Intracoronary Application of C1 Esterase Inhibitor Improves Cardiac Function and Reduces Myocardial Necrosis in an Experimental Model of Ischemia and Reperfusion
Background Myocardial injury from ischemia can be aggravated by reperfusion of the jeopardized area. The precise underlying mechanisms have not been clearly defined, but proinflammatory events, including complement activation, leukocyte adhesion, and infiltration and release of diverse mediators, probably play important roles. The present study addresses the possibility of reducing reperfusion damage by the application of C1 esterase inhibitor (C1-INH).
Methods and Results Cardioprotection by C1-INH 20 IU/kg IC was examined in a pig model with 60 minutes of coronary occlusion, followed by 120 minutes of reperfusion. C1-INH was administered during the first 5 minutes of coronary reperfusion. Compared with the NaCl controls, C1-INH reduced myocardial injury (48.8±7.8% versus 73.4±4.0% necrosis of area at risk, P≤.018). C1-INH treatment significantly reduced circulating C3a and slightly attenuated C5a plasma concentrations. Myocardial protection was accompanied by reduced plasma concentration of creatine kinase and troponin-T. C1-INH had no effect on global hemodynamic parameters, but local myocardial contractility was markedly improved in the ischemic zone. In the short-axis view, 137° of the anteroseptal region showed significantly improved wall motion at early and 29° at late reperfusion with C1-INH treatment.
Conclusions C1-INH significantly protects ischemic tissue from reperfusion damage, reduces myocardial necrosis, and improves local cardiac function.
Reperfusion injury may be broadly considered as the conversion of reversible to irreversible cell injury on perfusion of a previously ischemic area. The major underlying assumption is that a fraction of the ischemic myocytes is still viable at the time of reperfusion but that the cells succumb to one or several additional noxious agents to which they are exposed shortly after reperfusion of the area at risk.1 Interest currently centers on activated granulocytes as a major source of detrimental mediators because reperfusion injury is characterized by infiltration of afflicted areas with neutrophils2 3 and because suppression of this cellular infiltration4 or application of scavengers of reactive oxygen metabolites diminishes the extent of damage.5 6 7
Factors responsible for neutrophil influx into reperfused tissues are being investigated.8 One candidate is complement activation, which may occur when plasma gains contact with intracellular constituents (eg, mitochondria) that are released from dead cells.9 10 11 Additionally, downregulation of the complement-inhibitory molecules (eg, CD 59) on the surface of ischemic cells may play a role.12 13 Previous studies have already shown that complement is regularly activated to completion in infarcted areas of human myocardium.14 15 Moreover, there is evidence to suggest a role for complement in mediating reperfusion damage.16 Early accumulation of the terminal complement complex was observed in ischemic myocardium after reperfusion.17 Furthermore, application of C1-INH in an animal model investigated by Buerke et al18 indicated that reduction of leukocyte infiltration reduced the size of MI in ischemic and reperfused feline hearts. The present investigation was undertaken to rigorously test the contention that application of C1-INH may represent a simple and effective means to reduce reperfusion damage. For a number of reasons, we elected to use pigs as experimental animals. Schaper et al19 emphasized the very low amount of intramyocardial collateral circulation in swine. Despite the suspected similarity of collateral blood flow, MI in relation to time of coronary occlusion develops much more slowly in cats than in pigs. The results after coronary occlusion in human myocardium within the critical time limit of 6 to 12 hours are comparable to the results in the experimental system of 1 hour of coronary occlusion in the pig.20
Here we present data showing a remarkable protective effect of C1-INH application in our experimental system.
Twelve Landrace pigs of either sex weighing 27±2 kg were randomized to intracoronary treatment with either purified human C1-INH or physiological NaCl infusion (vehicle). Human C1-INH was provided by Behringwerke as a solution containing 50 IU/mL (1 IU corresponds to 0.2 mg protein). Analysis of 20 μg C1-INH by SDS-PAGE under nonreducing conditions revealed the presence of a single protein band with the correct molecular weight of 105 kD. The physiological level of C1-INH in plasma is between 150 and 250 μg/mL; the plasma half-time has been shown to be 4.5 hours in the rat.21 The C1-INH was applied over 5 minutes as a 15-mL bolus of an isoosmolar protein solution (in saline). The pigs were premedicated with the sedative azaperon intramuscularly. Anesthesia was initiated with an intravenous α-chloralose bolus and maintained with intravenous α-chloralose. After intubation, the pigs were mechanically ventilated with a Dra¨ger respirator AV-1 (O2 in room air: Fio2, 0.3; Pco2, controlled), and central venous and arterial lines were introduced. Before a midline thoracotomy, a 7.5-mg bolus of the analgeticum piritramid was given intravenously. The pericardium was opened and fixed to the border of the sternum. An LAP catheter was introduced.
