Time Course of Late Preconditioning Against Myocardial Stunning in Conscious Pigs
We have recently found in conscious pigs that a sequence of brief coronary occlusions induces severe myocardial stunning, but when the same sequence is repeated 24 hours later, the severity of stunning is markedly reduced (≈50%) (“late preconditioning against stunning”). As an initial step toward elucidating the mechanism and potential clinical significance of this powerful cardioprotective response, the present study was conducted to define the time course of late preconditioning against myocardial stunning. Conscious pigs underwent a sequence of ten 2-minute coronary occlusion/2-minute reperfusion cycles and then a second identical sequence at 6 hours (group I, n=7), 12 hours (group II, n=6), 24 hours (group III, n=10), 3 days (group IV, n=10), or 6 days (group V, n=11) after the first. Systolic wall thickening (WTh) in the ischemic/reperfused region remained significantly depressed for at least 3 hours after the 10th reperfusion of the first sequence, indicating myocardial stunning. When the second sequence of coronary occlusions was performed 6 hours after the first (group I), the recovery of WTh was similar to the first. In contrast, when the second sequence was repeated 12 hours after the first (group II), the recovery of WTh was improved, though not consistently, and the total deficit of WTh decreased by 41% (P<.05) compared with the first sequence. When the second sequence was repeated 24 hours (group III) and 3 days (group IV) after the first, the recovery of WTh was substantially enhanced, with 52% and 49% reductions in the total deficit of WTh, respectively (P<.01 versus the first sequence). When the second sequence was repeated 6 days later (group V), the recovery of WTh was indistinguishable from the first sequence. Thus, late preconditioning against myocardial stunning requires >6 hours to develop, lasts for at least 60 hours after its appearance (with the most effective protection present at 24 hours and 3 days), and disappears within 6 days after the preconditioning ischemia, a time course that is consistent with the synthesis and degradation of cardioprotective proteins. In view of its sustained duration, this endogenous cardioprotective mechanism is of potential clinical importance.
Ischemic preconditioning is the phenomenon whereby a brief episode of ischemia increases the resistance of the heart to a subsequent ischemic insult.1 2 3 4 It is now apparent that there is an early and a late phase of ischemic preconditioning. The early phase is powerful but short-lived and wanes quickly, lasting for only ≈1 hour.2 3 5 6 This phase is effective in limiting myocardial necrosis1 5 6 7 and decreasing the incidence of arrhythmias8 9 but has generally failed to attenuate myocardial stunning.10 11 12 13 Recently, however, we have described a new form of ischemia-induced protection against stunning. Using conscious pigs, we have found that a sequence of ten 2-minute coronary occlusion/2-minute reperfusion cycles induces severe myocardial stunning, but when the same sequence is repeated 24 hours later, the severity of stunning is markedly reduced (by ≈50%).14 15 We have termed this phenomenon “late preconditioning against stunning.”14
The time course of late preconditioning against stunning is unknown. Although it has been found that the protection is no longer present 10 days after the preconditioning ischemia,14 no data are available on when it begins and ends and how its intensity changes over time. Obviously, the clinical importance of this phenomenon will depend, among other things, on its duration. The time course of late preconditioning against stunning is also important from a mechanistic standpoint, since any proposed pathogenetic theory must account for the temporal course of the protection. For example, it has been suggested that the synthesis of some cardioprotective protein(s) is involved in late preconditioning against stunning.14 Before concluding that the induction of any protein is responsible for this phenomenon, it must be shown that the time course of this induction parallels that of the functional protection.
Thus, before one can elucidate the mechanism and potential clinical significance of late preconditioning against myocardial stunning, it is necessary to determine when this protective effect appears and how long it lasts. Accordingly, the goal of the present study was to carefully define the time course of late preconditioning against myocardial stunning, focusing on the following questions: How soon after ischemia does the protective effect develop? When does it disappear? Is the magnitude of the protection constant at different time points? To address these issues, the presence of protection was systematically investigated at different time intervals ranging from 6 hours to 6 days after the preconditioning ischemia using the same conscious pig model in which late preconditioning against stunning was previously described.14 15 As in previous studies,14 15 meticulous attention was paid to the variables that govern myocardial stunning to ensure that any changes in postischemic recovery of contractility after preconditioning could not be ascribed to favorable modifications of the extrinsic determinants of postischemic dysfunction.16
Materials and Methods
A total of 53 pigs were used for the present study. The experimental preparation and techniques have been previously described in detail.14 15 The study was performed in accordance with the guidelines of the Committee on Animals of Baylor College of Medicine and with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, Publication No. [NIH] 86-23).
