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(Circulation Research. 1996;79:407-414.)
© 1996 American Heart Association, Inc.

Articles

No Prevention of Ischemic Preconditioning by the Protein Kinase C Inhibitor Staurosporine in Swine

Christian Vahlhaus, Rainer Schulz, Heiner Post, Raouf Onallah, Gerd Heusch

Abteilung fur Pathophysiologie, Zentrum fur Innere Medizin des Universitatsklinikum Essen (Germany).

Correspondence to Prof Dr Gerd Heusch, FESC, FACC, Abteilung fur Pathophysiologie, Zentrum fur Innere Medizin, Universitatsklinikum Essen, Hufelandstraße 55, 45122 Essen, Germany.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The delay of infarct size development by ischemic preconditioning involves the activation of protein kinase C in rats and rabbits. In dogs the role of protein kinase C in ischemic preconditioning is controversial. We investigated whether or not the activation of protein kinase C is a prerequisite for ischemic preconditioning in swine. Swine were used, since they are large mammals and since infarct development in this species, due to the lack of an innate collateral circulation, is similar to that in humans. In 20 enflurane-anesthetized swine, the proximal left anterior descending coronary artery was cannulated and perfused from an extracorporeal circuit. The impact of continuous intracoronary infusion of 10^-7 mol/L staurosporine, a potent protein kinase C inhibitor, on global and regional myocardial function (sonomicrometry), subendocardial blood flow (ENDO, microspheres), and infarct size (IS, triphenyltetrazolium chloride staining after 120 minutes of reperfusion) was analyzed. Staurosporine (10^-7 mol/L) abolished the 1.6-fold increase in coronary arterial resistance in response to 10^-6 mol/L IC 4ß-phorbol 12-myristate 13-acetate, a potent protein kinase C activator. In the presence of staurosporine, 90 minutes of low-flow ischemia at an ENDO of 0.05±0.04 (mean±SD) mL·min^-1·g^-1 resulted in an IS of 12.5±8.6% (n=10) of the area at risk. Also, in the presence of staurosporine, ischemic preconditioning by a cycle of 10 minutes of low-flow ischemia followed by 15 minutes reperfusion before the 90 minutes sustained ischemic period (ENDO, 0.05±0.03 mL·min^-1·g^-1) reduced IS to 3.3±3.4% (n=10, P<.05). The protein kinase C inhibitor staurosporine does not prevent ischemic preconditioning in swine.

Key Words: myocardial ischemia • reperfusion • ischemic preconditioning • protein kinase C • staurosporine • swine

	Introduction

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Ischemic preconditioning, ie, the delay of infarct size development during prolonged and severe myocardial ischemia by one or more preceding short episodes of ischemia and reperfusion,¹ is the most powerful endogenous cardioprotective effect observed thus far. Therefore, the analysis of the signal transduction cascade mediating ischemic preconditioning and its potential pharmacological exploitation have attracted great interest. Endogenous activation of the adenosine A₁ receptor in rabbits² and dogs,³ the ₁-adrenergic receptor in dogs,⁴ the bradykinin B₂ receptor in rabbits,^{5 6} and the opioid receptor in rats⁷ is involved in the infarct size–reducing effect of ischemic preconditioning. Also, pharmacological activation of the adenosine A₁ receptor in rabbits,⁸ dogs,³ and swine,⁹ the ₁-adrenergic receptor in rabbits¹⁰ and dogs,⁴ the bradykinin B₂ receptor in rabbits,^{5 6} the muscarinic M₂ receptor in rabbits¹¹ and dogs,¹² and the angiotensin II AT₁ receptor in rabbits¹³ mimics the infarct size reduction by ischemic preconditioning.

The infarct size reduction by pharmacological activation of adenosine A₁ receptors is completely abolished by blockade of ATP-dependent potassium channels in anesthetized dogs¹⁴ and swine,⁹ suggesting that the cardioprotection by adenosine is mediated through activation of ATP-dependent potassium channels. A potential link between the activation of a receptor system and opening of ATP-dependent potassium channels is the activation of protein kinase C, as observed in isolated rabbit^{15 16} and human¹⁶ ventricular myocytes with pharmacological activation of protein kinase C. In the isolated rabbit heart subjected to regional ischemia,¹⁷ as well as in human atrial trabeculae subjected to simulated ischemia (hypoxic superfusion in combination with rapid pacing),¹⁸ ischemic preconditioning is mediated through activation of protein kinase C and subsequent activation of ATP-dependent potassium channels.

