Ann Thorac Surg 2002;73:1236-1245
© 2002 The Society of Thoracic Surgeons
Original article: cardiovascular
Protective effects of protein kinase C during myocardial ischemia require activation of phosphatidyl-inositol specific phospholipase C
Mamoru Munakata, MDa,
Christof Stamm, MDa,
Ingeborg Friehs, MDa,
David Zurakowski, PhDb,
Douglas B. Cowan, PhDc,
Hung Cao-Danh, PhDa,
Francis X. McGowan, Jr, MDc,
Pedro J. del Nido, MD*a
a Department of Pediatric Cardiac Surgery, Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
b Department of Biostatistics, Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
c Department of Pediatric Anesthesiology/Critical Care, Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
Accepted for publication November 28, 2001.
* Address reprint requests to Dr del Nido, Department of Cardiac Surgery, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115 USA
e-mail: pedro.delnido{at}tch.harvard.edu
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Abstract
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Background. Protein kinase C (PKC) activation during myocardial ischemia is thought to be cardioprotective. However, the mechanism of ischemia-induced PKC activation remains unclear. We hypothesized that ischemic PKC activation occurs through activation of phosphatidyl-inositol specific phospholipase C (PI-PLC) and protects the heart from ischemic injury.
Methods. Isolated rabbit hearts were subjected to 20 minutes of normothermic ischemia and reperfusion. The PI-PLC inhibitor U73122 (0.5 µmol/L), its inactive analogue U73343 (0.5 µmol/L), or the PKC inhibitor chelerythrine (2 µmol/L) were given just before ischemia. Another group received U73122 plus the direct PKC activator phorbol 12-myristate-13-acetate (PMA, 10 pmol/L). Measurements included contractile function, intracellular calcium, PI-PLC activity, and translocation of PKC isoforms.
Results. PI-PLC activity increased during myocardial ischemia and was inhibited by U73122. PI-PLC inhibition prevented the ischemic translocation of PKC-
, PKC-
, and PKC-
, and impaired cardiac recovery and cytosolic calcium regulation without significant changes in energy metabolism. PMA restored both contractile function and PKC translocation pattern in U73122-treated hearts. Direct PKC inhibition with chelerythrine mimicked the effects of U73122.
Conclusions. PI-PLC mediates PKC translocation during myocardial ischemia. Inhibition of PI-PLC or PKC activation, or both, during ischemia significantly impairs postischemic myocardial recovery.
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Introduction
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Ischemia with or without myocardial protection is known to activate a variety of membrane-associated and intracellular enzymes in cardiomyocytes. Of particular importance is the activation of protein kinase C (PKC), a regulatory kinase that phosphorylates a number of target proteins that are involved in excitation-contraction coupling and myocardial contractility. Sarcolemmal membrane receptors including 1 adrenergic, endothelin-1, angiotensin-II, and bradykinin receptors have been shown to be coupled to phosphatidyl-inositol specific phospholipase C (PI-PLC), leading to phosphoinositide turnover when activated. Breakdown of phosphatidylinositol 4,5-bisphosphate by PI-PLC produces inositol 1,4,5-triphosphate and diacylglycerol (DAG). DAG then activates PKC by binding to a specific activation site, resulting in phosphorylation of ion channels, cell membrane receptors, and contractile proteins. In experimental models PKC activation can also be directly activated through exogenous administration of DAG-analogous phorbol esters. In nonischemic hearts infusion of phorbol esters at low concentrations results in impaired cardiac contractile function, affecting both the systolic and diastolic pressure-volume relationship [1–3]. The responsible mechanisms involve PKC phosphorylation of troponin T and troponin I, causing suppression of acto-myosin ATPase activity and thus decreasing contractile protein calcium sensitivity, as well as alterations in intracellular calcium handling [4]. Conversely, we have shown that in a transgenic mouse model with a loss-of-function mutation of the PKC phosphorylation site on troponin I, contractile protein sensitivity to calcium was increased, associated with impaired recovery of contractile function after ischemia and reperfusion [5].