Coronary occlusion by a snare was applied by tunneling the LAD with a monofil suture between the proximal and medial third behind the first diagonal branch. At the same level, the vena cordis magna was cannulated for blood analysis with a small catheter. A myocardial Po2 probe was then implanted into the expected center of the area at risk. A temperature probe was positioned next to the Po2 sensor.
Baseline values were acquired during a 1-hour preoperative period. Coronary occlusion was achieved for 60 minutes by tightening the snare around the LAD. The snare was then loosened, and a 120-minute reperfusion period followed. At the beginning of reperfusion, either 20 IU/kg body weight of C1-INH or vehicle was infused into the LAD in a blinded fashion under maintenance of constant flow and pressure. Hemodynamic and Po2 measurements and blood samples were obtained before; 5, 10, 20, 30, and 60 minutes after coronary occlusion; and after 5, 10, 20, 30, 60, 90, and 120 minutes of coronary reperfusion. Global and regional contractility were recorded by regional 2D ultrasound before, after 15 and 60 minutes of coronary occlusion, and after 15, 60, and 120 minutes of reperfusion. At the end of reperfusion, the pigs were killed, and their hearts were recovered for further analysis.
ECG, right atrial, pulmonary artery, and arterial pressure and LAP were recorded on a Siemens Sirecust 404-1 at different time points. Cardiac output was determined by thermodilution (5 mL NaCl 0.9%, room temperature) and by continuous measurement with a Baxter Vigilance monitor.
Myocardial Po2 Measurement
The Licox catheter probe measurement system22 was used in which the flexible Licox catheter Po2 microprobe is in direct contact with the myocardium. The microprobe averages the local Po2 values in tissue with a 90% response time of 60 to 90 seconds at body temperature. Measurements were recorded on-line after in vitro calibration and after a stabilization period of 60 minutes after implantation. Po2 readings were obtained by permanent compensation of the thermal Po2 probe drift.
Blood Gas and Lactate Analysis
One milliliter of heparinized venous and arterial blood was drawn with polypropylene syringes from the femoral artery and the vena cordis magna. Lactate and arteriovenous O2 differences were determined before, after 60 minutes of coronary occlusion, and after 10 and 120 minutes of reperfusion. Blood gas analysis was performed with the Radiometer Copenhagen Arterial Bloodgas Laboratory 3. For lactate determination, samples were centrifuged at 2000g (10 minutes at 4°C), plasma was decanted, and lactate was measured with the Lactate Analyzer model 23L from Yellow Springs Instrument Co Inc.
Measurements of C3a and C5a Plasma Concentrations
Blood was drawn from the vena cordis magna into polypropylene tubes containing EDTA. Plasma samples were obtained by centrifugation and immediately frozen in liquid nitrogen. C3a and C5a were determined by an ELISA with monoclonal antibodies against C3a and C5a described by Ho¨pken et al.23
Cardiac Enzyme Analysis
Venous blood samples (3 mL) were collected in polypropylene tubes containing citrate and were centrifuged at 2000g for 15 minutes at 4°C. Plasma creatine kinase activity was determined24 and expressed as international units per milliliter. Troponin-T was measured according to the method of Katus et al.25
Determination of Infarct Size
After 120 minutes of reperfusion, the LAD was reoccluded. Then 40 mL of Evan's blue (2% wt/vol solution) was injected into the pulmonary artery to stain perfused myocardium. Unstained myocardium was defined as the area at risk. After cardioplegia with 20 mL potassium chloride IV (20%), the heart was excised. The right ventricle, the large vessels, and fat tissue were removed. The left ventricle was then sliced perpendicular to the axis of the left side of the heart from the apex to the AV groove in 4-mm slices. The unstained part of the left ventricular myocardium was separated from the Evan's blue–stained portion and immersed in a 0.09-mol/L sodium phosphate buffer, pH 7.4, containing 1% triphenyltetrazolium chloride (Sigma Chemie GmbH) and 8% dextran (molecular weight, 77.800) for 20 minutes at 37°C. The tetrazolium dye forms a dark-red formazan complex in the presence of viable myocardial cells that contain active dehydrogenases and cofactors.26 Dead cells remained unstained.
