Does Ischemic Preconditioning Trigger Translocation of Protein Kinase C in the Canine Model?
Background Brief episodes of ischemia protect or “precondition” the heart and reduce the size of infarcts caused by subsequent sustained coronary artery occlusion, yet the mechanisms responsible for this cardioprotection remain unresolved. We tested the theory that translocation of protein kinase C (PKC) to the myocyte membranes, initiated in response to brief preconditioning ischemia and manifest during the initial minutes of the sustained occlusion, mediates this phenomenon by attempting to (1) blunt the cardioprotective effects of preconditioning by administration of the PKC inhibitors H-7 and polymyxin B, (2) visualize by fluorescence staining and confocal microscopy changes in the amount or location of PKC, and (3) quantify by incorporation of 32P into PKC-specific peptide changes in the subcellular distribution of PKC in preconditioned versus control hearts.
Methods and Results In the first three limbs of this study, anesthetized open-chest dogs underwent four 5-minute episodes of preconditioning ischemia or a comparable control period before 1 hour of sustained occlusion and 4 to 5 hours of reperfusion. Collateral blood flow was assessed with radioactive microspheres; area at risk (AR) was delineated by injection of blue dye; and the area of necrosis (AN) was measured by tetrazolium staining. AN/AR was smaller in preconditioned versus control dogs that received no treatment (6±2% versus 19±3%, P<.01), H-7 (2±2% versus 14±5%, P<.02), or polymyxin B (10±3% versus 29±5%, P<.01) during the preconditioning or control period. Additional dogs underwent four 5-minute episodes of ischemia, with biopsies obtained at baseline and after the first and fourth occlusions. Frozen sections were stained with a fluorescent probe for active PKC and viewed with confocal microscopy. No differences in the intensity or distribution of fluorescence staining were observed after brief ischemia compared with baseline. Finally, myocardial samples were obtained from dogs subjected to four 5-minute episodes of preconditioning ischemia and time-matched sham-operated controls. Incorporation of 32P into PKC-specific peptide revealed no quantitative difference in the subcellular distribution of PKC between control and preconditioned cohorts.
Conclusions H-7 and polymyxin B did not blunt the reduction in infarct size achieved with ischemic preconditioning. Neither fluorescence staining and confocal microscopy nor biochemical quantification revealed evidence of preconditioning-induced translocation of PKC to the cell membranes. These results fail to support the hypothesis that translocation of PKC, triggered by preconditioning ischemia, is an important mechanism for the reduction in infarct size seen with preconditioning in the dog model.
In 1986, Murry and colleagues1 made the seminal observation that brief periods of ischemia paradoxically protect or “precondition” the canine heart and limit the size of infarcts resulting from a subsequent episode of sustained coronary artery occlusion. Numerous studies have since confirmed this phenomenon in every species tested,2 3 4 5 and recent indirect evidence suggests that brief antecedent ischemia may also protect the human heart.6 7 Despite intensive investigation, however, the mechanisms responsible for the cardioprotective effects of preconditioning remain unresolved.
One theory, derived largely from indirect evidence in the rabbit model,8 9 suggests that brief preconditioning ischemia triggers a sequence of events that initiate the activation and subsequent translocation of protein kinase C (PKC) from the cytosol to the myocyte membranes. PKC is proposed to remain on cell membranes during the intervening brief reperfusion,8 where it would then be strategically positioned during the early minutes of the subsequent sustained occlusion to facilitate phosphorylation of an as-yet-unidentified membrane-bound effector protein and thereby elicit the ultimate reduction in infarct size.8 9 That is, cardioprotection achieved with preconditioning has been proposed to be critically dependent on the presence of PKC on myocyte membranes early during the sustained ischemic insult.
Using the anesthetized canine model, we tested this hypothesis that translocation of PKC to the cell membranes, initiated in response to brief ischemia and manifest during the initial stages of the sustained occlusion,8 9 plays a pivotal role in the reduction in infarct size seen with ischemic preconditioning. Specifically, we sought to (1) blunt the cardioprotective effects of preconditioning by administration of the PKC inhibitors H-7 or polymyxin B; (2) visualize, by fluorescence staining and confocal microscopy, changes in the intensity, pattern, or distribution of PKC staining in response to brief preconditioning ischemia; and (3) quantify, by incorporation of 32P into PKC-specific peptide, changes in the subcellular distribution of PKC during the initial minutes of the sustained occlusion.
This study was approved by the Institutional Animal Care and Use Committee of the Hospital of the Good Samaritan and conforms to the position of the American Heart Association on research animal use.
The study consisted of five discrete protocols: reduction in infarct size with preconditioning was confirmed; the effect of H-7 on infarct size was assessed in control and preconditioned groups; polymyxin B was administered to control and preconditioned cohorts; tissue was harvested from a fourth group of preconditioned dogs for fluorescence staining; and tissue blocks were sampled from control and preconditioned cohorts for biochemical quantification of PKC activity.
Protocol 1: Reduction of Infarct Size With Preconditioning
Twenty-four dogs were anesthetized with sodium pentobarbital (30 mg/kg), intubated, and ventilated with room air. After the left jugular vein (for administration of fluids and drugs) and the left carotid artery (for measurement of heart rate and arterial pressure) were cannulated, 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 regional myocardial blood flow (RMBF). A segment of the left anterior descending coronary artery (LAD) was isolated, usually distal to its first major diagonal branch, for later placement of occlusive vascular clamps; a second segment was isolated for placement of a Doppler flow probe for measurement of mean coronary blood flow (CBF).
After baseline hemodynamic measurements were obtained, the dogs were randomly assigned to undergo four 5-minute episodes of LAD occlusion, each interrupted by 5 minutes of reperfusion (preconditioned group) or a 40-minute no-intervention period (control group). All dogs then underwent 1 hour of sustained LAD occlusion followed by 5 hours of reperfusion.
Heart rate, arterial pressure, and CBF were monitored during the preconditioning or no-intervention period, 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 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 through the left atrial catheter to delineate the in vivo extent of the occluded LAD bed or area at risk of infarction (AR).2 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. The heart slices were then incubated for 10 minutes in a 1% solution of triphenyltetrazolium chloride at 37°C to distinguish necrotic from viable myocardium,2 10 rephotographed for later calculation of the area of necrosis (AN), and stored in formalin.