Domestic pigs of either sex (weight at surgery, 25.4±0.9 kg) were premedicated with ketamine hydrochloride (20 mg/kg IM) and atropine (0.04 mg/kg IM). Twenty to 30 minutes later, anesthesia was induced with methohexital sodium (7.5 mg/kg IV), after which the animals were intubated, and anesthesia was maintained with 0.5% to 1.0% methoxyflurane. A left thoracotomy was performed under sterile conditions at the level of the fifth intercostal space. Tygon catheters were placed in the left atrium and right ventricle, and an additional catheter was inserted into the femoral artery and advanced to the thoracic aorta. A hydraulic occluder and a Doppler flow velocity probe were implanted around the mid LAD. Two insulated copper wires were sutured to the right ventricle to record the electrocardiogram. To measure LV wall thickening, 10-MHz pulsed-Doppler ultrasonic crystals17 were sutured to the epicardial surface, three in the center of the region to be rendered ischemic and another in an area remote from it (posterior LV wall); each probe was sutured with four 6-0 prolene stitches penetrating 0.5 to 1.0 mm into the myocardium, thus producing minimal trauma. To avoid the “tethering” effect of nonischemic myocardium on adjacent ischemic/reperfused myocardium, the crystals were placed at least 1.0 cm inside the boundaries of the ischemic region, which were identified by occluding the LAD for 30 seconds. All wires and catheters were tunneled under the skin and exteriorized through small incisions on the back. The chest was closed in layers, and a small tube was left in the thorax to drain air and fluid postoperatively. Antibiotics were administered intravenously before surgery and daily for 7 days thereafter (cefazolin, 30 mg/kg BID; gentamicin, 0.7 mg/kg BID). Arterial blood gases, hematocrit, rectal temperature, and heart rate were measured daily after instrumentation to ensure that the animals had fully recovered from the surgical procedure. The catheters were flushed daily until the end of the protocol. All pigs were allowed to recover for a minimum of 10 days after surgery and were trained for at least 6 days to lie quietly for 6 hours in a specially designed cage. The cage is constructed of wood and can be adjusted (length, 90 to 115 cm; width, 55 to 73 cm) to match the size of the pig.
Throughout the experiment, pigs were studied as they lay quietly in a cage in a quiet, dimly lit room. Aortic and left atrial pressures were measured with Statham P23 Db pressure transducers. All measured variables (aortic pressure, left atrial pressure, LAD blood flow velocity, WTh, and the electrocardiogram) were recorded simultaneously on an eight-channel direct-writing oscillograph (Gould Brush System 200).
Pigs were sedated with diazepam (initial dose, 1.5 to 2.5 mg/kg IV over 60 minutes; subsequent additional doses were given as needed to maintain sedation). Fifteen minutes after the initial dose of diazepam, pigs underwent a sequence of ten 2-minute LAD occlusion/2-minute reperfusion cycles, followed by a 5-hour observation period to monitor the recovery of myocardial function after the 10th reperfusion. Hemodynamic and WTh measurements were taken at the following time points: before administration of diazepam (baseline); 14 minutes after administration of diazepam, ie, immediately before LAD occlusion (preocclusion); 1 minute into the first and 10th LAD occlusions; 1 minute into each of the first nine reperfusions; and 5, 15, and 30 minutes and 1, 2, 3, 4, and 5 hours after the 10th reperfusion. To measure regional myocardial blood flow, radioactive microspheres were injected via the left atrial catheter, as previously described,18 at 30 to 60 seconds into the fifth LAD occlusion.