The ischemic but nonnecrotic, red-stained tissue was separated from the unstained, infarcted tissue. The three tissue sections—nonischemic (area not at risk), ischemic nonnecrotic (vital [V]), and ischemic necrotic tissue (MI)—were weighed. The following definition was made: AR=V+MI.
Data were expressed as LVMM, AR, AR as a percent of LVMM, MI as a percent of LVMM, MI as a percent of AR, and total amount of infarcted tissue.
Global Myocardial Parameters
The arterial pressure-rate product (mean arterial pressure times heart rate) was taken as a global parameter of myocardial contractility. The AF was calculated as a global echocardiography parameter in the ischemic apical and the nonischemic basal region as the difference of end-diastolic (EDA) and end-systolic (ESA) areas according to the formula AF (%)=(EDA−ESA/EDA)×100.
Regional Wall Motion Analysis
A 2D cardiac ultrasound from Hewlett Packard (Sonos 500) equipped with a 5-MHz transesophageal echo probe was used. The transesophageal echo probe provided flexible movement with constant pressure and contact to the myocardial surface. Markers were set in the ischemic and nonischemic areas to constantly achieve the same 2D view. ECG was recorded simultaneously. End diastole was defined according to the recommendations for quantification of the left ventricle by 2D echocardiography published by the American Society of Echocardiography.27 A short-axis view from the lateral wall of the left ventricle was used. The quantitative analysis was performed from digitized frames of recorded videotapes at different times and analyzed with a semiautomatic computer system (Cardio 500, Kontron). Extrasystolic and postextrasystolic cycles were excluded from analysis.
For quantitative regional wall motion analysis, the fixed centerline method, which is based on a system of automatic diastolic center point determinations28 (Cardio 500, Kontron), was used. Motion was measured along 100 chords drawn perpendicularly to a centerline constructed midway between the end-diastolic and end-systolic contours. The measured motion of each chord was normalized for heart size by dividing through the length of the end-diastolic perimeter. This results in a dimensionless “shortening fraction”. The starting and end points of the diastolic and systolic circumference were the transition between the posterior septum and the posterior free wall.
Values before coronary occlusion in each group served as controls. Radiant shortening fraction before coronary occlusion conformed to a normal distribution. Therefore, each chord of either group was compared for significant wall motion differences between the C1-INH– and vehicle-treated groups.
Differences between the two experimental groups at baseline and after ischemia and reperfusion were determined with unpaired Student's t test. Paired Student's t test was applied on effects before and after 120 minutes of reperfusion. If values did not show a normal distribution, the Wilcoxon or Mann-Whitney test was performed. For repeated measures (C3a, C5a, troponin-T, creatine kinase, and cardiac output), ANOVA with the multiple comparison method (Student-Newman-Keuls test) was used.
Differences of wall motion analysis were determined with unpaired Student's t test. Data of corresponding chords of the C1-INH– and vehicle-treated groups were compared. Statistical significance was accepted at a value of P≤.05 between groups.
Average values in text and figures are mean±SEM.
The key hemodynamic parameters arterial pressure, pulmonary artery pressure, LAP, central venous pressure, and heart rate displayed no significant differences between groups at any time of the experiments. There was no increase in LAP or central venous pressure during the experiments. Neither C1-INH nor NaCl treatment had any detectable effect on left or right ventricular preload.
Cardiac output was not significantly different between the vehicle and C1-INH groups at any time of the experiment. Fig 1A⇓ gives the measured values for cardiac output.
Pressure-rate product, expressed as mean arterial blood pressure times heart beats per minute, was 5502±532 mm Hg×bpm in the C1-INH group and 7234±869 mm Hg×bpm in the NaCl group at baseline. No significant differences of pressure-rate product were observed at any time point between groups.
Myocardial Oxygen Pressure
Intramyocardial Po2 served as a sensitive and rapid indicator of myocardial ischemia and successful reperfusion (Fig 1B⇑). The mean Po2 of the myocardium of all 12 pigs before coronary occlusion was 30.3±4.7 mm Hg. During the first 5 minutes of coronary occlusion, the Po2 dropped dramatically. After 60 minutes of coronary occlusion, Po2 had decreased to 1.5±1.3 mm Hg. Myocardial Po2 started to increase within the first minutes of reperfusion. Five minutes after reperfusion, Po2 was 2.4±0.9 mm Hg; 10 minutes after reperfusion, it was 3.8±1.4 mm Hg. Compared with the Po2 after 60 minutes of myocardial ischemia, the recovery at 20 minutes after reperfusion was significant (18.9±6.1 mm Hg, P≤.05). At the end of reperfusion, Po2 had increased to 80.1±6.4 mm Hg. Compared with data under control conditions, there was a significant increase in Po2 60 minutes after reperfusion (65.9±7.7 mm Hg, P≤.05) that was still present at the end of the experiment in all 12 pigs.
