia, respectively, and decreases in heart rate of 34±5 bpm (P<.05) and 58±6 bpm (P<.05 versus normothermia), respectively. Fifteen-minute CAO further decreased mean arterial blood pressure slightly (7±3 mm Hg, P<.05), whereas MAO caused an increase in mean arterial blood pressure (18±2 mm Hg, P<.05). These responses were not different from those observed in the absence of hexamethonium in protocol 1 (Table 3⇓).
Heart rate did not change in either group during the experimental protocol. After onset of permanent MAO, mean arterial blood pressure increased by 14±3 mm Hg (P<.05) during normothermia and by 16±5 mm Hg (P<.05) during hypothermia but returned to baseline levels during the 60-minute CAO (Table 4⇓).
Lack of Effect of Systemic Hemodynamics on Infarct Size
Multivariate regression analysis of the groups in protocol 1 demonstrated that in the two sham and two MAO groups, AR explained 99% (r2=.99) and 93% (r2=.93), respectively, of the variability in IA, with no contributions of temperature or rate-pressure product measured at the onset of 60-minute CAO. In both the CAO and RAO groups, AR explained 88% and 90% of IA variability, respectively, whereas AR together with temperature explained 94% and 94%, respectively, of the variability of IA, with no contribution of the rate-pressure product. Similarly, ANCOVA (with temperature as independent factor and AR and the rate-pressure product as covariants) demonstrated that temperature but not different hemodynamic conditions explained the enhanced protection in the hypothermic RAO and CAO groups. There was no correlation between the mean arterial blood pressure response to the preconditioning stimuli and IA/AR in the CAO, MAO, or RAO groups, indicating that the pressure responses to 15-minute CAO, MAO, or RAO were not responsible for the decrease in IA/AR. This is supported by the observation that despite a pressor response to the permanent MAO, this stimulus failed to limit infarct size.
In both the hexamethonium-treated sham and MAO groups, AR explained 99% of the variability in IA, with no contribution of either temperature or rate-pressure product measured at the onset of 60-minute CAO. In the CAO groups, AR explained 82% of the IA variability, whereas AR together with temperature explained 97% of IA variability, again with no contribution of the rate-pressure product. Taken together with the observation that hexamethonium had no effect on the relation between IA and AR or IA/AR despite the decreases in heart rate and arterial blood pressure, the data clearly indicate that AR, temperature, and brief remote organ ischemia but not hemodynamic conditions were determinants of IA.
Until now, the protective effects of ischemic preconditioning have been investigated only in models in which the preconditioning stimulus was applied to the organ that was also subjected to the prolonged period of ischemia.1 2 3 4 5 The present study investigated whether ischemia in an organ other than the heart could limit infarct size produced by a sustained CAO and examined the mechanism(s) leading to cardioprotection. For this purpose, we first investigated whether protection by remote organ ischemia could be organ specific and evaluated the effects of ischemia in two different organs, the kidney and small intestine. Second, we performed our studies at two different body temperatures (36.5°C to 37.5°C and 30°C to 31°C). This approach was chosen because earlier studies have shown that infarct size development depends on temperature7 8 and also because the ability of the adenosine deaminase inhibitor pentostatin to limit myocardial infarct size was observed only at lower body temperature.9 The major results of the first part of the present study were that (1) ischemia in remote organs can limit myocardial infarct size as effectively as ischemic myocardial preconditioning with 15-minute CAO, since 15-minute MAO limited infarct size to the same extent as ischemic myocardial preconditioning during normothermia; (2) the degree of protection depends on body temperature, since 15-minute RAO failed to protect the myocardium during normothermia but was protective during hypothermia; and (3) the protection by ischemic myocardial preconditioning was more pronounced during hypothermia than during normothermia, although infarct size produced by 60-minute CAO per se was not different during normothermia and hypothermia. Because of the results of the first part of the study, we selected the 15-minute MAO stimulus to examine its mechanism of protection. To investigate the involvement of a neurogenic pathway, we examined the