After recovering from the first sequence of LAD occlusions, pigs underwent a second identical sequence of ten 2-minute LAD occlusion/2-minute reperfusion cycles at 6 hours (group I), 12 hours (group II), 24 hours (group III), 3 days (group IV), and 6 days (group V) after the first sequence. The earliest time point for repeating the second sequence was chosen at 6 hours, because it is known that WTh recovers completely by this time.14 15
Postmortem Tissue Analysis
At the end of the study, the pigs were given heparin (5000 U IV), anesthetized with sodium pentobarbital (35 mg/kg IV), and killed with a bolus of saturated KCl solution. The heart was excised, and the size of the occluded coronary vascular bed was determined by ligating the LAD at the site of the previous occlusion and perfusing the aortic root for 2 minutes with a 0.5% solution of Monastral blue dye in 6% dextran 70 in normal saline at a pressure of 100 mm Hg.14 The rationale for using dextran in the perfusate was to prevent myocardial edema, which may hinder perfusion and cause underestimation of myocardial blood flow as determined by the microsphere technique. The heart was then cut into 1.0-cm-thick transverse slices and incubated for 20 minutes at 38°C in a 1% solution of triphenyltetrazolium chloride to verify the absence of infarction. The portion of the LV supplied by the occluded coronary artery (occluded bed) was identified by the absence of blue dye and separated from the rest of the myocardium. Both stained and nonstained specimens were weighed to express occluded bed size as a percentage of total LV weight. Four transmural specimens, each weighing 1 to 2 g, were then obtained from both the ischemic and nonischemic regions. To avoid admixture of ischemic and nonischemic tissue, the ischemic specimens were obtained at least 0.5 cm inside the boundary of the occluded bed. Each specimen was divided into endocardial and epicardial halves, weighed, and placed in scintillation vials containing 10% neutral buffered formalin. Regional myocardial blood flow was calculated by standard methods.19
Measurement of Regional Myocardial Function
Regional myocardial function was assessed as systolic thickening fraction using a pulsed-Doppler probe, as previously described.14 15 17 18 19 20 21 22 23 The beginning of systole was determined from the peak of the QRS complex on the right ventricular electrogram and the end of systole from the onset of the rapid rise in LAD blood flow velocity after systole, as previously described.14 15 Percent systolic thickening fraction was calculated as the ratio of net systolic WTh to end-diastolic wall thickness, multiplied by 100.19 The total deficit of WTh after reperfusion (an integrative assessment of the severity of postischemic dysfunction) was calculated by measuring (in arbitrary units) the area between the WTh-versus-time line and the baseline (100% line) during the recovery phase14 15 22 23 ; the recovery phase was defined as the interval between the 10th reperfusion and the time when thickening fraction returned to values >90% of preocclusion values. As discussed previously,14 15 the total deficit of WTh is a measure of the overall severity of myocardial stunning during the 5-hour recovery period. In all animals, measurements from at least 10 beats were averaged at baseline and preocclusion; measurements from at least five beats were averaged at all subsequent time points. As indicated above, three thickening Doppler probes were implanted in the potentially ischemic region. The measurements used for this study are those derived from the probe that gave the lower values of WTh (ie, the most severe degree of myocardial stunning) after reperfusion.
Data are reported as mean±SEM. Hemodynamic variables and WTh were analyzed by a two-way repeated measures ANOVA (time and sequence) to determine whether there was a main effect of time, a main effect of sequence, or a sequence-by-time interaction. If the global tests showed a significant main effect or interaction, post hoc contrasts between different time points of the same sequence or between different sequences at the same time point were performed with paired Student's t tests.24 All statistical analyses were performed using the SAS software system.25 Two-way ANOVA was performed using the procedure GLM (General Linear Models).25 Statistical significance was defined as P≤.05.
Exclusions and Histochemical Analysis
A total of 53 pigs were entered into the study. Of the 10 pigs assigned to group I (6-hour group), 3 were excluded: 2 developed VF upon reperfusion (1 during the seventh and 1 during the eighth reperfusion of the second sequence), and 1 did not complete the first sequence because the balloon burst during the 10th occlusion. Of the 10 pigs assigned to group II (12-hour group), 4 were excluded: in 2, VF developed upon reperfusion (1 during the third and 1 during the seventh reperfusion of the second sequence); in 1, the balloon burst during the first occlusion of the second sequence; and in 1, the occluder failed to function. Of the 11 pigs assigned to group III (24-hour group), 1 was excluded because of VF during the ninth occlusion of the first sequence. Of the 11 pigs assigned to group IV (3-day group), 1 was excluded because the balloon burst during the fifth occlusion of the first sequence. None of the 11 pigs assigned to group V (6-day group) was excluded. Therefore, a total of 7, 6, 10, 10, and 11 pigs were included in groups I, II, III, IV, and V, respectively.
Postmortem tissue staining with tetrazolium demonstrated the absence of infarction in all pigs, indicating that the injury associated with the sequence of ten 2-minute occlusion/2-minute reperfusion cycles was completely reversible. In all animals, postmortem perfusion confirmed that the Doppler ultrasonic crystals were at least 1 cm away from the boundaries of the ischemic region.