Liu et al¹⁹ first proposed a unifying hypothesis of ischemic preconditioning, with protein kinase C activation playing a pivotal role. This hypothesis has been supported by several studies in rats^{20 21} and rabbits^{6 8 13 17 22} in vivo, as well as in human isolated atrial¹⁸ and ventricular²³ cardiomyocytes. In anesthetized dogs, the role of protein kinase C in ischemic preconditioning remains controversial. There are two studies using the same protein kinase C inhibitor (polymyxin B) and an identical preconditioning protocol (four cycles of 5 minutes of ischemia and 5 minutes of reperfusion), which come to opposite conclusions.^{24 25} In one study, neither was protein kinase C activated by ischemic preconditioning nor did intravenous pretreatment with polymyxin B (50 mg·kg^-1) alter infarct size reduction by ischemic preconditioning²⁴ ; however, in the other study protein kinase C was activated by ischemic preconditioning, and intracoronary pretreatment with polymyxin B (300 µg/kg) abolished the infarct size reduction by ischemic preconditioning.²⁵ In swine, which are large mammals like dogs but without an innate collateral circulation and thus like rats and rabbits,²⁶ protein kinase C is activated during ischemia.²⁷ Ischemic preconditioning, however, is not abolished by intramyocardial microinfusion of either of the two potent protein kinase C inhibitors, staurosporine or bisindolylmaleimide, during the preconditioning ischemia.²⁷ The intramyocardial microinfusion technique is obviously hampered by an inhomogeneous distribution of the protein kinase C inhibitors, and sufficient blockade of protein kinase C throughout the perfusion territory cannot be ensured. More important, staurosporine only prevents ischemic preconditioning when it is administered until 10 minutes into the sustained ischemia.²⁸

Therefore, in the present study we reexamined the effect of blockade of protein kinase C on ischemic preconditioning using an established model in swine.^{29 30 31} We hypoperfused the left anterior descending coronary artery at constant flow to guarantee a homogeneous distribution and continuous intracoronary delivery of the protein kinase C inhibitor staurosporine throughout the perfusion territory from before the preconditioning ischemia until the end of the sustained ischemia and to define the relation between infarct size and subendocardial blood flow as the most valid end point of ischemic preconditioning. Finally, the effectiveness of staurosporine for blocking protein kinase C activation was tested by intracoronary infusion of the protein kinase C activator 4ß-phorbol 12-myristate 13-acetate at the end of each experiment.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
The experimental protocols used in the present study were approved by the bioethical committee of the district of Dusseldorf, and they adhere to the guiding principles of the American Physiological Society.

Experimental Model
Twenty-five Gottinger miniswine (20 to 40 kg) of either sex were initially sedated using ketamine hydrochloride (1 g IM) and then anesthetized with thiopental (Trapanal, 500 mg IV). Through a midline cervical incision, the trachea was intubated for connection to a respirator (Drager). Anesthesia was then maintained using enflurane (1% to 1.5%) with an oxygen/nitrous oxide mixture (40%:60%). Arterial blood gases were monitored frequently in the initial stages of the preparation until stable and then periodically throughout the study (Radiometer). Rectal temperature was monitored and maintained between 37°C and 38°C by the use of a heated surgical table and drapes.

Through the cervical incision, both common carotid arteries and internal jugular veins were isolated. The arteries were cannulated with polyethylene catheters, one to measure arterial pressure and the other to supply blood to the extracorporeal circuit. The jugular veins were cannulated for volume replacement using warmed 0.9% NaCl and for the return of blood to the animal from the coronary venous line (see below).

A left lateral thoracotomy was performed in the fourth intercostal space, and the pericardium was opened. A micromanometer (model P7, Konigsberg Instruments) was placed in the left ventricle through the apex, together with a saline-filled polyethylene catheter (used to calibrate the micromanometer in situ). Ultrasonic dimension gauges were implanted in the left ventricular (LV) myocardium to measure the thickness of the anterior and posterior (control) wall (System 6, Triton Technologies, Inc).