Blood Gas and Lactate Analysis
There was a significant drop in AV Do2 in the C1-INH– and NaCl-treated groups 10 minutes after reperfusion (P≤.05). At the end of reperfusion, the decrease in AV Do2 was still significant (P≤.05), however, without any difference between the groups.
AV lactate difference decreased during ischemia in both groups. At early reperfusion (10 minutes after coronary occlusion) AV lactate difference was significantly different between the vehicle- (−1.15±0.5 mmol/L) and the C1-INH– treated group (0.58±0.4 mmol/L, P≤.05) with a higher lactate production in the NaCl group. Values at the end of reperfusion time remained significantly different from baseline values (Fig 1C⇑).
Plasma Anaphylatoxin Levels
Levels of C3a and C5a in the two groups did not differ significantly before ischemia and after 60 minutes of coronary occlusion (Fig 2⇓). C3a levels increased in the vehicle-treated animals significantly after 20 minutes and remained elevated throughout the experiment (Fig 2A⇓). C1-INH treatment reduced the increase in C3a; at the end of the reperfusion period, C3a plasma levels in the C1-INH group remained significantly lower than in the vehicle group (P≤.05; Fig 2A⇓).
C5a determinations revealed a significant increase in C5a at 90 and 120 minutes of reperfusion compared with baseline and ischemic values (60 minutes of coronary occlusion, P≤.05) in the vehicle-treated group. However, these increases in C5a levels were much smaller than those found for C3a, and differences between the two groups were not significant (Fig 2B⇑).
Cardiac Creatine Kinase and Troponin-T
Plasma creatine kinase activity remained constant in both groups during the whole ischemic period. The washout of creatine kinase into the circulating blood occurred during the first minutes of reperfusion and was significantly higher in the group receiving vehicle, with maximum reached 10 minutes after reperfusion (P≤.05). Fig 3A⇓ gives the measured values for creatine kinase.
Troponin-T was also released into the circulation at the beginning of reperfusion. C1-INH–treated pigs had significantly lower values compared with the vehicle-treated group. This effect remained until the end of the observation period and was statistically significant 90 and 120 minutes after ischemia (P≤.05) compared with untreated pigs (Fig 3B⇑).
Total LVMM and total wet weight of the AR were determined at the end of the experiment. Wet weight of LVMM in the C1-INH group (67.4±2.5 g) was not significantly different from that of the NaCl group (70.8±4.2 g; Fig 4A⇓). Neither AR (C1-INH, 21.4±2.8 g; NaCl, 25.0±1.8 g; Fig 4B⇓) nor the wet weight of the AR expressed as a percentage of LVMM (C1-INH, 31.4±3.2%; NaCl, 35.9±2.9%; Fig 4C⇓) showed a significant difference. This showed that the region of myocardial ischemia was comparable in size in both groups.
In the NaCl-treated group, 73.4±4.0% of the ischemic AR became necrotic (Fig 4D⇑). In contrast, C1-INH treatment reduced the necrotic area expressed in relation to AR (48.8±7.8%, P≤.018; Fig 4D⇑) or LVMM (14.5±2.1%; NaCl, 26.4±2.6%; P≤.005; Fig 4E⇑) or as total necrotic tissue (9.9±1.7 g wet weight; NaCl, 18.2±1.1 g wet weight; P≤.002; Fig 4F⇑). Thus, C1-INH markedly reduced the size of infarctions.
Area Ejection Fraction
The AF was measured as a global parameter of contractility.
In the apical infarcted area, the C1-INH–treated group had an AF of 74.6±1.9% and the vehicle-treated group had an AF of 68.9±3.8% before coronary occlusion (Fig 5A⇓). There was no significant difference between groups. During ischemia, there was a significant decrease in AF (C1-INH, 32.3±4.5%, P≤.05; NaCl, 25.8±5.4%, P≤.05) with no statistical difference between C1-INH and vehicle. With reperfusion and drug treatment, AF increased significantly in both groups until the end of the experiment. After 120 minutes of reperfusion, AF was 53.4±2.6% in the C1-INH–treated group and 39.2±4.7% in the vehicle-treated group. In both groups, a significant reduction persisted after 120 minutes of reperfusion (C1-INH and NaCl, P≤.05). The recovery of AF was statistically better with C1-INH treatment 15, 60, and 120 minutes after coronary occlusion than with vehicle treatment (Fig 5A⇓).
