Is Calcium a Mediator of Infarct Size Reduction With Preconditioning in Canine Myocardium?
Background The cellular mechanisms by which brief episodes of ischemia protect or “precondition” the heart and limit infarct size caused by a later period of sustained coronary artery occlusion remain unresolved. We propose that calcium may be an important mediator in eliciting this cardioprotection.
Methods and Results To test this hypothesis, anesthetized dogs received a 15-minute intracoronary infusion of 20 mmol/L CaCl2 or saline before undergoing 1 hour of coronary occlusion and 4 hours of reperfusion (protocol 1). Collateral blood flow during occlusion was measured with radiolabeled microspheres, area at risk of infarction (AR) was delineated by injection of blue dye, and area of necrosis (AN) was determined by tetrazolium staining. AN/AR was reduced from 20±5% in the saline-treated controls to 9±3% in CaCl2-treated dogs (P<.05). Additional animals underwent 10 minutes of preconditioning ischemia or a comparable waiting period before the 1-hour test occlusion (protocol 2). Administration of 5-(N,N-dimethyl)-amiloride (an inhibitor of calcium influx via Na+-H+ and Na+-Ca2+ exchange) before the preconditioning stimulus attenuated the protective effect of ischemic preconditioning: AN/AR was 12±1%, larger than the value of 4±1% observed in preconditioned dogs that received saline (P<.05) and comparable to the values of 12±3% and 14±3% seen in saline- and dimethylamiloride-treated controls.
Conclusions Brief intracoronary infusion of CaCl2 mimicked, whereas treatment with dimethylamiloride blocked, infarct size reduction with preconditioning, thereby implicating calcium as a mediator of preconditioning in this canine model.
There is no doubt that one or more episodes of brief ischemia protect or “precondition” the heart and reduce infarct size caused by a subsequent sustained period of coronary artery occlusion. However, the cellular mechanism(s) responsible for this paradoxical protection remain controversial. For example, activation of the adenosine-regulating enzyme ectosolic 5′ nucleotidase and translocation of PKC to myocyte membranes at the onset of sustained occlusion1 2 3 4 have both been proposed to play important roles in eliciting cardioprotection, yet data from our laboratory and others5 6 7 8 have failed to support these hypotheses.
Regulation of ionic homeostasis, particularly calcium, is a crucial determinant of myocyte viability. Considerable evidence indicates that brief, nonlethal episodes of myocardial ischemia are associated with a brief, transient, and reversible increase in cytosolic calcium concentrations, whereas a massive increase in total calcium content is a hallmark of myocytes irreversibly injured by prolonged ischemia/reperfusion.9 10 11 12 13 14 15 This focal role of calcium led us to postulate that a brief, transient, and modest increase in intracellular calcium concentration during the preconditioning stimulus may be an important component of the second-messenger pathway ultimately responsible for the reduction of infarct size seen with ischemic preconditioning. If so, then brief intracoronary infusion of calcium in lieu of brief ischemia should mimic the protective effects of preconditioning. Moreover, because calcium influx during ischemia/reperfusion occurs at least in part as a secondary consequence of intracellular acidosis and resultant activation of Na+-H+ and Na+-Ca2+ exchange,11 12 13 14 15 we further proposed that the selective Na+-H+ exchange inhibitor DMA,16 administered during the preconditioning stimulus, should attenuate the reduction of infarct size seen with preconditioning. Using the anesthetized canine model, we sought to determine whether calcium may serve as a mediator of infarct size reduction with preconditioning by evaluating each of these two corollaries.
This study was approved by the Institutional Animal Care and Use Committee of the Hospital of the Good Samaritan (an American Association for Accreditation of Laboratory Animal Care–certified institution) and conforms to the Position of the American Heart Association on Research Animal Use (Circulation. 1985;71:849).