The left anterior descending coronary artery was dissected over a distance of 1.5 cm, ligated, cannulated (within 40 seconds at the latest), and perfused from an extracorporeal circuit. Before coronary cannulation, the swine were anticoagulated with 20 000 IU sodium heparin; additional doses of 10 000 IU were given at hourly intervals. The system included a roller pump, windkessel, and two side ports, one for the injection of radiolabeled microspheres and the other for the administration of the protein kinase C inhibitor staurosporine (Sigma-Aldrich Chemie GmbH) or the protein kinase C activator 4ß-phorbol 12-myristate 13-acetate (Sigma-Aldrich Chemie GmbH). Staurosporine and 4ß-phorbol 12-myristate 13-acetate were prepared in a 1:1:2 solution of polyethylene glycol (PEG 400, Sigma-Aldrich Chemie GmbH), ethanol (Sigma-Aldrich Chemie GmbH), and saline solution. Coronary arterial pressure was measured from the side arm of a polyethylene "T" connector (Cole-Parmer) used as a catheter tip with an external transducer (Bell and Howell Type 4-327I). Minimal coronary arterial pressure was held above 70 mm Hg by adjusting the roller pump of the extracorporeal circuit to avoid hypoperfusion before ischemia. Therefore, mean coronary arterial pressure exceeded peak LV pressure. The large epicardial vein parallel to the left anterior descending coronary artery was dissected and cannulated. Coronary venous blood was drained to an unpressurized reservoir and then returned to a jugular vein through use of a second roller pump. Heart rate was controlled throughout the study by left atrial pacing (Hugo Sachs Elektronik type 215/T).

Regional Myocardial Function
End diastole was defined as the point at which LV dP/dt started its rapid upstroke after crossing the zero line. Regional end systole was defined as the point of maximal wall thickness within 20 milliseconds before peak negative LV dP/dt.³² Systolic wall thickening was calculated as end-systolic wall thickness minus end-diastolic wall thickness divided by the end-diastolic wall thickness. In view of the ventricular asynchrony observed during regional ischemia, a "work index" (WI) was calculated. To estimate regional myocardial work, the sum of the instantaneous LV pressure–wall thickness product over the time of the cardiac cycle was calculated using the following equation:

where ed is end diastole of the cardiac cycle, n is the actual cardiac cycle, m is the sampling point within cardiac cycle n at a sampling frequency of 5 milliseconds, LVP_n,m is LV pressure within cardiac cycle n at sampling point m, LVP_min is minimum LV pressure, WTh_n,m is anterior wall thickness within cardiac cycle n at sampling point m, and WTh_n,m-1 is anterior wall thickness within cardiac cycle n at the sampling point 5 milliseconds before sampling point m. The maximum work value during systole is referred to as WI.³³

Regional Myocardial Blood Flow
Radiolabeled microspheres (15-µm diameter, ¹⁴¹Ce, ¹¹⁴In, ¹⁰³Ru, ⁹⁵Nb, or ⁴⁶Sc; NEN, DuPont Co) were injected into the coronary perfusion circuit (1 to 2x10⁵ suspended in 1 mL saline) to determine the regional myocardial blood flow and its distribution throughout the left anterior descending coronary artery perfusion bed. This procedure for the determination of blood flow has been validated extensively.³⁴ Blood flow to the tissue at the site of the ultrasonic crystals is reported, and this piece of tissue was divided into transmural thirds of approximately equal thickness. In addition, subendocardial blood flow to the entire left anterior descending coronary artery–perfused territory was measured and related to myocardial infarct size.

4ß-Phorbol 12-Myristate 13-Acetate
In preliminary studies in 10 swine also undergoing 90 minutes of ischemia and 120 minutes of reperfusion, 4ß-phorbol 12-myristate 13-acetate at a concentration of 10^-6 mol/L was infused into the left anterior descending coronary artery at the end of the reperfusion period. A flow-constant coronary perfusion was chosen to avoid hypoperfusion secondary to coronary vasoconstriction. 4ß-Phorbol 12-myristate 13-acetate (10^-6 mol/L) increased coronary arterial resistance within 5 minutes after the onset of infusion from 2.92±0.97 to 4.55±1.81 mm Hg·mL^-1·min^-1 (P<.05, paired t test). This increase in coronary arterial resistance was maintained over at least 15 minutes. Systolic wall thickening was further decreased from 2.46±4.19% to -0.04±3.41% (P<.05, paired t test), whereas heart rate and LV peak pressure remained unchanged.