Arterial Blood Gases, Hematocrit, and Temperature
As in our previous studies,14 15 arterial pH, Po2, hematocrit, and rectal temperature were within physiological limits throughout the study in all groups (data not reported for the sake of brevity). The doses of diazepam used to induce and maintain sedation were similar between the first and second sequences of LAD occlusion/reperfusion cycles in all groups (data not shown).
The administration of diazepam did not produce significant hemodynamic alterations, as indicated by the comparison of baseline (before diazepam) and preocclusion (after diazepam) measurements (Table 1⇓). With few exceptions, all measured variables (heart rate, systolic arterial pressure, rate-pressure product, left atrial pressure, and LAD blood flow) remained stable within each day of the protocol and between the first and second sequences. That is, except at sporadic time points, these variables did not change significantly from preocclusion values throughout the sequence of LAD occlusions and the subsequent 5 hours of reperfusion and did not differ between the first and second sequences of occlusions within the same group (Table 1⇓).
After the 10th reperfusion, the hyperemic response was similar between the first and second sequences in group I (56.5±11.4 versus 60.1±12.1 mL/min, respectively, at 1 minute; 32.1±5.8 versus 37.5±6.9 mL/min at 2 minutes; 23.2±4.8 versus 25.0±4.5 mL/min at 3 minutes; and 18.3±3.6 versus 20.3±3.3 mL/min at 4 minutes), group II (65.7±10.7 versus 71.0±9.6 mL/min, respectively, at 1 minute; 41.3±8.2 versus 49.5±10.3 mL/min at 2 minutes; 30.0±5.3 versus 35.8±8.5 mL/min at 3 minutes; and 23.4±4.3 versus 26.2±5.7 mL/min at 4 minutes), group III (51.4±10.8 versus 47.3±8.8 mL/min, respectively, at 1 minute; 39.8±7.1 versus 37.4±8.0 mL/min at 2 minutes; 30.7±6.4 versus 28.8±6.0 mL/min at 3 minutes; and 24.4±5.9 versus 23.3±4.9 mL/min at 4 minutes), group IV (70.3±12.1 versus 70.3±15.5 mL/min, respectively, at 1 minute; 48.1±9.8 versus 47.2±13.1 mL/min at 2 minutes; 33.8±7.3 versus 32.2±9.4 mL/min at 3 minutes; and 26.2±5.3 versus 24.7±6.8 mL/min at 4 minutes), and group V (71.4±9.5 versus 71.6±9.4 mL/min, respectively, at 1 minute; 46.9±7.7 versus 48.1±7.8 mL/min at 2 minutes; 33.3±5.7 versus 35.8±6.0 mL/min at 3 minutes; and 26.0±4.1 versus 27.5±5.0 mL/min at 4 minutes). Similarly, there were no appreciable differences in coronary vascular resistance between the two sequences at these time points in any group (data not shown). Thus, the development of preconditioning had no effect on the hyperemic response of the coronary vasculature to the sequence of occlusion/reperfusion cycles.
Occluded Bed Size and Regional Myocardial Blood Flow
The size of the occluded/reperfused vascular bed was similar among the five groups: 20.3±2.9 g (19.4±2.2% of LV weight) in group I, 19.0±2.1 g (17.0±2.0% of LV weight) in group II, 21.5±1.8 g (20.4±1.4% of LV weight) in group III, 20.8±2.0 g (17.7±1.3% of LV weight) in group IV, and 19.5±1.9 g (17.7±1.8% of LV weight) in group V.
The measurements of regional myocardial blood flow are summarized in Table 2⇓. In all five groups, blood flow to the ischemic region (measured during the fifth LAD occlusion) was virtually zero during both the first and second sequences of LAD occlusions. There were no statistically significant differences with respect to epicardial, endocardial, or mean transmural flow to the nonischemic zone between the two sequences in any group (Table 2⇓).
Regional Myocardial Function
The administration of diazepam had no significant effect on thickening fraction on any day of the protocol, either in the region to be rendered ischemic (Figs 1 through 5⇓⇓⇓⇓⇓) or in the nonischemic (control) region (Table 1⇑). Systolic thickening fraction in the nonischemic region remained stable within each day of the protocol during the sequence of LAD occlusions and the subsequent 5 hours of reperfusion (Table 1⇑). In addition, thickening fractions in the nonischemic zone did not differ significantly between the two sequences within the same group at any time point (Table 1⇑). These data indicate that the effects of repetitive coronary occlusions were evaluated in a preparation in which regional myocardial function was otherwise stable.