The basal AF in the nonischemic area was not different in both groups (Fig 5B⇑). During ischemia, there was a significant drop in the baseline AF in both groups (C1-INH and NaCl, P≤.05). After reperfusion, AF increased in both groups, and no statistical difference was observed between the C1-INH and vehicle treatment at any time point (Fig 5B⇑).
Regional Wall Motion Analysis
Regional wall motion before coronary occlusion showed normal distribution with no significant difference in both groups (Fig 6A⇓). After 15 minutes of coronary occlusion, wall motion dropped significantly in the anteroseptal region in both groups (Fig 6B⇓). At 60 minutes of coronary occlusion, the decrease was still significant without differences between groups (Fig 6C⇓). Fifteen minutes after reperfusion, a significant difference in wall motion was detectable in the anteroseptal region and the posterior sector. Wall motion in the NaCl-treated group was significantly lower from chord 36 to 74, including a region of profound dyskinesia (136.8° of the anteroseptal area of 360° of the short-axis view; Fig 6D⇓). In the posterior region, wall motion was significantly increased from chord 7 to 13 as a sign of compensatory hyperkinesia in the NaCl-treated group.
Sixty minutes after reperfusion, the region with significantly lower wall motion in the vehicle-treated group went from chord 42 to 75, representing 118.8° of the anteroseptal area of the short-axis view (Fig 6E⇑). The area of dyskinesia became smaller. Posterior wall motion was significantly better from chord 10 to 13 and from chord 19 to 20 compared with the C1-INH group.
At the end of reperfusion (120 minutes), wall motion was significantly higher in the C1-INH–treated group from chord 65 to 72. This represents 28.8° of the total left ventricular circumference (Fig 6F⇑). There was no differentiation between groups in posterior wall motion at that time point.
Control pigs without ischemia showed no statistical differences in global cardiac function and regional wall motion at different time points throughout the 3-hour observation period.
Two major questions were addressed in this study. First, does C1-INH application reduce the size of the infarcted area? Second, does application of C1-INH improve cardiac function? The affirmative answers to both questions raise the possibility that intracoronary application of C1-INH may represent a simple and safe measure to improve reperfusion therapeutic strategies.
Complement activation occurs to completion in ischemic and infarcted tissues,9 10 14 16 and several studies indicate that classic pathway activation through C1 fixation is one indicating mechanism.29 Thus, it has been shown that myocardial ischemia causes release of subcellular constituents that bind C1 in vivo.9 10 An accumulation of C1 in ischemic canine muscle has been demonstrated immunohistochemically.29 Furthermore, application of C1-INH reduced C1 deposition and leukocyte infiltration in reperfused feline myocardium.18
The potential importance of neutrophil infiltration and activation in the pathogenesis of reperfusion damage is undisputed.30 In reperfused myocardium, neutrophil accumulation was observed preferentially in the subendocardial region.3 Neutrophils may contribute to reperfusion damage by releasing reactive oxygen metabolites,31 lipid mediators, and proteases.32 33
A correlation exists between reperfusion-dependent neutrophil infiltration and infarct size,6 and neutrophil activation in reperfusion areas appears to have detrimental consequences on cardiac function.34 Complement anaphylatoxins now represent logical candidates as initiators of the detrimental processes.
We used pigs as experimental animals. This system provided the following advantages over previously used models. First, there is little collateral blood flow from other coronary arteries to the vena cordis magna, which drains the blood of the LAD.19 Second, hemodynamic parameters are obtainable in exactly the same fashion as in humans. Third, myocardial and coronary anatomies are very similar in pigs and humans. In these first experiments, we applied C1-INH intracoronarily to guarantee rapid access of the inhibitor in high concentration to the AR. The dose of 20 IU/kg body weight was selected on the basis of pilot experiments that had indicated this dose to be effective. Possible dose-related toxic effects of C1-INH have not been studied to date. Such studies would have to be performed before therapeutic trials could be conducted in humans. Of note, the applied dose is not particularly high. The physiological concentration of C1-INH in serum is ≈1 IU (0.2 mg)/mL; hence, application of 20 IU/kg will lead to a maximal systemic increase of <50% of C1-INH plasma levels.