Staurosporine
In previous studies in conscious rats, the intravenous infusion of staurosporine caused systemic vasodilation.³⁵ Therefore, to avoid an initial subendocardial hypoperfusion under control conditions secondary to coronary vasodilation and a consequent decrease in coronary arterial pressure, the intracoronary staurosporine infusion was started at a constant coronary arterial pressure perfusion. To prevent alterations in systemic hemodynamics secondary to staurosporine infusion, coronary venous blood from the cannulated coronary vein was collected during the staurosporine infusion and centrifuged, and the concentrated erythrocytes were redissolved in saline solution and reinfused intravenously, whereas the supernatant was discarded. A total of 163±25 mL (range, 145 to 180 mL) was discarded over a time frame of 90 minutes in group 1, resulting in a decrease in plasma protein concentration from 74±13 to 64±13 g/L, as measured using a modified Lowry's method.³⁶ In group 2, 165±49 mL (range, 130 to 200 mL) serum was removed in 115 minutes, resulting in a decrease in plasma protein concentration from 81±5 to 73±3 g/L. The influence of such plasma removal on infarct development was studied in three control animals undergoing 90 minutes of severe ischemia with a decrease in plasma protein concentration from 71±14 to 61±8 g/L; these three animals developed the same amount of infarction for a given subendocardial blood flow as historic control animals³⁰ without plasma removal. The infusion of staurosporine at a concentration of 10^-7 mol/L significantly decreased coronary arterial resistance in group 1 from 3.75±0.64 to 2.14±0.59 mm Hg·mL^-1·min^-1 (P<.05, paired t test). In group 2, coronary arterial resistance was decreased from 3.96±1.28 to 2.30±0.67 mm Hg·mL^-1·min^-1 (P<.05, paired t test); ie, at constant coronary arterial pressure, mean transmural blood flow was raised to 1.7-fold in group 1 and 1.5-fold in group 2.

In the present study, the effective blockade of protein kinase C by staurosporine was tested at the end of the protocol. At a concentration of 10^-7 mol/L, staurosporine blocked the protein kinase C activation in response to 4ß-phorbol 12-myristate 13-acetate infusion; ie, coronary arterial resistance and systolic wall thickening remained constant (1.37±0.50 mm Hg·mL^-1·min^-1 before versus 1.29±0.40 mm Hg·mL^-1·min^-1 after administration and -1.75±1.86% before versus -1.74±1.78% after administration of 4ß-phorbol 12-myristate 13-acetate, respectively; P=NS, paired t test). To exclude a simple delay of the 4ß-phorbol 12-myristate 13-acetate–related vasoconstrictor effect, coronary arterial pressure was monitored for 20 minutes.

To estimate the dilution of the intracoronary infusion of staurosporine by native drug-free flow, collateral blood flow was measured in seven animals (three control animals and two animals each from the staurosporine-treated groups) using left atrial infusion and arterial reference withdrawal of colored microspheres³⁷ during total coronary occlusion at the end of the experiment. Subendocardial collateral blood flow was 0.018±0.007 mL·min^-1·g^-1, ie, 2.1±0.8% of control zone flow (0.889±0.177 mL·min^-1·g^-1) and on the average 36% of the native subendocardial blood flow during the sustained ischemia (Table).

View this table: [in this window] [in a new window]   Table 1. Systemic Hemodynamics, Regional Myocardial Function, and Blood Flow

Morphology
At the end of each study, the heart was removed and sectioned from base to apex into five transverse slices in a plane parallel to the atrioventricular groove. The tissue slices were immersed in a 0.09 mol/L sodium phosphate buffer (pH 7.4) containing 1.0% triphenyltetrazolium chloride (Sigma-Aldrich Chemie GmbH) and 8% dextran (molecular weight, 77 800) for 20 minutes at 37°C to identify infarcted tissue. The amount of infarcted tissue is expressed as a percentage of the LV area at risk, as determined by the radioactive microsphere technique.³⁸

Group 1: Staurosporine and 90 Minutes of Severe Ischemia
In 10 swine, under control conditions, microspheres were injected into the left anterior descending coronary artery perfusion system for the measurement of regional myocardial blood flow, and systemic hemodynamic and regional myocardial dimension data were recorded. After control measurements, staurosporine (10^-7 mol/L) was infused intracoronarily at constant coronary arterial pressure. Fifteen minutes after the start of the staurosporine infusion in a steady state of all recorded parameters, regional myocardial blood flow was once more measured, and systemic hemodynamic and regional myocardial dimension data were recorded. Thereafter, animals were subjected to 90 minutes of ischemia in the presence of staurosporine. Blood flow to the left anterior descending coronary artery was reduced to a level sufficient to reduce the regional myocardial work index by at least 90%. The staurosporine infusion was reduced in proportion to the reduction of coronary blood flow to maintain a constant concentration of 10^-7 mol/L. At 5 minutes and 90 minutes of ischemia, additional sets of measurements were obtained before the myocardium was reperfused for 120 minutes. With the onset of reperfusion, the staurosporine infusion was stopped.