Protocol 2: Effect of H-7 on Infarct Size
Twenty-five dogs were assigned to undergo either four 5-minute episodes of preconditioning ischemia or a 40-minute control period. In this limb of the study, all dogs received H-7 (Calbiochem) dissolved in sterile water.
Fifteen dogs (9 control and 6 preconditioned) received 4 mg/kg IV H-7, given as 1 mg/kg bolus at 5 minutes before the preconditioning or no-intervention period, 2 mg/kg infusion throughout the preconditioning or no-intervention period and continued for the first 10 minutes of the sustained LAD occlusion, and 1 mg/kg IV bolus immediately before the sustained ischemia. Because inhibition of PKC has been associated with relaxation of vascular smooth muscle, our dose was based on preliminary experiments that showed that 4 mg/kg H-7 produced consistent alterations in mean arterial pressure and CBF. All dogs then underwent 1 hour of sustained LAD occlusion and 4 hours of reperfusion.
Ten dogs were preconditioned while receiving a continuous intracoronary infusion of H-7. A 4F catheter was introduced through the right femoral artery and positioned so that the catheter tip was immediately distal to the site of subsequent LAD occlusion (n=2), or the first side branch of the LAD was directly cannulated with a 24-gauge catheter and the tip advanced into the lumen of the main LAD (n=8). Five minutes after the onset of treatment, the dogs underwent four 5-minute episodes of LAD occlusion, with continuous H-7 infusion into the LAD bed throughout each brief ischemia and reperfusion period. All dogs then underwent 1 hour of sustained LAD occlusion and 4 hours of reperfusion, with infusion of H-7 terminated 10 minutes into the sustained ischemia. All dogs received 20 mg H-7 dissolved in 20 mL sterile water.
For all dogs in protocol 2, heart rate, arterial pressure, and CBF were monitored before and during sustained LAD occlusion and throughout reperfusion, whereas RMBF was assessed 30 minutes into the sustained ischemic insult. AR and AN were measured as described for protocol 1.
Protocol 3: Effect of Polymyxin B on Infarct Size
Nineteen dogs received a continuous IV infusion of polymyxin B sulfate (Sigma Chemical Co; 50 mg/kg dissolved in 10 mL ethanol), an agent reported to block the cardioprotective effects of preconditioning in the rabbit model.9
Five minutes after the onset of infusion, the dogs were randomly assigned to either control or preconditioned groups as detailed for protocols 1 and 2, followed by 1 hour of sustained LAD occlusion and 4 hours of reperfusion. Infusion of polymyxin B was terminated 10 minutes into the sustained occlusion. Hemodynamics, CBF, RMBF, AR, and AN were measured as described previously.
Protocol 4: Direct Visualization of PKC
Six dogs underwent four 5-minute episodes of LAD occlusion, each interrupted by 5 minutes of reperfusion. Multiple (two to four) transmural needle biopsies were taken from the center of the LAD bed at baseline and 5 minutes after the final brief ischemia (ie, the time at which we would begin the sustained occlusion in our typical preconditioning protocols), immediately frozen in liquid nitrogen, and subdivided into endocardial and epicardial halves. Because the reduction in infarct size achieved with preconditioning in this model is manifest in the subendocardium,1 2 the endocardial halves were stored at −80°C until processed, and the epicardial samples were discarded. To determine whether brief ischemia triggered an early but transient translocation of PKC, biopsies were also obtained in 4 of 6 dogs after the first 5-minute occlusion. In 4 of the 6 dogs, additional biopsies were taken at baseline and incubated for 10 minutes in 200 nmol/L phorbol myristate acetate (PMA, a potent activator of PKC)11 or PBS before freezing.
All animals were euthanitized immediately after the preconditioning regimen. Hemodynamics, CBF, and RMBF were not assessed in this limb of the study, but the severity of epicardial cyanosis and incidence of ventricular ectopy were noted for each animal.
Protocol 5: Biochemical Quantification of PKC
Eight dogs were randomly assigned to undergo four 5-minute episodes of LAD occlusion, each interrupted by 5 minutes of reperfusion; 7 sham-operated controls received an equivalent 40-minute period of uninterrupted perfusion. Five minutes after the final brief ischemia (ie, at what would be the start of the sustained occlusion), the dogs were euthanitized under deep anesthesia and the hearts were rapidly excised. Transmural tissue blocks (each weighing ≈2 g) were cut from the center of the LAD bed and remote normally perfused circumflex territory, immediately frozen in liquid nitrogen, and stored at −80°C until processed.
Eight dogs were randomized to undergo brief preconditioning ischemia (n=3) or no intervention (n=5) as described above. To determine whether PKC was translocated during the early minutes of what would be the subsequent sustained occlusion, the LAD was then ligated in all dogs, and 10 minutes into occlusion, tissue blocks were rapidly harvested and frozen.
An additional 2 dogs received injections of PMA (30 mmol/L) directly into the left ventricular (LV) wall: 13 to 18 intramyocardial injections,12 each with a volume of 0.15 mL, were tightly clustered into an area occupying ≈2.5 to 4 cm2. One animal received one set of injections; in the second dog, PMA was injected into three separate regions of the anterior LV wall. At 40 minutes after injection, transmural tissue blocks from the center of each injection site were rapidly excised and frozen as described above.
Hemodynamics, CBF, and RMBF were not assessed in this protocol, but for all dogs undergoing ischemia and reperfusion, the severity of epicardial cyanosis and incidence of ventricular ectopy were noted.
AR and Infarct Size (Protocols 1 through 3)
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 approximately 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. All measurements were made without knowledge of the treatment group.
RMBF (Protocols 1 through 3)
After LV weights were 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 with the standard technique of Domenech et al.13
Hemodynamics and CBF (Protocols 1 through 3)
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 percent of CBF measured at baseline.