effect of ganglion blockade on the protection by 15-minute MAO as well as 15-minute CAO. Ganglion blockade abolished the protection by 15-minute MAO during both normothermia and hypothermia but had no effect on infarct size produced by 60-minute CAO and protection by ischemic myocardial preconditioning at either temperature. These results demonstrate the involvement of a neurogenic pathway in the protection by 15-minute MAO, indicating that protection by remote organ ischemia may be different from that by ischemic myocardial preconditioning. However, our data do not exclude a common intramyocardial end point for the mechanism of protection by remote organ ischemia and ischemic myocardial preconditioning, eg, activation of protein kinase C.13 14 15
The final question we addressed was whether activation of the neurogenic pathway occurred during MAO or in the ensuing 10 minutes of reperfusion. The observation that permanent MAO failed to limit myocardial infarct size produced by 60-minute CAO indicates that reperfusion of the small intestine was mandatory to activate the neurogenic pathway. These data could be interpreted to suggest that at reperfusion, substances released in the mesenteric bed (eg, oxygen-derived free radicals,16 cytokines17 ) stimulate afferent neurofibers. From the present study, it cannot be determined whether these neurofibers are activated within or outside the mesenteric bed. Future studies should therefore be directed at examining the factors involved in activating the neurogenic pathway at release of the MAO and how this activation results in limitation of myocardial infarct size.
The finding that 15-minute MAO protected the myocardium during both normothermia and hypothermia, whereas the 15-minute RAO was protective only during hypothermia, might suggest that 15-minute RAO resulted in a subthreshold stimulus during normothermia. The renal and mesenteric arteries are of similar size in terms of amount of total blood flow, but whereas the anterior mesenteric flow is considered almost completely nutrient flow, <10% of renal blood flow is nutrient flow.18 Consequently, the amount of tissue that became ischemic during RAO was less than during mesenteric artery occlusion, so that the stimulus by RAO may have been below threshold. We can therefore not exclude the possibility that multiple or a single longer renal occlusion or bilateral RAO could have protected the myocardium. In this respect, it should be kept in mind that in ischemic myocardial preconditioning, the severity of ischemia appears to be more important than its duration in eliciting cardioprotection.19 Nevertheless, brief interruption of renal artery blood flow protected the heart during hypothermia. There have been preliminary reports that an RAO may reduce infarct size produced by a CAO.20 21 In our earlier study,21 we did not control temperature as rigorously as in the present study. In subsequent experiments, we observed that without appropriate measures, temperature can decrease easily by as much as 3°C to 4°C during the course of surgical instrumentation. We can therefore not exclude that in our earlier study, the effects of the 15-minute RAO occlusions were due to the presence of hypothermia.
An intriguing finding in the present study was that hypothermia per se had no effect on infarct size in the control rats but unmasked a protective effect by 15-minute RAO. In contrast, the protection by 15-minute MAO tended to be enhanced, but this was not statistically significant. The lack of effect of hypothermia on infarct size in the control rats seems at variance with previous observations in rabbits.7 However, in that study a different temperature range (35°C to 42°C) was used, and the sustained CAO lasted only 30 minutes. At 30 minutes of occlusion, infarction progresses rapidly in the rabbit, so that a small delay of infarction by a decrease in body temperature may have a greater effect on infarct size. It is possible that in rats, infarcts produced by 60-minute CAO are less susceptible to the delay in infarction produced by a decrease in body temperature. This hypothesis is supported by two recent studies in swine. Whereas Duncker et al8 observed that a decrease in body temperature from 39°C to 35°C reduced infarct size produced by 45-minute CAO by more than 80%, McClanahan et al9 reported that a decrease in temperature from 37°C to 35°C had no effect on infarct size after 60-minute CAO. These findings indicate that the effect of temperature on infarct size may depend critically on the experimental model, including the duration of the sustained CAO.