Seventy-four mongrel dogs weighing between 14 and 32 kg were anesthetized with sodium pentobarbital (30 mg/kg), intubated, and ventilated with room air. After cannulation of the left jugular vein (for administration of fluids and supplemental anesthesia) and the left carotid artery (for measurement of heart rate and arterial pressure), the heart was exposed through a left lateral thoracotomy and suspended in a pericardial cradle. A fluid-filled catheter was positioned in the left atrium for later injection of radiolabeled microspheres (141Ce, 103Ru, or 95Nb) for measurement of RMBF, and a microtipped pressure transducer was positioned in the LV cavity via the left atrial appendage for measurement of LV pressure and its first derivative, LV dP/dt. A segment of the LAD was isolated, usually distal to its first major diagonal branch, for later placement of occlusive vascular clamps, and a second segment was isolated for placement of a Doppler flow probe for measurement of mean CBF.
Protocol 1: Intracoronary Calcium Infusion
In the 28 dogs enrolled into the first limb of the study, a proximal branch of the LAD was cannulated with a 24-gauge catheter, and the tip was advanced into the lumen of the main LAD. Each dog was randomized to receive a 15-minute IC infusion of either CaCl2 (concentration of 20 mmol/L infused at a rate of 0.5 mL/min, ie, 1.47 mg/min) or saline (0.5 mL/min) into the soon-to-be-ischemic LAD bed, followed by a 10-minute saline “washout” at 0.5 mL/min (Fig 1⇓). This “dose” was selected, on the basis of work by Ito et al,17 such that a small but consistent increase in LV dP/dt (suggestive of a modest increase in intracellular calcium concentration) was obtained during CaCl2 infusion compared with baseline but with no significant differences between the saline- and calcium-treated groups. All dogs then received a prophylactic dose of lidocaine (≈1.5 mg/kg IV bolus) and underwent 1 hour of sustained LAD occlusion followed by 4 hours of reperfusion. Hemodynamics and CBF were monitored before and during saline or calcium infusion, before and throughout sustained LAD occlusion, and after sustained reflow. In addition, the severity of ischemia was assessed in all dogs by measurement of RMBF at 30 minutes into the prolonged LAD occlusion.
At the end of the protocol, the LAD was ligated at the site of previous occlusion, and Unisperse blue pigment (0.25 to 0.5 mL/kg) was injected into the coronary vasculature via the left atrial catheter to delineate the in vivo extent of the occluded LAD bed, or AR. Under deep anesthesia, cardiac arrest was produced by intracardiac injection of KCl. The hearts were rapidly excised, cut into five to seven transverse slices, and photographed for later measurement of AR. To distinguish necrotic from viable myocardium, the standard method of incubation for 10 minutes in a 1% solution of triphenyltetrazolium chloride at 37°C was used,5 8 and the heart slices were rephotographed for later calculation of the AN and stored in formalin.
Protocol 2: Effect of DMA on Ischemic Preconditioning
Of the 46 animals entered into the second limb of the study, 20 were assigned to undergo one 10-minute episode of preconditioning ischemia and 30 minutes of reperfusion before the 1-hour sustained test occlusion (Fig 1⇑). Immediately before preconditioning, either DMA (400 μmol/L, n=12) or saline (n=8) was administered directly into the soon-to-be-ischemic LAD territory via ≈30 intramyocardial injections (0.15 mL each) into the subendocardium at a depth of 8 to 10 mm, an approach successfully used by our laboratory and others to facilitate local drug delivery in the absence of deleterious and confounding hemodynamic consequences.5 7 18 The remaining 26 dogs served as controls and received ≈30 intramyocardial injections of DMA (n=15) or saline (n=11) followed by an equivalent 40-minute waiting period before the 1-hour sustained occlusion. Hemodynamics and CBF were monitored at frequent intervals throughout the protocol, RMBF was measured at 30 minutes into the sustained LAD occlusion, and infarct size was delineated as described in protocol 1.