Fluorescence Microscopy (Protocol 4)
Frozen subendocardial samples were cut in cross section at a thickness of 5 mm, and the sections (two to eight per sample) were thawed on albumin-coated glass slides. The sections were then stained with a bisindolylmaleimide PKC inhibitor conjugated to fluorescein (fim, Teflabs), a fluorescent probe recently developed and validated for visualization of PKC in cell monolayers.14 Specifically, the sections were fixed for 10 minutes in 3.75% paraformaldehyde, permeabilized for 10 minutes in methanol at −20°C, washed twice in PBS, incubated for 2 hours in fim (200 nmol/L), washed four times in PBS, and covered with a coverslip and an aqueous nonbleaching mounting medium. We made only one minor modification to the original method: we increased the duration of incubation in fim from 30 minutes for the cell monolayers14 to 2 hours for our cryosections. To avoid heterogeneity in staining intensity, all samples from each dog were processed and stained together.
The sections were viewed under argon laser illumination by use of a Zeiss confocal laser scanning microscope. Fluorescent images for semiquantitative analysis were obtained from fields of view in which the myocytes (≈20 per field) appeared in cross section, and hard copies were generated at magnifications of 900× to 1100× with a video printer.
The fluorescent images were graded in terms of the overall brightness of fluorescence and the pattern of fluorescence within the myocytes and on the outer myocyte membranes. First, the intensity of fluorescence in each preconditioned and PMA-incubated sample was graded on a scale of −1 to +4 with respect to the brightness of the corresponding baseline samples; the maximum score of 4 was assigned to the brightest of all samples obtained in the study; 0 denoted no change in brightness; and −1 denoted a decrease in the intensity of fluorescence compared with baseline. Each image obtained from the baseline, preconditioned, and PMA-incubated samples was then reevaluated for the pattern of fluorescence. Fluorescence within the myocytes was assigned a score of 0 if absent, 1 if present as diffuse particles, 2 if seen as a mixture of particles and confluent strands, or 3 if present as confluent filaments. Similarly, the pattern of fluorescence on the myocyte membranes was graded as 0 if absent, 1 if present but diffuse, 2 if present and moderate, or 3 if present and confluent around the margins of the cells.
PKC Assay (Protocol 5)
Tissue was processed as described by Hattori et al.15 Specifically, all samples were rinsed and homogenized in a buffer containing 50 mmol/L Tris, pH 7.5; 1 mmol/L EDTA; 2 mmol/L EGTA; 10 mmol/L benzaminidine; 0.3% β-mercaptoethanol; and 50 mg/mL PMSF. Four milliliters of buffer was used per 1 g tissue. Samples were homogenized (VirTis Homogenizer, model 23) at 4°C with five 5-second pulses at maximum speed, each separated by 10 seconds. Homogenates then underwent ultracentrifugation for 1 hour at 105 000g with a 70.1 titanium rotor. The obtained supernatant (the cytosolic fraction) was stored overnight in 50% glycerol at −20°C. The sediment was dispersed in a buffer containing all components described above, with the addition of 0.3% Triton X-100. Samples were then solubilized for 1 hour by applying continuous stirring at 4°C and separated by 1 hour of ultracentrifugation at 105 000g. The obtained supernatant (the particulate fraction) was then stored overnight in 50% glycerol at −20°C. The purity of the cytosolic and particulate fractions was confirmed by microscopic examination. In addition, we confirmed in three samples that the activity of lactate dehydrogenase (LDH, a cytosolic enzyme) was, as expected, virtually entirely present in the cytosolic fraction: of the 360±43 U · min−1 · g−1 (mean±SD) LDH initially measured in the whole-tissue homogenates, 330±10 U · min−1 · g−1 was subsequently detected in the cytosolic fractions. The protein yield in each fraction was determined with a standard kit (procedure 690, Sigma Chemical Co).
PKC was assayed in both the cytosolic and particulate fractions with a commercial kit (Protein Kinase C Enzyme Assay System, Amersham). The fractions were diluted with buffer containing all components described above, except for the ion chelators EDTA and EGTA. Aliquots of the diluted fractions corresponding to 0.65 mg tissue were incubated for 15 minutes with 0.2 μCi of 32P-γ-ATP (specific activity, 3000 Ci/mmol/L; Amersham) according to the Amersham protocol. Separation of peptide-bound radioactivity was performed by use of SpinZyme Basic Separation Units (Pierce). Radioactivity was determined with a Beckman liquid scintillation counter, and blanks for calcium phospholipid-independent PKC activity and endogenous phosphorylation were subtracted from the total counts for each sample. Duplicate aliquots were assayed for all samples. Results were expressed both as picomoles of 32P incorporated into the threonine group of PKC-specific peptide per minute per gram of tissue and as picomoles per minute per milligram of protein. Assays were performed without knowledge of the treatment groups.
For protocols 1 through 3, dogs were excluded from analysis according to the following prospective criteria: (1) high collateral blood flow during LAD occlusion, defined as values of RMBF in the “ischemic” subendocardium >0.20 mL · min−1 ·g−1; (2) a small AR, defined as <10% of the LV; or (3) obvious epicardial cyanosis during dissections of the LAD or placement of the coronary flow probe. For protocols 4 and 5, the prospective exclusion criteria were the absence of obvious epicardial cyanosis, dyskinesis, and ectopy during the preconditioning regimen. For all protocols, dogs that developed ventricular fibrillation were not resuscitated.
Because protocols 1 through 3 were performed discretely (ie, dogs were assigned to one of two groups and not one of six interventions), statistical analysis was performed separately for each limb of the study. In each protocol, RMBF, AR, and infarct size were compared between the corresponding control and preconditioned groups by unpaired t tests. ANCOVA was used to determine whether the relation between infarct size and collateral blood flow differed between control and preconditioned groups. Hemodynamic parameters were compared at baseline, before sustained occlusion, 30 minutes into sustained occlusion, and 4 hours after reperfusion with 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 with Tukey’s test.
For protocol 4, semiquantitative scores for the brightness and patterns of fluorescence in the baseline, preconditioned, and PMA-incubated samples were compared with the Friedman nonparametric repeated-measures test.