Group 2: Staurosporine, Ischemic Preconditioning, and 90 Minutes of Severe Ischemia
After control measurements in 10 swine, the infusion of staurosporine was started at a concentration of 10^-7 mol/L. After additional measurements with staurosporine, the swine were subjected to an initial period of 10 minutes of ischemia, in which blood flow was decreased to reduce the regional myocardial work index by at least 90%. Again, the staurosporine infusion was decreased in proportion to the reduction in coronary blood flow. After 5 minutes of ischemia, measurements were repeated. After 15 minutes of reperfusion at constant perfusion pressure, when reactive hyperemia had nearly subsided, a fourth set of measurements of systemic hemodynamic and regional myocardial dimension data was obtained. Thereafter, the swine were subjected to 90 minutes of ischemia with severity identical to that of the first ischemic period. At 5 minutes and 90 minutes of ischemia, further sets of measurements were obtained before the myocardium was reperfused for 120 minutes. Again, with the onset of reperfusion, the staurosporine infusion was stopped.

Two additional animals received intracoronary staurosporine at a concentration of 10^-6 mol/L. One animal receiving 10^-6 mol/L staurosporine developed an immediate low cardiac output failure and did not survive the initial preconditioning period. The other animal also experienced a marked reduction in ventricular function but then survived until the end of the experiment; this animal developed only a small infarction (infarct size, 3.0% of the area at risk) at a subendocardial flow of 0.08 mL·min^-1·g^-1. This animal was therefore not included in the data analysis.

At the end of each study, the digital reading of the roller pump was calibrated by collecting arterial blood in a graduated cylinder.

For reference purposes, the infarct size reduction in the present study with staurosporine was compared with that in historic controls without drug administration.³⁰ The historic control group undergoing 90 minutes of ischemia only was supplemented with the three additional animals of the present study that underwent plasma removal during 90 minutes of ischemia (see above).

Data Analysis and Statistics
Hemodynamic data were recorded on an eight-channel recorder (Gould MK 200A), simultaneously digitized at 200 Hz, and directly stored to the hard disk of an AT-type computer. Systemic hemodynamic and regional myocardial dimension parameters were recorded and digitized over a 20-second period during each microsphere injection (33 consecutive beats over at least two complete respiratory cycles) using CORDAT II software.³⁹ Hemodynamic parameters reported are heart rate, LV end-diastolic and peak pressure, LV dP/dt_max, and mean coronary arterial pressure. Regional wall function is presented as systolic wall thickening and the WI described above. Calculation of all systemic hemodynamic parameters was performed on a beat-to-beat basis, and data were then averaged.

All data are reported as mean±SD, and a value of P<.05 was accepted as indicating a significant difference in mean values. Statistical analysis was performed using SYSTAT software. Systemic hemodynamic, wall function, and blood flow data were subjected to a two-way ANOVA for repeated measures, accounting for the two groups of swine and the time course of the experiment. When significant differences were detected, individual mean values were compared using Tukey's post hoc tests. Area at risk and infarct size were analyzed by a one-way ANOVA comparing a historic control group undergoing 90 minutes of ischemia only (supplemented with the three additional animals of the present study), a historic ischemic preconditioning group, and the two staurosporine groups of the present study. Linear regression analyses between subendocardial blood flow at 5 minutes of ischemia³⁸ in the LV area at risk and infarct size (expressed as percentage of the area at risk) were compared by ANCOVA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heart rate was held constant by left atrial pacing, and systolic wall thickening of the posterior control wall remained stable throughout the experimental protocol.

Systemic Hemodynamics and Myocardial Blood Flow
Intracoronary infusion of staurosporine tended to slightly decrease LV peak pressure (P=NS) and LV dP/dt_max (P=NS). At constant coronary arterial pressure, regional myocardial blood flow was increased. In group 2, during the preconditioning ischemic period, the decrease in coronary inflow reduced mean coronary arterial pressure and regional myocardial blood flow. LV end-diastolic pressure slightly increased (P=NS), whereas LV peak pressure and LV dP/dt_max decreased. In both groups of swine, the decrease in coronary inflow during the prolonged ischemic period reduced mean coronary arterial pressure and regional myocardial blood flow. LV end-diastolic pressure increased (P=NS), whereas LV peak pressure and LV dP/dt_max decreased. Prolonging ischemia to 90 minutes did not result in further significant changes in LV pressure, LV dP/dt_max, or regional myocardial blood flow.