Dogs from either protocol were excluded from analysis according to the following standard prospective criteria: (1) high collateral blood flow, defined as values of RMBF to the “ischemic” subendocardium >0.20 mL·min−1·g tissue−1; (2) a small AR, defined as AR <10% of the LV; or (3) intractable VF, unresponsive to cardioversion with low-energy (20- to 30-J) DC pulses applied directly to the heart.
AR and infarct size. After fixation, right ventricular tissue was trimmed from each heart slice, and the remaining LV tissue was weighed. Photographic images of the heart slices were projected and traced at magnifications of ≈×2 to ×4. The extent of the AR and AN in each heart slice was quantified by computerized planimetry, corrected for the weight of the tissue slice, and summed for each heart.
Regional myocardial blood flow. After LV weights had been obtained, tissue blocks were cut from the center of the previously ischemic LAD bed and remote, normally perfused circumflex bed and divided into subendocardial, midmyocardial, and subepicardial segments. RMBF was then quantified by the standard method described previously.5 8
Hemodynamics and CBF. Heart rate and arterial pressures were measured and averaged over five continuous cardiac cycles in sinus rhythm for each sample period. Mean CBF was also recorded during these same five cardiac cycles and expressed as a percentage of CBF measured at baseline.
Because protocols 1 and 2 were conducted consecutively and not concurrently, statistical analyses were performed separately for each limb of the study. In protocol 1, RMBF, risk region, and infarct size in calcium- and saline-treated animals were compared by unpaired t tests, and the incidence of VF was compared by Fisher’s exact test. ANCOVA was used to determine whether the relationship between infarct size and collateral blood flow differed between the two groups. Hemodynamic parameters were compared at baseline, at 15 minutes into CaCl2/saline infusion, before sustained occlusion, at 30 minutes and 1 hour into sustained occlusion, and at 15 minutes, 1 hour, and 4 hours after reperfusion by two-factor ANOVA (for treatment and time) with repeated measures across the second factor, and if significant F ratios were obtained, subsequent pairwise comparisons were made by Tukey’s test. Statistical comparisons for protocol 2 were done in a similar manner, except discrete variables (RMBF, risk region, infarct size) were compared by ANOVA followed by Tukey’s test. All data are expressed as mean±SEM, and values of P<.05 were considered to be statistically significant.
Mortality and exclusions. Of the 28 dogs enrolled in protocol 1, 14 were assigned to each of the IC CaCl2 and saline infusion groups (Table 1⇓). Seven saline-treated controls and 4 calcium-treated dogs developed VF (P=NS between groups), with successful cardioversion achieved in 2 animals (1 saline and 1 calcium). In addition, 3 dogs that received IC CaCl2 were excluded on the basis of high collateral blood flow. Data are therefore reported for the 8 saline- and 8 calcium-treated dogs that successfully completed the protocol.
Hemodynamics. Baseline values of heart rate, arterial pressure, and dP/dt were similar in both groups (Table 2⇓). Comparison of hemodynamic parameters measured at the end of the 15-minute IC infusion with their respective baseline values revealed, as expected, a small but consistent increase in LV dP/dt in the CaCl2-treated group (1618±159 versus 1416±139 mm Hg/s, P<.01). There was, however, no significant difference with saline infusion (1488 versus 1454 mm Hg/s) and no significant difference between groups. When data obtained throughout infusion, occlusion, and reperfusion were included in the statistical analysis, this modest increase in LV dP/dt with CaCl2 infusion did not achieve significance.
Coronary blood flow remained stable at ≈100% of baseline throughout IC infusion of calcium or saline. All dogs were hyperemic upon reperfusion, with CBF remaining comparable between the groups throughout the 4 hours of reflow.
Regional myocardial blood flow. Collateral blood flow during sustained LAD occlusion was similar in saline- and calcium-treated animals: ie, subendocardial RMBF was 0.06±0.02 and 0.05±0.02 mL·min−1·g−1, respectively (Table 3⇓). In addition, RMBF measured in the normally perfused circumflex bed during LAD occlusion did not differ between groups.