For protocol 5, differences in PKC activity between control and preconditioned groups were compared by use of three-factor ANOVA for treatment (control versus preconditioned), site (LAD versus circumflex territory) and tissue fraction (cytosol versus particulate). Differences in PKC activity in the LAD bed among dogs that received preconditioning or no intervention alone, preconditioning or no intervention followed by 10 minutes of sustained coronary occlusion, and PMA injection were compared by use of two-factor ANOVA (for treatment and fraction). If significant F ratios were obtained, pairwise comparisons were made with Tukey’s test.
All data are expressed as mean±SEM.
Mortality and Exclusions
Of the 24 dogs in protocol 1, 1 preconditioned dog succumbed to ventricular fibrillation on the first brief reflow, 4 died during sustained coronary occlusion (3 control dogs and 1 preconditioned dog), 1 control dog died at the time of sustained reperfusion, and 1 preconditioned dog fibrillated 2 hours after reflow (Table 1⇓). In addition, 1 control dog was excluded because of possible inadvertent “preconditioning” (ie, severe and sustained cyanosis) on placement of the coronary flow probe. Data are therefore reported for the 9 control and 7 preconditioned dogs that successfully completed the protocol.
Heart rate and arterial pressure did not differ between the two groups at baseline and remained comparable throughout the preconditioning or no-intervention period, during sustained coronary occlusion, and after reperfusion (Table 2⇓).
Coronary Blood Flow
In the control group, CBF remained constant during the no-intervention period. As expected, however, CBF was reduced to 0 during each episode of brief preconditioning ischemia, rebounded to >100% of baseline values during each period of brief reflow, and remained hyperemic at the onset of sustained coronary occlusion (Table 2⇑). During the 4 hours of sustained reperfusion, CBF remained comparable in both control and preconditioned dogs.
Regional Myocardial Blood Flow
Both groups were equally ischemic during sustained coronary occlusion, with subendocardial RMBF reduced to 0.06±0.02 and 0.09±0.03 mL · min−1 · g−1 tissue in the control and preconditioned dogs, respectively (Table 3⇓). RMBF measured in the normally perfused circumflex bed during LAD occlusion was also similar in both groups.
AR and Infarct Size
AR did not differ between the two groups, averaging 19±2% versus 19±1% of the LV in control and preconditioned dogs, respectively (Fig 1⇓). As expected, however, the extent of necrosis was significantly smaller in preconditioned compared with control dogs: mean infarct size was 6±2% versus 19±3% of the AR in the two groups (P<.01), and when collateral blood flow was incorporated as a covariate, the relation between AN/AR and subendocardial collateral blood flow was shifted downward for preconditioned versus control dogs (P<.01).
Mortality and Exclusions
Of the 15 dogs that received 4 mg/kg IV H-7, 1 preconditioned dog developed ventricular fibrillation at the second brief reflow, 2 control dogs died during sustained coronary occlusion, and 1 control dog fibrillated at the time of sustained reperfusion (Table 1⇑). Of the 10 preconditioned dogs that received intracoronary H-7, 1 died at the first brief reflow, 1 died during sustained occlusion, 2 fibrillated at the time of sustained reperfusion, and 1 was excluded on the basis of high collateral blood flow. Thus, 6 control dogs treated with 4 mg/kg IV H-7, 5 preconditioned dogs that received 4 mg/kg IV H-7, and 5 preconditioned dogs that received intracoronary H-7 are included in the analysis.
H-7 treatment was associated with modest bradycardia: heart rate decreased significantly in the intravenous H-7 preconditioned group between baseline and preocclusion and tended to decrease in both the control and preconditioned dogs that received intracoronary H-7 (Table 2⇑).
Intravenous administration of H-7 markedly reduced arterial pressure in both the control and preconditioned cohorts, with mean arterial pressure averaging 99±15 and 102±11 mm Hg at baseline versus 73±15 and 70±9 mm Hg before sustained occlusion in the two groups, respectively (P<.01 versus baseline for each group; P=NS between the two groups). This effect was reversed on termination of the H-7 infusion, and by 30 minutes into the sustained coronary occlusion, mean arterial pressure had recovered to baseline values. As expected, however, mean arterial pressure was not decreased by local intracoronary delivery of H-7.
Coronary Blood Flow
In dogs that received intracoronary H-7, CBF immediately increased to 188±15% of baseline within seconds of the onset of infusion. CBF also tended to increase, albeit less dramatically, during the 40-minute preocclusion period in control dogs treated with 4 mg/kg IV H-7, averaging 132±16% of baseline before the onset of the sustained coronary occlusion (Table 2⇑).
Regional Myocardial Blood Flow
Control and preconditioned dogs underwent a similar ischemic insult during sustained coronary occlusion: subendocardial RMBF in the LAD bed was 0.06±0.02, 0.09±0.03, and 0.07±0.03 mL · min−1 · g−1 in the control group, preconditioned dogs treated with 4 mg/kg IV H-7, and preconditioned dogs that received intracoronary H-7, respectively (Table 3⇑). RMBF in the normally perfused circumflex bed was also comparable among the control and preconditioned groups.
AR and Infarct Size
AR averaged 22±2%, 24±1%, and 20±3% of the total LV weight in control dogs, preconditioned dogs that received intravenous H-7, and preconditioned dogs that received intracoronary H-7 (Fig 2⇓).
Mean infarct size in control animals that received H-7 was 14±5% of the AR. In contrast, infarct size in preconditioned dogs treated with H-7 was significantly smaller (P<.02), averaging only 3±3% and 2±2% of the AR in the cohorts that received intravenous and intracoronary H-7, respectively. This persistent reduction in infarct size in preconditioned dogs despite treatment with H-7 was further confirmed by ANCOVA; the regression relation for the preconditioned dogs differed significantly from that of the control group (P<.02).
Mortality and Exclusions
Of the 19 dogs entered into the third limb of the study, 1 preconditioned dog died during sustained coronary occlusion, 5 preconditioned dogs fibrillated on sustained reperfusion, and 1 preconditioned dog developed asystole and died 5 minutes after reflow. Thus, 6 polymyxin-treated control dogs and 6 preconditioned dogs that received polymyxin completed the protocol.