The baseline (prediazepam) systolic thickening fraction in the region to be rendered ischemic was 30.5±3.3%, 32.3±4.4%, 29.8±2.9%, 31.1±2.6%, and 30.3±2.5% in groups I, II, III, IV, and V, respectively, on the day of the first sequence and 28.2±2.9%, 26.3±4.8%, 28.1±3.2%, 32.1±2.9%, and 29.6±2.7%, respectively, on the day of the second sequence. After administration of diazepam (preocclusion measurements), the values of thickening fraction averaged 29.6±3.1%, 31.8±4.5%, 30.8±2.5%, 30.8±2.8%, and 31.8±2.5%, respectively, on the day of the first sequence and 29.8±3.1%, 29.7±4.0%, 29.1±2.9%, 30.5±2.3%, and 29.6±2.9%, respectively, on the day of the second sequence. There were no significant differences between baseline and preocclusion values in any group. In addition, there were no significant differences in baseline or preocclusion values between the two sequences within the same group.
The extent of paradoxical systolic wall thinning during ischemia did not change significantly with subsequent occlusions, so that it was similar during the 10th occlusion and first occlusion (Figs 1 through 5⇑⇑⇑⇑⇑). The extent of paradoxical systolic thinning during the first or 10th coronary occlusion was also similar during the first and second sequences in all groups (Figs 1 through 5⇑⇑⇑⇑⇑). During each sequence, the recovery of systolic WTh decreased progressively with subsequent occlusion/reperfusion cycles (Figs 1 through 5⇑⇑⇑⇑⇑). (The values at 1 minute after the 10th reflow are not shown; at this time point, the thickening fraction was similar to that recorded at 1 minute after the ninth reflow but then deteriorated over the ensuing 4 minutes.) The values of thickening fraction during the first nine reperfusions were not significantly different between the two sequences within any group. The recovery of thickening fraction after the 10th reperfusion is described separately for each group.
Group I (6-Hour Group)
Fig 1⇑ illustrates systolic WTh in group I, in which the second sequence of LAD occlusions was repeated 6 hours after the first. After the first sequence, regional myocardial function remained significantly depressed for 3 hours, with thickening fraction averaging 12.8±10.0% of preocclusion values at 30 minutes (P<.01 versus preocclusion), 32.2±9.1% at 1 hour (P<.01), 65.8±6.4% at 2 hours (P<.01), and 76.9±4.7% at 3 hours (P<.01) after the 10th reperfusion. Thus, the first sequence of ten 2-minute occlusion/2-minute reperfusion cycles resulted in severe myocardial stunning that lasted, on average, 3 hours. After the second sequence, there was a similar delay in the recovery of contractile function, with WTh averaging 13.7±11.1% of preocclusion values at 30 minutes (P<.01 versus preocclusion), 22.5±11.5% at 1 hour (P<.01), 57.0±11.9% at 2 hours (P<.01), and 73.1±6.9% at 3 hours (P<.01) (Fig 1⇑). The total deficit of WTh was similar to that measured after the first sequence (203±33 arbitrary units after the second sequence versus 190±18 arbitrary units after the first, P=NS) (Fig 6⇓), confirming that the severity of myocardial stunning was similar. These results demonstrate that 6 hours after the preconditioning ischemic insult, no protection had yet appeared against myocardial stunning.
Group II (12-Hour Group)
Fig 2⇑ illustrates WTh in group II, in which the second sequence of LAD occlusions was repeated 12 hours after the first. After the 10th reperfusion of the first sequence, the recovery of myocardial function was delayed, with thickening fraction averaging 24.1±14.6% of preocclusion values at 1 hour (P<.01 versus preocclusion), 50.7±18.0% at 2 hours (P<.01), and 72.1±15.3% at 3 hours (P<.01) (Fig 2⇑). After the 10th reperfusion of the second sequence, 3 of the 6 pigs included in this group displayed a large improvement of recovery of WTh vis-a`-vis the first sequence, with a decrease of >50% in the total deficit, whereas 3 pigs showed a slight improvement of WTh, with a decrease of <20% in the total deficit. Despite this variability, the average WTh after the second sequence was significantly improved compared with the first sequence at 5 minutes (20.5±21.4% after the second sequence versus −9.3±14.8% after the first, P<.01), 15 minutes (32.4±24.4% versus −5.2±11.5%, P<.05), and 3 hours (92.8±3.1% versus 72.1±15.3%, P<.05) (Fig 2⇑). The total deficit of WTh was significantly reduced (137±53 arbitrary units after the second sequence versus 214±33 arbitrary units after the first, P<.05) (Fig 6⇑). Thus, the first sequence of ischemic episodes provided some protection against stunning 12 hours later, although this response was not consistent in all animals.