Risk region and infarct size. AR tended to be larger in the calcium-treated group than in the saline controls (23±2% versus 19±1% of the total LV weight, P=.07; Fig 2A⇓). Nonetheless, infarct size in dogs treated with IC CaCl2 averaged only 9±3% of the AR (or 2.1% of the total LV), significantly smaller than the value of 20±5% (or 3.9% of the total LV) observed in animals that received IC saline (P<.05; Fig 2A⇓). This reduction in infarct size with IC CaCl2 was confirmed by ANCOVA: when collateral blood flow was incorporated as a covariate, the relationship between AN/AR and subendocardial RMBF was shifted downward for calcium-treated animals versus saline controls (P<.03; Fig 2B⇓).
Mortality and exclusions. Of the 46 dogs entered into the second limb of the study, 8 received intramyocardial saline and 12 received intramyocardial DMA before 10 minutes of preconditioning ischemia, and 26 controls (11 treated with saline and 15 with DMA) received intramyocardial injections before the 40-minute waiting period (Table 1⇑). Of the 2 saline-preconditioned, 6 DMA-preconditioned, 8 saline control, and 6 DMA control dogs that developed VF (P=NS), both saline-preconditioned, 4 DMA-preconditioned, 2 saline control, and 2 DMA control dogs were successfully resuscitated. Three DMA control dogs were excluded because of high collateral blood flow, and 1 DMA-preconditioned animal was excluded because of technical difficulties. Thus, a total of 5 saline control, 8 DMA control, 8 saline-preconditioned, and 9 DMA-preconditioned animals completed the protocol.
Hemodynamics. Animals assigned to the DMA-preconditioned group tended to have higher values of heart rate, mean arterial pressure, and LV dP/dt at baseline (ie, before randomization), and for both heart rate and arterial pressures, this trend persisted throughout much of the protocol (Table 2⇑). However, only two treatment effects attained statistical significance: heart rate was transiently increased by 16 to 19 bpm with intramyocardial saline injections (P<.05 for the saline-preconditioned group), and peak positive dP/dt in the DMA control cohort increased from 1281 mm Hg/s at baseline to 1500 mm Hg/s immediately before sustained occlusion (P<.05).
Coronary blood flow increased to ≈130% of baseline values in all groups in response to intramyocardial injection of either DMA or saline. CBF returned to 96% to 101% of baseline in all groups before the onset of sustained occlusion, and all dogs exhibited hyperemia upon reperfusion.
Regional myocardial blood flow. Collateral blood flow during sustained occlusion was similar in all groups, ie, mean subendocardial RMBF in the LAD bed ranged from 0.02 to 0.05 mL·min−1·g tissue−1 (Table 3⇑). RMBF in the normally perfused circumflex bed was also comparable among the four cohorts.
Risk region and infarct size. AR in the four groups averaged 18% to 21% of the total LV weight (P=NS; Fig 3A⇓).
Mean infarct size in the saline and DMA control groups was 12±3% and 14±3% of the AR, respectively (P=NS). As expected, AN/AR was significantly smaller in the saline-preconditioned group, averaging only 4±1% (P<.01; Fig 3A⇑), and ANCOVA revealed a significant downward shift in the regression relationship between infarct size and subendocardial collateral blood flow for saline-preconditioned dogs compared with controls (P<.05 versus saline controls; P<.02 versus DMA controls; P<.01 versus all controls; Fig 3B⇑). In contrast, infarct size in the DMA-preconditioned cohort was 12±1%, significantly greater than the value of 4% obtained in saline-preconditioned animals and comparable to the infarct sizes of 12% and 14% seen in the saline and DMA controls (Fig 3A⇑). Moreover, the regression relationship for the DMA-preconditioned group was shifted upward compared with the saline-preconditioned group (P<.01; Fig 3B⇑) and did not differ from that of the controls (P=.9).