Polymyxin B had a profound and sustained bradycardic effect in all dogs (Table 2⇑). Heart rate was reduced from 147±12 and 157±10 beats per minute (bpm) at baseline to 112±7 and 106±6 bpm before sustained occlusion in the control and preconditioned dogs, respectively (P<.01 versus baseline for each group; P=NS between groups). Moreover, heart rate remained significantly depressed with respect to baseline throughout the entire protocol.
Mean arterial pressure in both the control and preconditioned groups treated with polymyxin B tended to decrease between baseline and preocclusion and differed significantly with respect to baseline 30 minutes into the sustained occlusion. This dilator response persisted into the first 30 minutes of reflow but had resolved by 4 hours after reperfusion. There were, however, no differences in mean arterial pressure between the control and preconditioned groups at any time during the protocol.
Coronary Blood Flow
Infusion of polymyxin profoundly decreased coronary blood flow (Table 2⇑). In control animals, CBF measured before sustained occlusion was reduced to 66±13% of baseline values (P<.05 versus baseline), whereas in the preconditioned group, CBF measured preocclusion (ie, during the last brief reperfusion) averaged only 88±23%. That is, polymyxin ablated the hyperemic response typically observed after brief preconditioning ischemia.
Regional Myocardial Blood Flow
All dogs treated with polymyxin B were rendered severely ischemic during LAD occlusion: subendocardial blood flow averaged only 0.01±0.01 and 0.02±0.01 mL · min−1 · g−1 in the control and preconditioned cohorts (Table 3⇑). However, RMBF was comparable between the two polymyxin-treated groups in both the LAD and circumflex beds.
AR and Infarct Size
AR was similar in both the control and preconditioned groups, averaging 24±2% and 21±2% of the LV, respectively (Fig 3⇓).
Infarct size in control animals averaged 29±5% of the AR. In contrast, infarct size in the preconditioned group was significantly smaller at 10±3% of the myocardium at risk (P<.01 by t test and P=.02 by ANCOVA versus control dogs). That is, preconditioning continued to limit infarct size despite infusion of polymyxin B.
Mortality and Exclusions
All 6 dogs entered into protocol 4 successfully completed the four brief episodes of ischemia/reperfusion, and all dogs exhibited obvious epicardial cyanosis and dyskinesis during the LAD occlusions (Table 1⇑). Therefore, samples from all 6 dogs are included in the analysis.
All baseline samples were consistently characterized by the presence of particulate and/or strand-like fluorescence within the myocytes (Fig 4⇓, left). This strandlike staining pattern was described previously in contractile cells and is thought to represent myofibrillar or cytoskeletal localization of PKC.14 16 17 Baseline biopsies from 5 of the 6 dogs were assessed for fluorescence on the outer sarcolemmal membranes (cross-sectional fields of view were not obtained for 1 dog); regions of particulate fluorescence were evident on the outer membranes of virtually all myocytes in the baseline samples.
All biopsies incubated for 10 minutes in 200 nmol/L PMA exhibited intense fluorescence relative to baseline, with a mean brightness score of 3.0±0.58 (P<.05 versus the arbitrary baseline value of 0; Fig 5⇓). In addition, incubation in PMA tended to increase the confluence of strandlike fluorescence within the myocytes; the mean score for the pattern of fluorescence within the cells averaged 2.63±0.24 compared with 1.92±0.08 in the baseline samples (P=.07; Fig 5⇓). We did not, however, observe an increase in the confluence of staining on the outer myocyte membranes with 10 minutes of PMA incubation (score, 0.67±0.17 versus 1.10±0.24 at baseline; P=NS; Fig 5⇓).
In contrast, all samples obtained after four 5-minute episodes of ischemia (ie, at what would be the start of the sustained occlusion) appeared qualitatively similar in terms of both intensity and pattern of fluorescence to those obtained at baseline (Fig 4⇑, right). This was confirmed by semiquantitative analysis: the mean brightness score was 0.33±0.49 versus the arbitrary value of 0; the grade for confluence of fim staining within the myocytes remained unchanged at 1.92±0.08; and the confluence of fluorescent staining on the outer myocyte membranes was 1.17±0.38 versus 1.10±0.24 for the preconditioned versus control images, respectively (P=NS; Fig 5⇑). Biopsies obtained after the first cycle of brief ischemia, although not graded semiquantitatively, also showed no differences from either the control or preconditioned samples. Thus, we observed no change in the intensity or location of fluorescence staining after either one or four 5-minute episodes of brief preconditioning ischemia.
Mortality and Exclusions
Of the 15 dogs randomized to receive four episodes of brief ischemia or no intervention, 2 preconditioned dogsdied of ventricular fibrillation (1 during the first occlusion and 1 in the fourth reperfusion; Table 1⇑). All surviving preconditioned dogs exhibited obvious epicardial cyanosis and dyskinesis during coronary occlusion. Thus, PKC activity after the preconditioning regimen was assessed in 6 dogs subjected to repeated brief ischemia and 7 sham-operated controls. Of the 8 dogs assigned to undergo either preconditioning or no intervention followed by “sustained” ischemia, 1 control dog fibrillated during occlusion, leaving 4 control and 3 preconditioned dogs for analysis. All tissue samples were successfully harvested from dogs receiving intramyocardial injections of PMA.
In control dogs that received 40 minutes of uninterrupted LAD perfusion, total PKC activity in the LAD bed was 4559±353 pmol · min−1 · g−1 tissue (Fig 6⇓). As expected, PKC activity was significantly higher in the cytosol versus the particulate fraction: 3409±236 versus 1150±162 pmol · min−1 · g−1, respectively; P<.0001. No change in the amount or distribution of PKC in the LAD bed was observed after four 5-minute episodes of preconditioning ischemia (at what would be the start of the sustained occlusion): total PKC activity was 5110±684 pmol · min−1 · g−1, with 3924±502 pmol · min−1 · g−1 present in the cytosolic fraction and 1186±217 pmol · min−1 · g−1 in the particulate compartment (P=NS versus sham-operated control dogs). Similar values were obtained in both groups in the normally perfused circumflex territory. Protein yield was comparable among samples and between groups, and there was no difference in PKC activity between the control and preconditioned cohorts in either the LAD or circumflex beds when the data were expressed per milligram of protein (data not shown).