Regional Myocardial Function
Intracoronary infusion of staurosporine slightly decreased anterior systolic wall thickening (P=NS) and WI (P=NS). During the 10 minutes of preconditioning ischemia in group 2, anterior systolic wall thickening and WI decreased by >90% and remained depressed until the end of the 15-minute reperfusion period. At 5 minutes of the prolonged ischemic period, anterior systolic wall thickening and WI of the anterior wall decreased by >90% in both groups of swine and were slightly further decreased when ischemia was prolonged to 90 minutes.

Infarct Size
The area at risk was comparable between all groups of swine (Fig 1). After 90 minutes of severe ischemia with staurosporine infusion (group 1), 12.5±8.6% of the area at risk was infarcted. With ischemic preconditioning and staurosporine infusion (group 2), infarct size was reduced to 3.3±3.4% of the area at risk (P<.05 versus group 1). This was comparable to the infarct size reduction achieved by ischemic preconditioning without any drug infusion in historic groups (14.5±10.5% in nonpreconditioned versus 2.6±3.0% in preconditioned animals). Infarct size for any given subendocardial blood flow was significantly reduced in group 2 compared with group 1 at 5 minutes of ischemia (regression lines: group 1, y=-210x+22, r=-.86; group 2, y=-46x+5, r=-.41; P<.05, ANCOVA; Fig 2).

View larger version (33K): [in this window] [in a new window]   Figure 1. Area at risk (AAR, left bars) and infarct size (right bars) in group 1 (staurosporine and 90 minutes of ischemia) and group 2 (staurosporine and ischemic preconditioning and 90 minutes of ischemia). For comparison, data of a historic control group (supplemented with three additional animals of the present study) and a historic preconditioned group are presented. The AAR was comparable between the groups. Staurosporine did not affect the infarct size reduction achieved by ischemic preconditioning. LV indicates left ventricular. *P<.05 vs 90 minutes of ischemia; +P<.05 vs staurosporine and 90 minutes of ischemia.

View larger version (20K): [in this window] [in a new window]   Figure 2. Relationship between subendocardial blood flow at 5 minutes of ischemia in the area at risk and infarct size expressed as percentage of area at risk. In the presence of staurosporine, for any given subendocardial blood flow, infarct size was reduced in the preconditioned hearts compared with control hearts undergoing 90 minutes of ischemia only.

For a subendocardial blood flow <0.07 mL·min^-1·g^-1 (group 1), 16.6±6.7% (n=7) of the area at risk was infarcted. Infarct size in the group with ischemic preconditioning was reduced to 4.1±3.6% (n=7, P<.05 versus group 1). Also, infarct size for any given subendocardial blood flow was significantly reduced in group 2 compared with group 1 at 5 minutes of ischemia (regression lines: group 1, y=-243x+23, r=-.66; group 2, y=-54x+6, r=-.27; P<.05, ANCOVA; Fig 3).

View larger version (19K): [in this window] [in a new window]   Figure 3. Relationship between subendocardial blood flow at values <0.07 mL·min-1·g-1 at 5 minutes of ischemia in the area at risk and infarct size expressed as percentage of area at risk. In the presence of staurosporine, for any given subendocardial blood flow, infarct size was reduced in the preconditioned hearts compared with control hearts undergoing 90 minutes of ischemia only.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Staurosporine completely blocked the 4ß-phorbol 12-myristate 13-acetate–induced coronary constriction seen in preliminary studies. At this concentration, staurosporine did not prevent the infarct size reduction achieved by ischemic preconditioning. Therefore, activation of protein kinase C appears not to be involved in ischemic preconditioning in anesthetized swine in situ.

Comparison With Previous Studies
In anesthetized rabbits, the infarct size reduction by ischemic preconditioning is abolished by protein kinase C inhibitors (polymyxin B and staurosporine).²² Supporting the hypothesis that protein kinase C activation is a central step in the intracellular signal transduction of ischemic preconditioning, as first proposed by Liu et al,¹⁹ the infusion of the protein kinase C activators 4ß-phorbol 12-myristate 13-acetate and 1-oleoyl-2-acetylglycerol reduces infarct size.²² Subsequent studies confirmed the importance of activation of protein kinase C in ischemic preconditioning in rats,^{20 21} rabbits,^{6 8 13 17} and human isolated cardiomyocyte preparations.^{18 23}

In anesthetized dogs in situ, the role of activation of protein kinase C in ischemic preconditioning is controversial. There are two studies using an identical preconditioning protocol (four cycles of 5 minutes of ischemia and 5 minutes of reperfusion), which yield opposite results. In the study of Przyklenk et al,²⁴ protein kinase C is neither translocated nor activated by ischemic preconditioning, and the infarct size reduction by ischemic preconditioning is not blunted by two protein kinase C inhibitors, H-7 and polymyxin B. In contrast, in the study of Kitakaze et al,²⁵ the membrane fraction of protein kinase C is activated by the same preconditioning protocol, and polymyxin B, a potent protein kinase C inhibitor, abolishes the infarct size reduction by ischemic preconditioning.