Group III (24-Hour Group)
Fig 3⇑ illustrates WTh in group III, in which the second sequence was repeated 24 hours after the first. The severity of myocardial stunning after the first sequence was comparable to that of the other groups (Fig 3⇑). Unlike group II, however, all pigs in group III consistently showed a marked enhancement in the recovery of contractile function after the second sequence, with statistically significant differences present at 5 minutes (28.6±7.9% after the second sequence versus 6.3±9.7% after the first, P<.05), 15 minutes (36.1±8.2% versus 15.6±10.4%, P<.05), 30 minutes (63.5±5.9% versus 29.3±11.4%, P<.01), 1 hour (55.9±10.3% versus 25.5±8.1%, P<.01), 2 hours (79.1±4.2% versus 55.7±6.2%, P<.01), and 3 hours (97.6±3.2% versus 75.6±4.9%, P<.01) (Fig 3⇑). The total deficit of WTh was markedly decreased (87±12 arbitrary units after the second sequence versus 197±23 arbitrary units after the first, P<.01) (Fig 6⇑). These results indicate that an effective and consistent protection against stunning developed 24 hours after the first sequence of ischemic insults.
Group IV (3-Day Group)
Fig 4⇑ illustrates WTh in group IV, in which the second sequence was repeated 3 days after the first. The recovery of contractile function after the second sequence was substantially improved at all time points from 5 minutes to 4 hours after the 10th reperfusion, with differences achieving statistical significance at 30 minutes (53.3±6.5% after the second sequence versus 31.1±8.5% after the first, P<.05), 1 hour (66.9±6.2% versus 45.2±9.0%, P<.05), 2 hours (83.7±3.7% versus 46.5±10.3%, P<.01), 3 hours (97.0±2.4% versus 80.9±5.5%, P<.05), and 4 hours (102.3±2.2% versus 90.8±3.8%, P<.05) (Fig 4⇑). The total deficit of WTh was markedly decreased (83±7 arbitrary units after the second sequence versus 180±25 arbitrary units after the first, P<.01) (Fig 6⇑). These changes were consistently observed in all pigs. Thus, the protection against stunning induced by the first sequence of LAD occlusions was still present 3 days later.
Group V (6-Day Group)
Fig 5⇑ illustrates WTh in group V, in which the second sequence was repeated 6 days after the first. The recovery of thickening fraction after the two sequences was similar (Fig 5⇑). The total deficit of WTh was also similar (198±18 arbitrary units after the second sequence versus 178±24 arbitrary units after the first, P=NS) (Fig 6⇑). These results indicate that the protection induced by the first sequence had disappeared 6 days later and that the myocardium had returned to its original (nonpreconditioned) state.
The goal of the present study was to define the time course of late preconditioning against myocardial stunning in the conscious pig. Our salient findings can be summarized as follows: (1) The overall severity of myocardial stunning after the first sequence of ten 2-minute occlusion/2-minute reperfusion cycles was similar among all groups and comparable to our previous observations,14 15 26 further confirming the reliability and reproducibility of this porcine model of postischemic dysfunction. (2) When a second identical sequence of occlusion/reperfusion cycles was repeated at a distance of 6 hours from the first, the severity of myocardial stunning observed after the second sequence was similar to that observed after the first, indicating that the first sequence of ischemic insults did not render the myocardium resistant to myocardial stunning 6 hours later. (3) When the second sequence was repeated at a distance of 12 hours, the severity of myocardial stunning was attenuated to a moderate extent, with a wide variability among animals, indicating that the preconditioning ischemia provided a partial protective effect against stunning at this time. (4) When the second sequence was repeated at a distance of 24 hours, the severity of myocardial stunning was markedly attenuated, indicating that a powerful protection was present. (5) When the second sequence was repeated at a distance of 3 days, the degree of attenuation of myocardial stunning was similar to that observed at 24 hours, indicating that the protective response afforded by late preconditioning persisted unabated. (6) Finally, when the second sequence was repeated at a distance of 6 days, the severity of myocardial stunning was indistinguishable from that observed after the first sequence, indicating that the protection afforded by late preconditioning had disappeared.