C1-INH inhibits C1s esterase at the site of its formation. Increasing C1-INH plasma levels will not be expected to affect overall CH50 complement titers; indeed, no such systemic effects were observed (not shown). However, at the sites of classic pathway complement activation, C1-INH would be expected to suppress formation of C3 convertase and thus attenuate C3a generation. We anticipate that the marked protective effects of C1-INH observed in this study were due to very high concentrations attained locally at the site of application. Because C3a would be liberated only locally, plasma samples were collected directly at the sites of blood efflux from the reperfused zones. The C3a determinations were significant for two important reasons. First, they provided unequivocal evidence that C3a is indeed generated in reperfused tissue; such direct evidence for anaphylatoxin generation during reperfusion had not been obtained before. Second, they showed that C3a generation was markedly suppressed when C1-INH was added to the reperfusion fluid; this supported the previous contention that C1 fixation is indeed a relevant pathway of complement activation during reperfusion. A similarly marked suppression of C5a generation by C1-INH could not be documented, however; because the increases in C5a levels during complement activation are much smaller compared with C3a, detection of significant differences will in general probably be difficult.
Myocardial damage was quantified by use of a double-staining technique. These experiments revealed a dramatic protective effect of C1-INH. Regardless of whether the infarcted areas were compared directly with each other or whether ratios were formed, eg, with the AR, highly significant differences between the C1-INH– and the vehicle-treated groups were observed. Parallel to these morphological criteria, plasma creatine kinase and troponin-T levels, two biochemical markers for myocardial necrosis, were measured. Again, clear attenuation of plasma levels of both markers was found in the C1-INH group.
Our observation that C1-INH reduces infarct size in reperfusion experiments can be compared with two related publications. A 44% reduction of myocardial necrosis was reported in a rat model of reperfusion injury after application of sCR1, which inhibits complement at the level of C3.16 A second study with a feline model recently reported 65% reduction in myocardial necrosis after application of C1-INH.18 Our results in the pig model are in line with these previous findings. Of note, the extent of infarction in the vehicle group varies considerably, depending on the animal species. Thus, only 28% of the ischemic myocardium became necrotic in the feline model despite longer periods of occlusion (90 minutes) and reperfusion (4.5 hours),18 whereas we observed 73% necrosis of the AR after only 60 minutes of occlusion. The differences in protection afforded by C1-INH should be considered in the light of these major initial differences. The reasons for such differing primary sensitivity toward reperfusion damage between cat and pig are not known in detail but probably derive from differences in capillarization and collateralization.19 20 35 36 37 In the feline model, C1-INH was administered intravenously before reperfusion. Experiments to determine the optimal time and mode of C1-INH administration in the pig model are currently underway.
Although documentation of reduced infarction area is in itself a relevant issue, functional aspects of any given therapeutic measure also need to be addressed. Selection of appropriate parameters is thereby of essence. For example, many studies use measurements of global parameters such as dP/dtmax and pressure-rate product. Improved dP/dtmax has been reported after inflammatory intervention in some reperfusion studies.18 38 Our data diverge from those findings in that no differences in any of the measured global cardiac parameters were discerned. However, the use of more sensitive assays revealed that C1-INH application had dramatic effects, specifically on the regional function of the ischemic myocardium.
The parameter used in our study was the assessment of wall motion combined with calculations of the AF, which are the criteria used in clinical ventriculography. Some studies compared wall thickening with wall motion analysis. We did not use the epicardial-endocardial Doppler crystal technique, which detects changes in regional thickening rather than net endocardial motion and does not measure the contribution of epicardial motion to endocardial excursion.39 This technique is also very vulnerable to disturbances such as those that occur during defibrillation. Instead, we elected to monitor the short-axis view; the accuracy of this approach has been validated for severe ischemic regional dysfunction and discussed by many investigators.40 41 42
With this technique, clear findings emerged. During coronary occlusion of the LAD, the left ventricle developed anteroseptal hypokinesia with a small area of dyskinesia. In the vehicle-treated group, there was a depression of anteroseptal cardiac function at early reperfusion with compensatory hyperkinesia in the posterior area. Anteroseptal wall motion improved slightly up to the end of reperfusion. This improvement was markedly enhanced by application of C1-INH.
In accordance with the wall motion data, the AF decreased in the ischemic part of the left ventricle. Application of C1-INH markedly improved the AF in the ischemic zone. The basal nonischemic area that also exhibited some wall thickening showed a reduction in AF during ischemia that was not affected by C1-INH application. Overall, we thus detected an improved local contractility of the ischemic myocardium that was dependent on the presence of C1-INH. In contrast, C1-INH had no effect on the function of nonischemic myocardium.