The mechanism of PKC activation during ischemia remains unclear, however. As the ischemic myocardium is not accessible for the above-mentioned circulating messenger molecules, the classic receptor-mediated pathway is unlikely to play an important role. Hence it is unclear whether PKC inhibition during ischemia occurs through PI-PLC activation. During myocardial ischemia and reperfusion PI-PLC activity is increased but the mechanism of ischemia-induced PI-PLC activation is not known [6–8]. There is also substantial evidence that activation/translocation of both Ca2+-dependent and Ca2+-independent isoforms of PKC occurs during ischemia or hypoxia and this effect is clearly independent of 1-adrenergic receptor activation [9–11]. Here, we sought to investigate whether PI-PLC activation is necessary for cardioprotective PKC activation/translocation during myocardial ischemia. To test this hypothesis we studied (1) whether inhibition of PI-PLC activity prevents ischemic translocation of PKC isoforms; and (2) whether suppression of either PI-PLC or PKC activity at the onset of ischemia results in impaired recovery of contractile function after reperfusion.
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Material and methods
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Isolated heart preparation
Animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources (NIH publication No. 86-23, revised 1985). New Zealand white rabbits (1.8 to 3.2 kg) were euthanized by intravenous bolus administration of ketamine (100 mg/kg), xylazine (2 mg/kg), and heparin (500 U/kg). Hearts were rapidly excised, placed in 4°C modified Krebs-Henseleit (KH) buffer, and perfused in the Langendorff mode at 80 mm Hg constant pressure with 37°C KH buffer (115 mmol/L NaCl, 26 mmol/L NaHCO3, 11 mmol/L glucose, 1.8 mmol/L MgSO4, 1.8 mmol/L KH2PO4, 2.7 mmol/L KCL, 1.25 mmol/L CaCl2, and 10 U/L insulin) that had been equilibrated with a 95% O2/5% CO2 gas mixture and passed through a 0.2-micron filter. The temperature of the hearts was monitored with a thermistor placed in the right ventricle and was maintained at 37°C. A fluid-filled latex balloon connected to a micromanometry catheter (Millar Instruments, Houston, TX) was placed in the left ventricle through the left atrium and was used for isovolumic measurement of left ventricular function. Caval and pulmonary veins were sutured closed. The pulmonary artery was cannulated but allowed to drain freely and samples were taken for measurement of myocardial oxygen consumption (MvO2), derived from the difference of O2 tension between aortic perfusate and coronary effluent. Coronary flow (CF) was measured by timed collection of the coronary effluent.
Experimental protocols
After 30 minutes stabilization, hearts were subjected to 20 minutes normothermic global ischemia followed by 30 minutes reperfusion. In one group of hearts (n = 9) the specific PI-PLC inhibitor U73122 (0.5 µmol/L), dissolved in dimethylsulfoxide (DMSO), was administered for 2.5 minutes just before the onset of ischemia. In a second group (n = 9) the direct PKC activator phorbol 12-myristate-13-acetate (PMA 10 pmol/L) was infused together with U73122 to counteract the effects of PI-PLC inhibition on PKC. Control hearts received vehicle only (DMSO, 0.05%). In an additional control group (n = 8), U73343 (the inactive analogue of U73122, 0.5 µmol/L) was administered for 2.5 minutes just before the onset of ischemia. To determine whether direct PKC inhibition mimics the effects of PI-PLC inhibition on postischemic recovery, chelerythrine (2 µmol/L), a specific PKC inhibitor, was administered for 15 minutes before the onset of ischemia in another group of hearts (n = 7). Both U73122 (0.5 µmol) and chelerythrine (2 µmol/L) were also given to nonischemic hearts for 2.5 minutes or 15 minutes, respectively (n = 5 each). The different infusion periods for U73122 and chelerythrine were chosen based on preliminary experiments to account for differences in lipid solubility and thus the rate of cellular uptake.