In swine with total occlusion of the distal left anterior descending coronary artery, protein kinase C is activated during the preconditioning ischemia.²⁷ The infarct size reduction achieved by ischemic preconditioning in this model, however, is not abolished by intramyocardial microinfusion of staurosporine or bisindolylmaleimide, two potent protein kinase C inhibitors.²⁷ The failure of staurosporine or bisindolylmaleimide to block the beneficial effect by ischemic preconditioning in this preparation could relate to the intramyocardial microinfusion technique, since it is hampered by an inhomogeneous distribution of the protein kinase C inhibitors; thus, sufficient blockade of protein kinase C throughout the perfusion territory cannot be ensured.²⁷ More important, in that study the protein kinase C inhibitors were infused only during the preconditioning ischemia.²⁷ However, staurosporine prevents the infarct size–reducing effect of ischemic preconditioning only when infused for at least 10 minutes into the sustained ischemia.²⁸ Finally, since the coronary artery was totally occluded, infarct size in that study could not be related to the major determinant of infarct size development, ie, ischemic myocardial blood flow.²⁷

Critique of Methods
The strengths and limitations of the present experimental preparation have been discussed in detail elsewhere.³¹ Using the same preparation, we have recently shown that the increased interstitial adenosine concentration²⁹ and activation of ATP-dependent potassium channels³⁰ are both essential for the infarct size reduction by ischemic preconditioning in anesthetized swine, as previously shown in rabbits^{2 17} and dogs.³ Swine, which are large mammals like dogs but without an innate collateral circulation and thus like rats and rabbits,²⁶ were studied because infarct development in this species most closely resembles that observed in humans.

In the present study, the proximal left anterior descending coronary artery was cannulated and hypoperfused at low flow, resulting in a large area at risk (on the average, 45% of the LV mass) and a small infarct size when expressed as a percentage of the area at risk (12.5±8.6% in group 1). However, infarct size expressed as a percentage of the total LV mass in the present study averaged 6% in group 1 and was thus comparable to that in a previous study using swine with a total occlusion of only one distal left anterior descending coronary arterial branch.⁴⁰

In the present preparation, a model of low-flow myocardial ischemia was used. Thus, staurosporine could be provided throughout the ischemic period by intracoronary infusion, ensuring a homogeneous delivery of the protein kinase C inhibitor staurosporine at a constant concentration. Also, infarct size development was related to subendocardial blood flow at 5 and 90 minutes of ischemia.

In order to study the role of endogenous activation of protein kinase C for ischemic preconditioning in swine, we used an antagonist approach. Therefore, the potent protein kinase inhibitor staurosporine was infused intracoronarily. Staurosporine has the lowest K_i (on the order of 1 to 3 nmol/L^{41 42 43} ) for any protein kinase inhibitor used thus far.⁴⁴ The dose of staurosporine in the present study was related to inflow (10^-7 mol/L blood) to account for large flow variations during ischemia and reperfusion.

The subendocardial drug-free native collateral blood flow was 0.018±0.007 mL·min^-1·g^-1. The infused concentration of 10^-7 mol/L staurosporine therefore does not reflect exactly the concentration of staurosporine in the ischemic microcirculation. However, the collateral drug dilution is probably overestimated, because collateral flow was measured during total coronary occlusion rather than low-flow hypoperfusion as used in the experimental protocol, and collateral flow will, despite plasma removal, still contain some staurosporine. In any event, higher concentrations of staurosporine could not be given, since profound ventricular pump failure led to the loss or instability of the experimental preparation.