C1 inhibitor exerts multiple functions. It not only inhibits C1s activity but also is effective against several other physiological serine proteases. Therefore, it influences the bradykinin-kinine system, the clotting cascade, and fibrinolysis. Our present study has addressed only its role in suppressing complement activation, and the results underline the important role of the classic complement pathway activation. They are in perfect agreement with recent data of Weiser et al,43 who showed that depletion of plasma immunoglobins or C4 also led to marked protection against reperfusion injury.43 Protection of C1-INH may emerge as a generally effective measure to protect against reperfusion injury in clinical settings.
Selected Abbreviations and Acronyms
|AF||=||area ejection fraction|
|AR||=||area at risk|
|C1-INH||=||C1 esterase inhibitor|
|LAD||=||left anterior descending coronary artery|
|LAP||=||left atrial pressure|
|LVMM||=||total left ventricular muscle mass|
Dr Bhakdi acknowledges support from the Deutsche Forschungsgemeinschaft (SFB 311, D10) and the Verband der Chemischen Industrie. We wish to thank A. Schollmayer, G. Sonntag, H. Erbes, M. Malzahn, L. Kopacz, and A. Schneider for their excellent laboratory and technical work. We gratefully acknowledge B. Eisele of Behringwerke AG for his critical comments and the supply of C1 esterase inhibitor Berinert. Data are from the doctoral thesis of O. Berg and P. Becker.
- Received April 25, 1996.
- Revision received August 29, 1996.
- Accepted September 9, 1996.
- Copyright © 1997 by American Heart Association
Entman ML, Michael LH, Rossen RD, Dreyer WJ, Anderson DC, Taylor AA, Smith CW. Inflammation in the course of early myocardial ischemia. FASEB J. 1991;5:2529-2537.
Dreyer WJ, Michael LH, West MS, Smith CW, Rothlein R, Rossen RD, Anderson DC, Entman ML. Neutrophil accumulation in ischemic canine myocardium: insights into time course, distribution, and mechanisms of localization during early reperfusion. Circulation. 1991;84:400-411.
Tanaka M, Brooks SE, Richard VJ, FitzHarris GP, Stoler RC, Jennings RB, Arfors K-E, Reimer KA. Effect of Anti-CD 18 antibody on myocardial neutrophil accumulation and infarct size after ischemia and reperfusion. Circulation. 1993;87:526-535.
Zweier JL. Measurement of superoxide derived free radicals in the reperfused heart: evidence for a free radical mechanism of reperfusion injury. J Biol Chem. 1988;263:1353-1357.
Amsterdam EA, Pan H-L, Rendig SV, Symons JD, Fletcher MP, Longhurst JC. Limitation of myocardial infarct size in pigs with dual lipoxygenase-cyclooxygenase blocking agent by inhibition of neutrophil activity without reduction of neutrophil migration. J Am Coll Cardiol. 1993;22:1738-1744.
Entman ML, Youker K, Shoji T, Kukielka G, Shappell SB, Taylor AA, Smith CW. Neutrophil induced oxidative injury of cardiac myocytes: a compartmented system requiring CD 11b/CD18-ICAM-1 adherence. J Clin Invest. 1992;90:1335-1345.
Rossen RD, Michael LH, Kagiyama A, Savage HE, Hanson G, Reisberg MA, Moake JN, Kim SH, Self D, Weakley S, Giannini E, Entman ML. Mechanism of complement activation after coronary artery occlusion: evidence that myocardial ischemia in dogs causes release of constituents of myocardial subcellular origin that complex with human C1q in vivo. Circ Res. 1988;62:572-584.
Crawford MH, Grover ML, Kolb WP, McMahan A, O'Rourke RA, McManus LM, Pinckard RN. Complement and neutrophil activation in the pathogenesis of ischemic myocardial injury. Circulation. 1988;78:144-148.
Tsao PS, Aoki N, Lefer DJ, Johnson G III, Lefer A. Time course of endothelial dysfunction and myocardial injury during myocardial ischemia and reperfusion in the cat. Circulation. 1990;82:1402-1412.
Scha¨fer HJ, Mathey D, Hugo F, Bhakdi S. Deposition of the terminal C5b-9 complement complex in infarcted areas of human myocardium. J Immunol. 1986;137:1945-1949.
Weismann HF, Bartow T, Leppo MK, Marsh HC, Carson GR, Concino MF. Soluble complement receptor type 1: in vivo inhibitor of complement suppressing post ischemic myocardial inflammation and necrosis. Science. 1991;249:146-151.
Mathey D, Schofer J, Scha¨fer HJ, Hamdoch T, Joachim HC, Ritgen A, Hugo F, Bhakdi S. Early accumulation of the terminal complement-complex in the ischemic myocardium after reperfusion. Eur Heart J. 1994;15:418-423.