Measurements of contractile function, coronary flow, and MvO2 were made at the end of the 30-minute equilibration period (just before ischemia) and 30 minutes after reperfusion. Left ventricular developed pressure was measured at a preischemic diastolic pressure of 5 mm Hg and the balloon volume to achieve this diastolic pressure was noted. The same balloon volume was later used to measure diastolic and developed pressure after 30 minutes of reperfusion. Because the hearts were not paced in these experiments, the rate-pressure product was also calculated to assess the treatment effects on left ventricular work. In the separate experiments where cytosolic calcium was measured, hearts were paced at 150 beats per minute by ventricular pacing after ablating the A-V node. When ventricular fibrillation occurred electrical defibrillation was performed. In nonischemic hearts measurements were obtained every 10 minutes over a period of 50 minutes, analogous to 20 minutes ischemia plus 30 minutes reperfusion in the ischemic model.
Metabolic measurements
In U73122 treated hearts, left ventricular tissue subjected to 20 minutes ischemia was obtained for lactate and adenosine triphosphate (ATP) measurements. Tissue samples were snap frozen in liquid nitrogen and homogenized in 6% perchloric acid solution. Lactate levels were obtained using an enzymatic assay (Sigma, St. Louis, MO) and expressed as mmol total lactate per gram dry weight. Myocardial ATP content was measured at end-ischemia by HPLC as described by Bernt and colleagues [12].
Calcium measurements
Measurement of cytosolic calcium levels was performed in a separate set of intact perfused hearts (n = 7 each) after 30 minutes reperfusion following 20 minutes ischemia as we have previously described in detail [13]. Briefly, after 12.5 minutes of postischemic reperfusion the calcium-sensitive fluorescent dye rhod-2 (0.5 mg; Molecular Probes, Eugene, OH) was loaded by injecting 0.5 mg cell-permeable rhod-2 acetoxymethyl ester dissolved in 0.25 mL anhydrous DMSO into the aortic root over 2.5 minutes, followed by a 15-minute washout period to remove nonhydrolyzed dye. The isolated perfused heart was placed in a modified spectrofluorometer (SLM-Aminco, Springfield, IL) and excitation light at 524 nm was guided to an 5 x 5 mm area of LV free wall that was gently immobilized behind a quartz window. Emission fluorescence (Fem) was collected in a high-voltage photomultiplier tube at 589 nm and plotted over time for recordings of intracellular calcium transients (time trace). Real-time acquisitions of 4 ms were digitized and stored. Because rhod-2 has no wavelength shift when it binds calcium, Fem must be corrected for changes in tissue rhod-2 concentration over time (eg, leakage) or differences in dye loading. The magnitude of light absorbance (A) at 524 nm indicates the amount of intracellular rhod-2 and was assessed by running an excitation scan immediately after every time trace. For these excitation scans scattered emission light was measured at 589 nm (Fex) while the excitation wavelength was varied between 500 and 600 nm in 1-nm increments. The ratio of scattered light at 524 nm (Fex(524)) and 589 nm (Fex(589)) was calculated to estimate the tissue dye concentration (where 524 nm is the peak rhod-2 absorbance in cardiac tissue and 589 nm is the reference wavelength at which rhod-2 does not absorb light). The calcium fluorescence signal was also corrected for tissue autofluorescence by deducting the emission light intensity measured with a time trace performed before rhod-2 loading (F0em). Cytosolic calcium values during the cardiac cycle were expressed as peak fluorescence intensity corrected for autofluorescence divided by the absorbance correction for dye concentration (F/A), using the following equation: 
Western immunoblotting
Hearts were snap frozen in liquid nitrogen and stored at -80°C. Left ventricular tissue was homogenized in buffer A (pH 7.5, 20 mmol/L Tris-HCl, 2 mmol/L EDTA, 0.5 mmol/L EGTA, 1 mmol/L PMSF, 25 µg/mL leupeptin, 0.33 mol/L sucrose) and centrifuged at 1,000g for 10 minutes to remove tissue debris. The supernatant (total cell homogenate) was then centrifuged at 100,000g for 1 hour. The supernatant was the cytosolic fraction. The pellet was resuspended in buffer B (buffer A with 1% Triton X 100, no sucrose) and incubated at 4°C for 1 hour followed by centrifugation at 100,000g for 1 hour. This resuspended pellet was the particulate (membrane containing) fraction. We have previously shown that the supernatant contains primarily cytosolic proteins, including troponin I, whereas the particulate fraction contains only membrane proteins such as Na-K ATPase and SERCA-2. Samples were stored at -80°C for later analysis. Protein samples of 25 µg each were separated by SDS-Page gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with primary rabbit polyclonal antibody against PKC-, PKC- (Upstate Biotechnology, Lake Placid, NY), PKC- (Calbiochem, San Diego, CA), and PKC-
(ICN, Costa Mesa, CA), followed by incubation with horseradish peroxidase-conjugated goat antirabbit IgG secondary antibody and detection using enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). Positive controls or molecular weight markers were used to identify the appropriate band. Laser densitometry was used to quantify the intensity of the bands.