The staurosporine-induced decrease in coronary arterial resistance in the present study was comparable to the previously reported decrease in total peripheral resistance following staurosporine infusion.⁴⁵ Finally, in the present study, the concentration of staurosporine used abolished the 4ß-phorbol 12-myristate 13-acetate–induced increase of coronary arterial resistance, thus demonstrating the effective blockade of vascular protein kinase C activity. We did not directly prove the effect of staurosporine on myocardial protein kinase C activity. To avoid uncontrolled bleeding in this fully heparinized preparation, we did not take myocardial biopsies. Nevertheless, there is good reason to assume that staurosporine does reach the myocardial cytosol, because it is lipophilic and its molecular weight (466.5) permits staurosporine to pass the membrane of endothelial and myocardial cells.⁴⁴ The fact that staurosporine is a potent, but not a selective, inhibitor of protein kinase C activity⁴⁴ is a potential conservative error with respect to the involvement of protein kinase C in ischemic preconditioning, since our data are negative.

In comparison with previous results from our laboratory in swine also undergoing 90 minutes of severe ischemia,³⁰ the relation between infarct size and subendocardial blood flow following staurosporine infusion in the present study was shifted somewhat leftward. The most likely reason for this shift is a slight reduction in systemic hemodynamics in the present study compared with our previous study. The effect of staurosporine on infarct size per se was not the subject of the present study.

Why No Effect of Staurosporine on Ischemic Preconditioning?
Results from previous experiments²⁷ and the data from the present study do not confirm the importance of protein kinase C activation for the infarct size reduction by ischemic preconditioning in swine, in contrast to its pivotal role in rats^{20 21} and rabbits.^{6 8 13 19 22} A possible explanation for these discrepant findings relates to species differences, although such an explanation is not very satisfying, when controversial data exist even in one species, ie, dogs.^{24 25}

Species differences might relate to the effectiveness of the blockade of different isoforms of protein kinase C by staurosporine. Several isoforms of protein kinase C have been detected in adult ventricular myocytes of rats (-isoform,^{46 47 48} ß-isoform,⁴⁸ -isoform,^{46 47 48 49} -isoform,^{46 47 48 49 50} and - and -isoforms^{46 48} ) and dogs (-, ß-, and -isoforms,⁵¹ -isoform,^{49 51} and -isoform⁴⁹ ). In rat⁵⁰ and dog⁴⁹ hearts, the -isoform of protein kinase C is the major isoform present, which is activated in response to 4ß-phorbol 12-myristate 13-acetate, epinephrine, and endothelin.⁵⁰ In anesthetized rats, the activation of the -isoform of protein kinase C during myocardial ischemia, as well as the activation of the -isoform during myocardial ischemia or during ₁-adrenoceptor activation, is associated with improved postischemic recovery of LV function by ischemic preconditioning, and this effect is reversed by staurosporine.⁴⁸ Whether or not staurosporine effectively inhibits the activity of the major isoform of protein kinase C in porcine cardiomyocytes remains unclear.

An alternative expression of species differences relates to different protein kinases, which are activated during ischemia. The G protein–coupled activation of bradykinin B₂ and muscarinic M₂ receptors results in the activation of both protein kinase C and protein tyrosine kinase.⁵² Also, the activation of G_i protein–coupled receptors stimulates mitogen-activated protein kinase and therefore shares a common pathway with the protein tyrosine kinase.⁵³ Pathways of different kinase systems obviously interact in a network of signal transduction.^{54 55} Therefore, the observation that blockade of protein kinase C activity does not prevent ischemic preconditioning in swine may relate to the activation of alternative pathways, such as the protein tyrosine kinase, which, once activated, protect the myocardium against infarction.

With respect to species differences, the most important data obviously arise from studies in human preparations. In cultured human ventricular myocytes, the injury (as assessed by trypan blue uptake) caused by 90 minutes of simulated ischemia and 30 minutes of reperfusion is reduced by a preconditioning cycle of 20 minutes of simulated ischemia and 20 minutes of reperfusion, and this effect involves the activation of protein kinase C.²³ Activation of protein kinase C is also essential for the improvement in functional recovery of atrial trabeculae subjected to 90 minutes of hypoxic substrate-free superfusion and rapid pacing at 3 Hz by a preceding episode of such simulated ischemia and reperfusion.¹⁸ However, these observations differ from classic ischemic preconditioning in their use of hypoxia rather than ischemia and the surrogate end points of trypan blue uptake or postischemic functional recovery rather than infarct size at a given flow. Therefore, the role of activation of protein kinase C in classic ischemic preconditioning in human ventricular myocardium is still not clear.

	Acknowledgments

 
This study was supported by the German Research Foundation (He 1320/8-2). Dr Vahlhaus was the recipient of a research fellowship from the German Research Foundation (Va 128/2-1). We thank Dr C. Martin for the chemical analysis and Petra Gres and Ursula Pragler for their technical support.

Received December 12, 1995; accepted May 9, 1996.

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