Buerke M, Murohara T, Lefer A. Cardioprotective effects of a C1 esterase inhibitor in myocardial ischemia and reperfusion. Circulation. 1995;91:393-402.
Schaper W. Experimental infarcts and the microcirculation. In: Hearse DJ, Yellon DM. Therapeutic Approaches to Myocardial Infarct Size Limitation. New York, NY: Raven Press; 1984:79-90.
De Smet BJGL, De Boer JP, Agterberg J, Rigter G, Bleeker WK, Hack CE. Clearance of human native, proteinase-complexed, and proteolytically inactivated C1-inhibitor in rats. Blood. 1993,81:56-61.
Boekstegers P, Trupkovic T, Krieger M, Weiss C. Intramyocardial oxgen partial pressure measurements by means of Po2 catheters: studies in acute myocardial ischemia, reperfusion and during selective ECG-synchronised suction and retroinfusion of coronary veins (SSR). In: Ehrly AM, Fleckenstein W, Landgraf H, eds. Clinical Pressure Measurement. Berlin, Germany: Blackwell Ueberreuter Wissenschaft; 1992;3:186-196.
Ho¨pken U, Stru¨ber A, Oppermann M, Mohr M, Mu¨cke KH, Burchardi H, Go¨tze O. Production and characterisation of peptide-specific monoclonal antibodies that recognise a neoepitope on hog C5a. In: Faist E, Meakins JL, Schildberg FW, eds. Host Defense Dysfunction in Trauma, Shock and Sepsis. Berlin, Germany: Springer Verlag; 1993.
Katus HA, Looser S, Hallermayer K, Remppis A, Scheffold T, Borgya A, Essig U, Geuß U. Development and in vitro characterization of a new immunoassay of cardiac troponin T. Clin Chem. 1992;38:386-393.
Sheehan FH, Bolson EL, Dodge HT, Mathey DG, Schofer J, Woo HW. Advantages and applications of the centerline method for characterizing regional ventricular function. Circulation. 1986;74:293-305.
Rossen RD, Swain JL, Michael LH, Weakley S, Giannini E, Entman ML. Selective accumulation of the first component of the complement and leucocytes in ischemic canine heart muscle. Circ Res. 1985;57:119-130.
Chatelain P, Latour JG, Tran D, De Lorgeril M, Dupras G, Bourassa M. Neutrophil accumulation in experimental myocardial infarcts: relation with extent of injury and effect of reperfusion. Circulation. 1987;75:1083-1090.
Freed MS, Needelman P, Dunkel CG, Saffitz JE, Evers AS. Role of invading leukocytes in enhanced atrial eicosanoid production following rabbit left ventricular myocardial infarction. J Clin Invest. 1989;83:205-212.
Henson PM, Johnston RB. Tissue injury in inflammation. J Clin Invest. 1987;79:669-674.
Shandeyla SML, Kuppusamy P, Weisfeldt ML, Zweier JL. Evaluation of the role of polymorphonuclear leukocytes on contractile function in myocardial reperfusion injury. Circulation. 1993;87:536-546.
Breisch EA, White FC, Nimmo LE, McKiran MD, Bloor CM. Exercise induced cardiac hypertrophy: a correlation of blood flow and microvasculature. J Appl Physiol. 1986;60:1259-1267.
Breisch EA, White FC, Nimmo LE, Bloor CM. Cardiac vasculature and flow during pressure overload hypertrophy. Am J Physiol. 1986;251:H1031-H1037.
Shandelya SML, Kuppusamy P, Herskowitz A, Weisfeldt ML, Zweier JL. Soluble complement receptor type 1 inhibits the complement pathway and prevents contractile failure in the postischemic heart. Circulation. 1993;88:2812-2826.
Lieberman AN, Weiss JL, Jugdutt BI. Two-dimensional echocardiography and infarct size: Relationship of regional wall motion and thickening to the extent of myocardial infarction in the dog. Circulation. 1981;63:739-746.
Moynihan PF, Parisi AF, Feldman CL. Quantitative detection of regional left ventricular contraction abnormalities by two-dimensional echocardiography, I: analysis of methods. Circulation. 1981;63:752-760.
Parisi AF, Moynihan PF, Folland ED, Feldman CL. Quantitative detection of regional left ventricular contraction abnormalities by two-dimensional echocardiography, II: accuracy in coronary artery disease. Circulation. 1981;63:761-767.
Weiser MR, Williams JP, Moore FD, Kobzik L, Ma M, Hechtman HB, Carroll M. Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement. J Exp Med. 1996,183:2343-2348.