Taken together, the results of the present study demonstrate that late preconditioning against myocardial stunning requires >6 hours to develop, lasts for at least 60 hours after its appearance, and disappears within 6 days from the preconditioning ischemia. The protective effect is partially developed at 12 hours and achieves full expression at 24 hours and 3 days. This time course is consistent with the upregulation of cardioprotective gene(s). Because of the prolonged duration of the protective effect, late preconditioning against stunning is of potential clinical importance.
The conscious pig model was used in the present study because this is the preparation in which the phenomenon of late preconditioning against myocardial stunning has been previously described.14 15 The chief advantage of the porcine model is the deficiency of coronary collateral vessels,27 28 29 30 31 32 which eliminates the variability in collateral flow that is inherent in canine models. Indeed, in the present study collateral flow was negligible (≤0.08 mL/min per gram) in all groups and was virtually identical between the first and second sequences of LAD occlusions (Table 2⇑). Since collateral flow is the major determinant of the severity of myocardial stunning,19 elimination of this variable should result in more reproducible postischemic dysfunction among different animals. Our results support this concept. For instance, the first sequence of occlusion/reperfusion cycles caused a comparable degree of myocardial stunning in all groups, and the second sequence caused a degree of stunning similar to the first in groups I and V (Fig 6⇑).
A sequence of ten 2-minute coronary occlusions was used because previous studies14 15 26 have demonstrated that this protocol causes an ischemic burden sufficient to produce significant myocardial stunning while minimizing the risk of VF. Furthermore, experimental models of repetitive coronary occlusions are relevant to many patients with coronary artery disease who experience recurrent bouts of myocardial ischemia in the same territory.33 34 Six hours was chosen as the earliest time point for repeating the second sequence of LAD occlusions, because we have previously found that WTh recovers completely by this time (but not before this time).14 15 26 Six days was chosen as the latest time point for repeating the second sequence, because a previous study14 has shown that the preconditioning effect disappears by 10 days; thus, in the present study, we sought to define more precisely the duration of the protection by examining the severity of stunning at 6 days.
As elaborated previously,14 15 sedation of the pigs with diazepam was necessary to ensure stable hemodynamic conditions within each sequence of occlusion/reperfusion cycles and between the two sequences. Administration of diazepam had no appreciable inotropic or hemodynamic effects (Table 1⇑). The doses of diazepam used were similar between the two sequences in all groups.
Possible Extrinsic Influences on Stunning
As a result of sedation with diazepam, the animals were in a stable state when the coronary occlusions were performed. Basic physiological variables such as arterial blood gases, hematocrit, and body temperature were within normal limits throughout the experimental protocol within each sequence. Similarly, hemodynamic variables such as heart rate, systolic arterial pressure, rate-pressure product, left atrial pressure, and LAD flow did not generally differ within each sequence or between the two sequences (Table 1⇑). The baseline and preocclusion measurements of thickening fraction in the LAD territory, as well as the degree of paradoxical wall thinning during coronary occlusion, were similar between the two sequences within each group (see “Results” and Figs 1 through 5⇑⇑⇑⇑⇑). The similarity of the measurements of thickening fraction in the nonischemic region at the various time points of a given sequence and between the two sequences of a given group (see “Results” and Table 1⇑) further corroborates the concept that regional myocardial function was stable throughout the study. In summary, none of the extrinsic variables known to modulate myocardial stunning can account for the differences in the severity of postischemic dysfunction between the two sequences of occlusion/reperfusion cycles. Therefore, we conclude that the attenuation of contractile dysfunction after the second sequence of LAD occlusions in groups II, III, and IV reflects late preconditioning against stunning.
Time Course of Late Preconditioning
Little information is available regarding the time course of the late phase of ischemic preconditioning. The only published study is a preliminary report by Baxter et al,35 who examined the duration of protection against infarction after a sequence of four 5-minute coronary occlusions in anesthetized rabbits. These authors found that infarct size was reduced at 24, 48, and 72 hours (but not at 96 hours) after the preconditioning ischemia, with the greatest protection observed at 48 and 72 hours. In general, these results are consistent with ours, except that Baxter et al did not investigate the earliest time point at which the late preconditioning effect appears, and the time of maximal protection was 48 hours (instead of 24 hours) after the preconditioning ischemia. On the basis of these findings and the present results, it would appear that the late phase of ischemic preconditioning has a similar time course for myocardial stunning and myocardial infarction. No data have been published regarding the time course of protection against arrhythmias.