The apparently chance trend of higher heart rates and arterial pressures in the DMA-preconditioned group raises the concern that the larger infarct sizes in these animals versus saline-preconditioned dogs were simply due to higher oxygen demand during occlusion. Importantly, however, when AN/AR for the DMA- and saline-preconditioned groups was plotted as a function of hemodynamic parameters measured midway during the sustained occlusion, we observed no correlation between infarct size and heart rate, mean arterial pressure, or their product (all P≥.44; Fig 4⇓). These results indicate that the larger infarct sizes obtained in DMA-preconditioned dogs were due to an attenuation of the preconditioning effect by DMA.
In the present study, we demonstrate that a brief 15-minute IC infusion of CaCl2 before 1 hour of sustained coronary occlusion significantly reduced infarct size compared with controls that received a comparable infusion of saline. In addition, intramyocardial injection of DMA essentially reversed the protective effects of a 10-minute episode of brief preconditioning ischemia. These results support the concept that calcium may serve as a cellular mediator of infarct size reduction with preconditioning in this canine model.
Brief Transient Calcium Infusion Protects the Heart Against Subsequent Sustained Ischemia
Our results showing infarct size reduction with brief infusion of CaCl2 are in agreement with preliminary reports in the dog model19 20 and recent evidence obtained in isolated buffer-perfused rat hearts21 : IC infusion of CaCl219 20 and CaCl2 added to the perfusate21 limited infarct size caused by subsequent sustained occlusion and enhanced the acute recovery of contractile function after relief of ischemia in the two models, respectively. Because 20 mmol/L CaCl2 produced only a modest (≈200 mm Hg/s) increase in LV dP/dt, our results indicate that the cardioprotective effects of brief preischemic administration of calcium are not simply due to relative demand-induced ischemia during infusion.
DMA Attenuates the Cardioprotective Effects of Ischemic Preconditioning
The obvious question arising from protocol 1 is, Does the cardioprotection seen with brief exogenous calcium infusion and ischemic preconditioning share a common pathway? Considerable evidence indicates that transient, nonlethal episodes of ischemia are associated with brief, transient, and reversible increases in cytosolic calcium concentrations.9 10 12 13 14 Calcium influx during brief ischemia is, at least in part, a secondary consequence of intracellular acidosis: the increase in intracellular concentrations of H+ stimulates Na+-H+ exchange, resulting in the extrusion of H+, influx of Na+, and subsequent increase in intracellular calcium via Na+-Ca2+ exchange.11 12 13 15 The importance of this pathway may diminish during prolonged ischemia, because extrusion of H+ and resultant extracellular acidosis serve to inhibit Na+-H+ exchange. However, reperfusion and the resultant washout of extracellular H+ rapidly reactivate Na+-H+ exchange, thereby restoring normal pH at the price of Na+ and subsequent Ca2+ influx.11 12 13 15
To evaluate the potential role of calcium influx via Na+-H+ exchange during the preconditioning stimulus in reduction of infarct size, we administered the selective Na+-H+ exchange inhibitor DMA16 22 by direct intramyocardial injection immediately before 10 minutes of preconditioning ischemia and 30 minutes of intervening reperfusion. Two aspects of our protocol design warrant comment. First, our decision to deliver DMA by intramyocardial injections was based on preliminary experiments in which continuous IC infusion of DMA appeared to precipitate VF (perhaps because of inhibited extrusion of H+ and exacerbated acidosis during brief ischemia/reperfusion), similar to previous observations