As expected, intramyocardial injection of PMA resulted in a marked redistribution of PKC. Total PKC activity was increased to 7352±708 pmol · min−1 · g−1(P<.01 versus dogs that received preconditioning or no intervention alone), with only 2422±203 pmol · min−1 · g−1 detected in the cytosol and 4930±726 pmol · min−1 · g−1 in the particulate fraction (P<.05, P<.01 versus the corresponding fractions in dogs that received preconditioning or no intervention alone).
In all dogs (both control and preconditioned groups) killed 10 minutes into sustained LAD occlusion, total PKC in the LAD bed was also increased, averaging 6854±297 and 6132±373 pmol · min−1 · g−1, respectively (P<.01 versus dogs that received preconditioning or no intervention alone; P=NS between groups). PKC in the cytosolic fraction was 4882±319 and 3943±319 pmol · min−1 · g−1 and did not differ significantly from that observed after 40 minutes of preconditioning or uninterrupted perfusion. Ten minutes of sustained occlusion produced a subtle but significant (P<.05) increase in PKC in the particulate fraction in all dogs (1972±86 and 2189±131 pmol · min−1 · g−1 in control and preconditioned groups, respectively) compared with dogs that received preconditioning or no intervention alone. Importantly, however, no difference in PKC was evident in the particulate fraction between the control and preconditioned cohorts killed 10 minutes into sustained coronary occlusion.
In this study, we confirm that brief preconditioning ischemia reduces infarct size resulting from subsequent sustained coronary artery occlusion in the canine model. However, neither H-7 nor polymyxin B, both PKC inhibitors, blunted the reduction in infarct size achieved with preconditioning. Moreover, neither fluorescence staining and confocal microscopy nor biochemical quantification revealed evidence of preconditioning-induced translocation of PKC to the cell membranes at the onset of or 10 minutes into the subsequent sustained coronary occlusion. These results fail to support the hypothesis that translocation of PKC, initiated by brief ischemia and manifest early during the sustained occlusion, is an important mediator of preconditioning in the dog model.
PKC Plays a Focal Role in the Regulation of Cellular Functions
PKC, a ubiquitous family of serine-threonine isoenzymes, has been implicated to play a pivotal role in signal transduction for the regulation of numerous cellular functions.11 18 19 Of particular interest, PKC mediates vascular tone16 20 and influences both inotropy and chronotropy in the heart.21
Enzymatically active PKC has been identified in both the cytosol and cell membranes of “resting” cardiac tissue.11 22 However, various physiological and pharmacological stimuli, including activation of α1-adrenergic receptors, stimulation of muscarinic receptors, administration of diacylglycerol analogues, and exposure to phorbol esters, result in an overall increase in PKC activity in both the cytosolic and membrane fractions. Activation of PKC is also manifest by the subsequent redistribution of isoenzymes from the cytoplasm to binding sites associated with the sarcolemma, cytoskeleton, perinuclear region and, in smooth muscle cells and cardiac myocytes, to sites associated with contractile proteins.11 14 16 17 18 19 20 21 22 23 Activation and subsequent translocation of PKC to membrane-associated sites, presumably in response to brief transient ischemia and manifest during the early minutes of the subsequent sustained occlusion, has been proposed to play a pivotal role in ischemic preconditioning.8 9
PKC and Preconditioning: Use of Inhibitors
Virtually all previous studies assessing the role of PKC in ischemic preconditioning used an indirect pharmacological approach; ie, they attempted to block the cardioprotective effects of brief antecedent ischemia by administration of PKC inhibitors. Staurosporine and polymyxin B, administered before the onset of sustained ischemia, were shown to block the reduction in infarct size achieved with preconditioning in both the in situ and isolated rabbit heart subjected to regional ischemia.8 9 24 Results from our laboratory and others using the in vivo rat model support these observations: calphostin-C25 and chelerythrine26 prevented the reduction in infarct size with preconditioning. Similarly, calphostin-C was reported to inhibit the “preconditioning effect” (ie, attenuation of cell death) of glucose-free incubation of isolated rabbit myocytes before centrifugation into an ischemic pellet,27 whereas preliminary data suggest that staurosporine and chelerythrine eliminated the enhanced recovery of left ventricular contractile function associated with preconditioning in the globally ischemic rat heart.28 In contrast, however, preliminary evidence from the pig model yielded the opposite result.29 The PKC inhibitors staurosporine and bisindolylmaleimide did not block the protective effects of preconditioning; rather, they further reduced infarct size, leading to the diametrically opposite hypothesis that inhibition (rather than activation) of PKC may protect the heart from sustained ischemia.29
We found that neither H-7 nor polymyxin B attenuated preconditioning in the canine model. The obvious question is, how can this apparent discrepancy with the rabbit and rat data be explained? First, it could be argued that the doses of H-7 and polymyxin B used in our study were insufficient to inhibit activation of PKC. Low concentrations of H-7 (≈10−7 to 10−6 mol/L) effectively inhibit PKC in cultured hamster smooth muscle cells30 and isolated perfused rat hearts,31 but to the best of our knowledge, there have been no previous attempts to administer this agent in vivo. PKC inhibition is associated with relaxation of vascular smooth muscle.16 20 We found from preliminary studies that lower intravenous doses of H-7 (0.1 to 1.5 mg/kg) given throughout brief preconditioning ischemia did not reduce mean arterial pressure and did not block preconditioning (AN/AR, 2±1%). However, the hemodynamic alterations observed with 4 mg/kg IV and 20 mg IC H-7 are consistent with (but not proof of) inhibition of PKC in vascular smooth muscle.