In contrast to the lack of effect of temperature on infarct size in the control rats, hypothermia markedly modified the efficacy of the preconditioning stimuli. With the exception of the mesenteric preconditioning stimulus, which was slightly but not significantly enhanced by the presence of hypothermia, protection by intramyocardial ischemic preconditioning was enhanced, and a cardioprotective effect of brief renal ischemia emerged. The mechanism of this synergistic (intramyocardial ischemia) or unmasking (renal ischemia) action of hypothermia is not readily explained. The present study excludes a contribution of activation of a neural pathway, because hexamethonium did not affect the enhanced protection of ischemic myocardial preconditioning by hypothermia. McClanahan et al9 reported that either mild hypothermia or adenosine deaminase inhibition alone had no effect on infarct size. In contrast, when these stimuli were combined, a significant reduction in myocardial infarct size was observed. Their findings are in agreement with the present study and suggest that body temperature, even when it does not alter infarct size by itself, can significantly modify the cardioprotective effects of other physiological or pharmacological interventions. The protection by 15-minute MAO was not significantly increased when experiments were performed at hypothermia, which might suggest that the stimulus was already maximally effective at normothermia.
In the present study, body core temperature was measured rectally. That this temperature reflects intrathoracic temperature was demonstrated in five rats in which simultaneous measurements of rectal and thoracic cavity temperatures were not different (see “Methods”). One might ask whether these rectal measurements reflect intramyocardial temperature. In a previous study in swine,8 rectal temperature exceeded myocardial temperature on average by only 0.3°C at baseline, whereas at the end of 45-minute CAO, intramyocardial temperature was 0.3°C lower than rectal temperature. Those findings suggest that although subtle differences in temperature of myocardium and rectum may have been present in this study, these were small compared with the 6°C rectal temperature difference in the normothermic and hypothermic groups.
An increase in myocardial stretch produced by rapid volume loading can limit infarct size produced by a 60-minute CAO in dogs.22 We did not measure LV diastolic volume or pressure and can therefore not exclude that MAO- or RAO-induced pressor responses produced stretch-mediated cardioprotection. However, such a mechanical pathway of protection appears unlikely, because ganglion blockade abolished the MAO-induced protection, even though the pressor response persisted.
The present study may have important clinical implications, because it suggests that ischemia in remote organs could result in cardioprotection when it precedes a coronary thrombotic event. Thus, patients suffering from abdominal angina or perhaps even intermittent claudication might conceivably have a longer time window for thrombolytic therapy to salvage ischemic myocardium. The present study also supports earlier reports that myocardium can be protected by stimuli that do not produce myocardial ischemia, such as myocardial stretch22 or ventricular pacing.23 Finally, the ability of such diverse stimuli, eg, nonischemic myocardial stimuli and nonmyocardial ischemic stimuli, to protect the myocardium may hamper the unequivocal demonstration of ischemic myocardial preconditioning in humans.24 25 26
The present study is the first to demonstrate that not only brief ischemia in an adjacent myocardial region but also a brief period of ischemia followed by reperfusion in a remote organ, such as small intestine or kidney, can protect the myocardium against irreversible damage produced by a prolonged CAO. MAO and reperfusion resulted in protection during both hypothermia and normothermia, whereas the protection by renal ischemia was apparent only under hypothermic conditions. The mechanism of protection by brief MAO involved a neurogenic pathway that required mesenteric artery reperfusion for its activation.
Selected Abbreviations and Acronyms
|AR||=||area at risk|
|CAO||=||left anterior descending coronary artery occlusion|
|LV||=||left ventricle, left ventricular|
|MAO||=||anterior mesenteric artery occlusion|
|RAO||=||left renal artery occlusion|
The research of Dirk J. Duncker was made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences.
- Received February 15, 1996.
- Revision received April 30, 1996.
- Accepted May 1, 1996.
- Copyright © 1996 by American Heart Association
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