with high-dose intracoronary infusion of amiloride in anesthetized dogs.23 Our laboratory and others have found intramyocardial injections or microinfusions to be an effective method to administer inotropic and vasoactive compounds such as phorbol myristate acetate, adenosine, and PKC inhibitors5 7 18 directly to the ischemic/reperfused myocardium in the absence of confounding systemic hemodynamic effects. It is important to note, however, that intramyocardial injections per se can initiate cardioprotection: we have found that direct intramyocardial injections of saline alone, made immediately before sustained coronary artery occlusion, significantly limited infarct size in both the rat and dog models because of local stretch and/or focal ischemia at the injection sites.18 Indeed, despite the difference in time course between the two studies (ie, injections made immediately before versus 40 minutes before sustained coronary occlusion), a persistent protective effect of the injections was also observed in the present protocol: mean infarct size in the saline and DMA controls was 12% to 14%, identical to the value of 12% obtained previously in the cohort of dogs in which subendocardial blood flow was <0.20 mL·min−1·g−1 during occlusion18 and smaller than the control values of ≈20% typically observed in our laboratory (and obtained in protocol 1) with 1 hour of LAD occlusion in this model.5 8
A second issue is our choice of 10 minutes of brief preconditioning ischemia followed by 30 minutes of intervening reperfusion. Our objective was to select a preconditioning regimen that, together with our choice of intramyocardial delivery of DMA, would provide a high probability of large interstitial concentrations of DMA being present throughout preconditioning ischemia and upon reperfusion. However, because considerable evidence indicates that amiloride and its analogues are highly cardioprotective if administered during a sustained period of regional or global myocardial ischemia,22 23 24 25 26 we used a prolonged 30-minute period of intervening reflow to facilitate diffusion and dissipation of DMA before the onset of the sustained test occlusion. As expected,27 10 minutes of ischemia plus 30 minutes of reflow was an effective preconditioning stimulus: this was confirmed in initial pilot experiments (ie, infarct size and subendocardial collateral blood flow averaged 5% and 0.02 mL·min−1·g tissue−1 in 3 dogs preconditioned with 10 minutes of ischemia that did not receive saline injections) and supported by the infarct size of 4±1% observed in our saline-preconditioned cohort.
Intramyocardial injections of DMA blocked the protective effects of preconditioning in our model: AN/AR in the DMA-preconditioned group was 12%, significantly larger than the value of 4% obtained in saline-preconditioned dogs and comparable to the infarct sizes of 12% to 14% in the saline and DMA control groups. The fact that infarct size was similar in both control cohorts may be interpreted to suggest that addition of DMA to the injection fluid failed to attenuate the protective effect of the injections per se, thereby implying that the mechanisms responsible for protection by ischemic preconditioning differ from those of local injections. An alternative explanation, however, is that despite the 40-minute interval between injection and the onset of coronary occlusion, residual amounts of DMA were present in the occluded LAD bed at the onset of sustained occlusion and exerted a modest protective effect (approximately equal in magnitude to that achieved by intramyocardial injections) during the prolonged ischemic insult. Although resolution of this latter issue is beyond the scope of this study, the results of protocol 2 nonetheless indicate that DMA abrogated the protection conferred by brief antecedent ischemia/reperfusion.