Tenfold higher concentrations of polymyxin B (ie, ≈10−5 mol/L) are required to inhibit PKC.31 Thus, in protocol 3, a dose of 50 mg/kg IV was used, comparable to the dose of 24 mg/kg reported to block preconditioning in the in vivo rabbit model.9 All dogs treated with polymyxin exhibited reductions in mean arterial pressure, but in contrast to the hemodynamic consequences of H-7, this systemic vasodilation was accompanied by significant and sustained bradycardia. Whether this profound reduction in heart rate was due to inhibition of PKC or some other action of polymyxin is unknown. Infusion of polymyxin also resulted in a decrease in coronary blood flow. Whether this represents a direct and paradoxical vasoconstrictive effect of PKC inhibition on the coronary vasculature or reflects the apparent reduction in myocardial oxygen demand associated with polymyxin treatment is unclear.
In any case, these marked alterations in hemodynamics and myocardial perfusion underscore the importance of monitoring these parameters and incorporating matched polymyxin- and H-7–treated control groups into the study. For example, infarct size in preconditioned dogs receiving polymyxin averaged 10±3% of the risk region, a value intermediate between the mean infarct sizes of 19±3% and 6±2% obtained in the control and preconditioned groups in protocol 1. This could be interpreted as evidence that polymyxin B partially attenuated the protective effects of preconditioning. However, comparison of the regression relations between infarct size and subendocardial blood flow for the two protocols (Figs 1⇑ and 3⇑) confirms that, although both control and preconditioned dogs receiving polymyxin tended to be more severely ischemic during sustained coronary occlusion than the untreated groups in protocol 1, polymyxin B did not blunt the cardioprotective effects of preconditioning.
Differences among species may provide a second explanation for the disparity among the dog, rabbit, and rat models. There are at least 10 recognized isoforms of PKC: 4 “conventional” isoforms that require calcium, phospholipids, and diacylglycerol or phorbol esters for activation and 6 “novel” isoforms that are calcium-independent.11 32 Not all isoforms are expressed in the heart,11 and although poorly characterized, differences also appear to exist among species in cardiac PKC isoform expression. For example, the calcium-dependent α isoenzyme is the most abundant form of PKC in bovine heart,33 whereas in adult rat ventricular myocytes, the calcium-independent isoform PKC-ε was predominant and PKC-α was not detected.17 32 Moreover, significant temporal changes in the amount and expression of PKC isoforms were described among fetal, neonatal, and adult rat hearts.32 These differences may be important because (1) the isoforms differ in their extent of activation by diglycerides and phorbol esters,11 (2) distinct isoforms of PKC are thought to translocate to distinct intracellular sites (ie, sarcolemma versus myofibrils versus perinuclear region) and phosphorylate distinct target proteins,23 32 and thus (3) different isoforms of PKC may mediate different cellular functions.11 32 Although there is a precedent for species differences with regard to the mediators of ischemic preconditioning (ie, adenosine and potassium-sensitive ATP channels appear to play a role in some species3 34 but not in others5 35 ), the questions of whether differences in PKC isoform expression exist among rat, rabbit, and dog myocardium and whether such differences might explain the apparent disparity in the role of PKC among models remain to be addressed.
PKC and Preconditioning: Direct Measurement of PKC Translocation
A second approach to evaluating the role of PKC in preconditioning is to attempt to directly detect the translocation of PKC to the cell membranes in response to repeated brief ischemia. We used two separate methods to address this issue: direct visualization of PKC by fluorescence staining and confocal microscopy and quantification of the subcellular distribution of PKC by incorporation of 32P into the threonine group of PKC-specific peptide. Neither method revealed evidence of preconditioning-induced translocation of PKC to the cell membranes at either what would be the onset of the sustained occlusion or 10 minutes into the sustained ischemic insult.
To visualize the amount and distribution of PKC in control and preconditioned myocardium, we used a novel approach, a newly available probe that binds to activated PKC (ie, a bisindolylmaleimide PKC inhibitor rendered fluorescent through conjugation to fluorescein)14 rather than the conventional immunofluorescence methods that do not distinguish between active and inactive PKC. Using this new and sensitive technique, we found no difference in the amount or distribution of PKC fluorescence with preconditioning in the canine model.
These results imply that the amount or subcellular distribution of PKC was not altered with preconditioning, but it could be argued that fim staining is ineffective in detecting PKC activation and translocation. Three pieces of evidence provide strong support for this method. First, fim was rigorously validated by the investigators who developed the technique.14 Second, fim has been shown to retain the potency and specificity of PKC inhibition exhibited by the parental compound; ie, 200 nmol/L fim inhibited phosphorylation of the specific PKC substrate protein MARKS,14 a protein recently identified as being phosphorylated in preconditioned rabbit myocardium.36 Finally, our “positive control,” PMA incubation of baseline samples, confirmed the expected profound increase in overall intensity of fim staining, consistent with phorbol ester–mediated activation of PKC, and produced the expected increase in the confluence of strandlike fluorescence within the myocytes, reflecting translocation of PKC to membranous cellular structures.11 21
Interestingly, using this method we were unable to discern translocation of PKC to the outer cell membranes with PMA incubation. Four factors may have contributed to this observation: (1) 10 minutes of incubation in PMA may have been insufficient to initiate translocation to the outer cell membranes (preliminary studies revealed considerable deterioration of the tissue samples with longer periods of incubation); (2) the cellular mechanisms required for PKC translocation to the outer membranes may no longer have been intact or operative in the excised biopsy samples; (3) the PKC isoforms activated by PMA in the canine model may translocate exclusively to membranous structures within the myocytes rather than the outer sarcolemmal membranes; and (4) incubation in PMA rendered the samples intensely fluorescent, so that considerable downward adjustment of the contrast and brightness settings of the confocal microscope were needed to interpret the images. This adjustment in the settings may have compromised our ability to resolve an increase in fluorescent staining localized on the outer cell membranes. Most importantly, however, fluorescence staining and confocal microscopy revealed no evidence of a change in the amount or distribution of PKC after either one or four cycles of brief preconditioning ischemia.