A previous study by Shen and Vatner36 in conscious pigs found that a 10-minute coronary artery occlusion did not precondition against the stunning induced by another 10-minute occlusion performed 48 hours later. These results are not necessarily in conflict with ours. The apparent discrepancy could be due to differences in experimental protocols, specifically to the use of a single 10-minute occlusion versus a sequence of ten 2-minute occlusion/2-minute reperfusion cycles. It is possible that the greater ischemic burden in the present study (20 versus 10 minutes) induced a preconditioning effect that would not have been inducible with only 10 minutes of ischemia. In support of this supposition is our recent finding (Y. Qiu and R. Bolli, unpublished data, 1996) in conscious rabbits that late preconditioning is induced by a sequence of three 4-minute coronary occlusions but not by one or two 4-minute occlusions, indicating that there is a “minimum dose” of ischemia that is necessary to precondition the heart. It is also possible that a cluster of several brief (2-minute) episodes of ischemia may be more effective in inducing late preconditioning than a single 10-minute ischemic episode. Indeed, if late preconditioning is mediated by activation of membrane receptors, as suggested by Baxter and colleagues,37 38 multiple bouts of ischemia would result in repetitive stimulation of these receptors, which could activate protecting signaling pathways more effectively than a single stimulation.
Possible Mechanism of Late Preconditioning Against Stunning
It is well established that the mechanism of early preconditioning against infarction involves the activation of adenosine receptors.2 7 39 Data are also available to suggest that this mechanism is involved in late preconditioning against infarction.37 38 However, unlike early and late preconditioning against infarction, late preconditioning against stunning does not appear to require activation of adenosine A1 receptors, since it is not prevented by blockade of these receptors.14 Recently, we have found that administration of antioxidants completely abolishes late preconditioning against stunning, indicating that the oxidative stress associated with the 10 occlusion/reperfusion cycles is necessary for the development of this endogenous cardioprotective response.15
The present findings that late preconditioning against stunning begins to appear at 12 hours and lasts for 3 days are consistent with the concept that it is due to the synthesis and degradation of proteins. The two major classes of cardioprotective protein presently known are antioxidant enzymes and HSPs. We have found that the myocardial levels of HSP70 are increased 24 hours after the initial ischemic stress in this porcine model of late preconditioning.14 Others have reported that a sequence of four 5-minute coronary occlusions results in increased myocardial levels, 24 hours later, of HSP70 and HSP60 in rabbits40 and of manganese-SOD activity in dogs,41 42 concomitant with increased resistance against cell death (late preconditioning against infarction).40 41 Brown and colleagues43 44 have reported that the hearts of rats given endotoxin43 or interleukin-1,44 which are thought to generate free radicals, exhibited increased resistance to ischemia/reperfusion injury 24 to 36 hours later and that this tolerance was associated with an increase in the myocardial activity of catalase. Thus, it seems plausible that the time course of late preconditioning against stunning may reflect the time course of synthesis and degradation of one or more HSPs and/or antioxidant enzymes. Besides antioxidant enzymes and HSPs, however, other proteins may also be induced and contribute to late preconditioning. Further complex studies addressing the time course of protein induction and the existence of a cause-effect relationship will be necessary to elucidate this issue.
Conclusions and Implications
In conclusion, definition of the chronological aspects of late preconditioning against stunning is an indispensable initial step toward unraveling the mechanism of this phenomenon. We have found in conscious pigs that the protection is absent at 6 hours after the preconditioning ischemia, is partially present at 12 hours, reaches maximal expression at 1 and 3 days, and disappears by 6 days. These results provide a framework for investigating the identity of the protein(s) responsible for late preconditioning against stunning, since knowledge of the time course of the protective effect will make it possible to compare the changes in the myocardial levels of a given protein with the changes in the severity of myocardial stunning. In view of the sustained nature of the protective response, which lasts for at least 60 hours, late preconditioning against stunning may have clinical importance.
Selected Abbreviations and Acronyms
|HSP||=||heat stress protein|
|LAD||=||left anterior descending coronary artery|
|LV||=||left ventricle, left ventricular|
This study was supported in part by National Institutes of Health grants R01 HL-43151 and R01 HL-55757 (Dr Bolli).
- Received February 1, 1996.
- Accepted May 22, 1996.
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