Comparison With Previous Studies
Several previous studies have sought to document favorable changes in calcium regulation, manifest after repeated brief ischemia or during a later test occlusion, in preconditioned hearts versus controls.28 29 30 31 32 33 In contrast, our specific objective in protocol 2 was to focus on the possible role of brief transient calcium influx via Na+-H+ exchange during the preconditioning stimulus on subsequent infarct size reduction. Our results differ from the one previous study that also endeavored to measure infarct size with administration of a Na+-H+ exchange inhibitor in the setting of brief preconditioning ischemia. In contrast to our observations, Bugge and Ytrehus33 found that ethyl isopropyl amiloride, preconditioning, and the combination of preconditioning plus the amiloride analogue were equally protective in an isolated rat heart model of regional ischemia. Moreover, when the duration of sustained occlusion was prolonged such that the benefits of preconditioning began to wane, concomitant treatment with ethyl isopropyl amiloride augmented (rather than blocked) the reduction in infarct size achieved with preconditioning.33
How can these divergent results be reconciled? Although possible differences in the amiloride analogues used (dimethyl versus ethyl isopropyl amiloride),16 differences between the in vivo and isolated buffer-perfused heart models,11 and/or differences in calcium handling among species34 35 may play a role, a more plausible explanation may lie in the timing of treatment. As discussed previously, we administered intramyocardial injections of DMA 40 minutes before the onset of the test occlusion in an effort to facilitate diffusion and dissipation of the DMA and thereby preclude the well-documented cardioprotection observed when amiloride or its analogues are present during a sustained ischemic insult.22 23 24 25 26 In contrast, in the study by Bugge and Ytrehus, ethyl isopropyl amiloride was added to the perfusate before and throughout the preconditioning stimulus and maintained for 5 minutes into the period of sustained regional ischemia.33 These data suggest an intriguing paradox: prolonged infusion of the amiloride analogue may have abrogated the benefits of preconditioning yet protected the hearts by inhibition of Na+-H+ exchange during sustained occlusion. The concept that an agent or mediator (in this case amiloride) may both initiate protection (when present during sustained occlusion) and have deleterious effects (if administered during the preconditioning stimulus) is not without precedent. For example, a brief increase in intracellular calcium concentration in neurons has been reported to result in a sustained and favorable increase in the excitability of postsynaptic cells,14 36 and formation of oxygen radicals, generally thought to be a deleterious process, has been shown to protect the porcine heart against postischemic dysfunction caused by a brief ischemic insult occurring 1 day later.37 This explanation is speculative, however, and definitive resolution of our findings with those of Bugge and Ytrehus33 awaits further prospective study.
Limitations and Unanswered Questions
Our results imply that brief exposure of myocytes to increased calcium concentrations protects the canine heart against a subsequent 1-hour sustained period of coronary artery occlusion. Direct measurement of intracellular calcium levels during CaCl2 infusion and during brief preconditioning ischemia/reperfusion would clearly strengthen this hypothesis but is beyond the scope of our in vivo model. The results obtained with DMA treatment further imply that in the setting of brief preconditioning ischemia, influx of calcium occurs in large part via Na+-H+ exchange. As with all other studies performed with these agents in intact heart models, this conclusion is potentially confounded by the fact that even “selective” analogues of amiloride, including DMA, can also act on other ion transport systems, protein kinases, and enzymes.16 Importantly, however, the concentrations of DMA required to inhibit the Na+ channel, Na+-Ca2+ exchange, kinase activity, etc, are 10- to 1000-fold higher than the 400 μmol/L DMA briefly present (before diffusion) at the focal intramyocardial injection sites.
Although the results from both protocols 1 and 2 are consistent with the concept that calcium plays a role in initiating cardioprotection, the present study (1) does not conclusively prove that the cellular mechanisms responsible for infarct size reduction with CaCl2 infusion are the same as those for infarct size reduction with ischemic preconditioning; (2) does not address the possible relationship between calcium and other known or suspected mediators of preconditioning; and (3) was not designed to identify the second messengers, cellular pathways, or end effectors involved in the ultimate limitation of infarct size with either brief calcium infusion or brief preconditioning ischemia. Although these important ancillary questions remain to be answered, our results nonetheless implicate calcium as an important mediator of infarct size reduction achieved with preconditioning in the canine model.
Selected Abbreviations and Acronyms
|AN||=||area of necrosis|
|AR||=||area at risk of infarction|
|CBF||=||coronary blood flow|
|LAD||=||left anterior descending coronary artery|
|PKC||=||protein kinase C|
|RMBF||=||regional myocardial blood flow|
Presented in part at the 45th Annual Scientific Sessions of the American College of Cardiology, Orlando, Fla, March 24-27, 1996, and the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 11-14, 1996, and published in abstract form (J Am Coll Cardiol. 1996;27[suppl A]:292A and Circulation. 1996;94[suppl I]:I-424).
- Received November 5, 1996.
- Revision received February 12, 1997.
- Accepted February 20, 1997.
- Copyright © 1997 by American Heart Association
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