To obtain quantitative information regarding the amount and subcellular distribution of PKC in control versus preconditioned myocardium, additional experiments were performed in which we measured the incorporation of 32P into the threonine group of PKC-specific peptide. This established method was proved capable of discerning both large changes in the amount and distribution of PKC (ie, the expected dramatic response to PMA) and subtle changes,15 29 37 such as the modest 10% to 20% redistribution of PKC from the cytosol to the particulate fraction described in isolated rat hearts in response to 10 minutes of sustained global ischemia37 and in vivo pig hearts subjected to 10 minutes of sustained coronary occlusion.29 In agreement with these previous results,29 37 we observed a subtle but significant increase in PKC in the particulate fraction in all dogs after 10 minutes of sustained occlusion compared with those killed immediately after four 5-minute episodes of preconditioning ischemia or no intervention. However, we found no difference in PKC activity between matched groups of control and preconditioned dogs at either what would be the onset of the long occlusion or at 10 minutes into sustained ischemia.
One preliminary report has sought to directly assess (by isoform-specific immunofluorescence) the subcellular distribution of PKC in control and preconditioned myocardium.28 Using the isolated rat heart, Mitchell et al28 reported that brief preconditioning ischemia resulted in the translocation of calcium-independent but not calcium-dependent PKC isoforms from the cytosol to the sarcolemma. The reason for the disparity between our results and those of Mitchell et al is unknown, but as discussed previously, differences among species in the expression and/or activation of various PKC isoenzymes may provide a possible explanation.
Critique of Methods
The fact that H-7 and polymyxin B failed to attenuate the reduction in infarct size achieved with preconditioning does not, in itself, provide conclusive proof that PKC is not important in preconditioning in the dog model. Alterations in hemodynamics associated with PKC inhibitor treatment represent only one potential consequence of PKC inhibition and may not reflect the doses of H-7 and polymyxin required to inhibit PKC on the myocyte membranes. In addition, we cannot be certain that the inhibitors effectively blocked all PKC isoforms. Importantly, however, the converse is also true: successful inhibition of preconditioning by PKC inhibitors would not conclusively identify PKC as the crucial mediator. The inhibitors H-7 and staurosporine are not selective for PKC, and others (ie, polymyxin and chelerythrine) have deleterious side effects (ruptured cell membranes and altered platelet function, respectively). Moreover, results obtained with inhibitors that must be dissolved in DMSO are further confounded by the fact that DMSO is a potent activator of PKC.38 39
For these reasons, we also used fluorescence microscopy and biochemical analysis to directly assess the subcellular distribution of PKC in control versus preconditioned myocardium. Results obtained by both methods argue strongly against the specific concept that translocation of PKC to cell membranes, initiated by brief ischemia and manifest during the early minutes of the long occlusion, is critical to the reduction in infarct size seen with preconditioning. However, four issues are worthy of comment. First, it could be argued that our methods were not sufficiently sensitive to detect a change in the amount or distribution of PKC with preconditioning. This is unlikely because we documented the expected profound changes with PMA incubation, discerned subtle differences within (but not between) the control and preconditioned groups by the fluorescence staining method, and detected the previously reported37 modest increase in PKC in the particulate fraction (by biochemical analysis) in response to 10 minutes of sustained ischemia. Second, because we focused our efforts on the measurement of PKC at the onset of and at 10 minutes into sustained occlusion, we might have missed an early and transient translocation at some point during the preconditioning regimen. Brief and transient translocation would be incongruous with the proposed hypothesis (ie, that preconditioning is critically dependent on the presence of PKC on the myocyte membranes early during the sustained occlusion), and it was beyond the objective of the present study to rigorously elucidate the time course of PKC translocation in response to ischemia in our model. We can state, however, that there was no evidence of preconditioning-induced translocation (by the fluorescent staining method) after one cycle of brief ischemia. Moreover, biochemical analysis of tissue from the preconditioned dog that fibrillated at the end of the fourth brief ischemia, although not included in the data analysis, showed that the amount and distribution of PKC (1199 and 3365 pmol · min−1 · g−1 in the particulate and cytosolic fractions, respectively) were identical to those from the control and preconditioned dogs that successfully completed the 40-minute control or preconditioning regimen. Third, although translocation of PKC from the cytosol to the particulate or membrane fractions is an accepted index,11 15 21 22 37 it may not reflect the total extent of PKC activation; it has been proposed that movement of some activated PKC isoforms from one cell compartment to another may not be associated with their binding to the particulate fraction.40 However, activation of PKC without its association to the myocyte membranes would, once again, be incongruous with the specific hypothesis we sought to test, and the resolution of this in-itself controversial concept was considered beyond the scope of the current study. Finally, our methods do not discern the translocation of specific isoforms of PKC to distinct subcellular sites. Using fluorescence staining, we assessed the total PKC present on the intracellular and outer myocyte membranes, whereas the biochemical assay quantified total PKC on the solubilized cytoskeletal, sarcolemmal, and sarcoplasmic reticular membranes and nuclear and mitochondrial fragments that compose the particulate fraction. A preconditioning-induced translocation of a specific PKC isoform to one distinct intracellular site, had it occurred, would presumably have been reflected as a trend toward a difference between preconditioned groups versus control groups by our methods. We would then have been mandated to pursue this issue using isoform- and site-specific techniques.40 However, we found no trend toward a difference in the amount or distribution of PKC, with either method and at any time point, between control and preconditioned groups.
In conclusion, neither fluorescence staining and confocal microscopy nor biochemical quantification revealed evidence of preconditioning-induced translocation of PKC to myocyte membranes at the onset of or at 10 minutes into the subsequent sustained coronary artery occlusion. In addition, administration of the PKC inhibitors H-7 and polymyxin B did not attenuate the reduction in infarct size achieved with preconditioning. Thus, our results fail to support the hypothesis that translocation of PKC to the cell membranes, initiated by brief ischemia and manifest during initial minutes of the sustained occlusion, is an important mechanism for the reduction in infarct size seen with preconditioning in the anesthetized open-chest dog.
We thank Peter Whittaker, PhD, for performing the intramyocardial injections of PMA, Stephen Bellows for assistance in the placement of intracoronary catheters in dogs that received intracoronary H-7, and Seda Dzhandzhapanyan for preparation of the cryosections and invaluable advice regarding the fluorescence staining.
- Received December 13, 1994.
- Revision received February 16, 1995.
- Accepted February 28, 1995.
- Copyright © 1995 by American Heart Association
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