Measurement of PI-PLC activity
To determine whether PI-PLC is activated during myocardial ischemia and to assess the effect of U73122 on PI-PLC activity, a separate set of hearts (n = 6) was perfused to obtain tissue samples for measurement of total PI-PLC activity as previously described by Schwertz and coworkers [5]. Myocardial tissue samples were taken from each heart before the onset of ischemia and at end-ischemia. Aliquots of left ventricular total tissue homogenate were incubated with a mixture of 3H-labeled (New England Nuclear, Boston, MA) and unlabeled phosphatidyl-inositol at a total concentration of 400 µmol/L in the presence of 1 mmol/L calcium for 30 minutes. Incubations were terminated by adding a CHCl3/MeOH/HCl mixture. CHCl3 and MeOH phases were separated, and the MeOH phase contained the reaction product, 3H-labeled inositol phosphate, which was measured by liquid scintillation counting. PLC activity was expressed as cpm/mg protein.
Statistical analysis
Continuous data are expressed as mean ± standard deviation. Analysis of variance (ANOVA) was used to determine the effects of U73343, U73122, chelerythrine, and U73122 plus PMA on left ventricular developed pressure and diastolic pressure between in nonischemic hearts. An overall F test was followed by the Dunnett procedure for multiple comparisons. Linear regression (least-squares) was used to evaluate the relationship between U73122 dose and developed and diastolic pressure. The F test was used to evaluate goodness-of-fit and the coefficient of determination (R2) was used to evaluate the amount of variation in pressure explained by dose. A paired t test was used to compare PLC activity measurements before and after ischemia. All reported p values are two-tailed using an alpha level of 0.05 for significance. Ventricular fibrillation rates between U73122 and U73122 plus PMA group were compared by 
2analysis. Analysis of the data were performed using the SPSS software package (version 11.0, SPSS Inc, Chicago, IL).
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Results
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PI-PLC activation during ischemia
PI-PLC activity was significantly increased at end-ischemia as compared with preischemic levels. However, administration of U73122 (0.5 µmol/L) before the onset of ischemia inhibited PI-PLC activation during ischemia (Fig 1).
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Fig 1. Phosphatidyl-inositol specific phospholipase C (PI-PLC) activity measured in left ventricular myocardium before (preischemia), after 20 minutes unmodified ischemia at 37°C (end-ischemia), and in hearts treated with the PI-PLC inhibitor U73122 just before ischemia (end-ischemia plus U73122). (Data are mean ± SD; n = 5 per group). Paired t-tests were used to assess changes in PI-PLC activity.
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PKC translocation in ischemic hearts
The total content of the , , , and isoforms of PKC in whole tissue homogenate was assessed by Western immunoblotting and was not different between nonischemic hearts, control (vehicle treated) ischemic hearts, U71322 treated hearts, or hearts treated with U73122 and PMA (Fig 2A).
However, PI-PLC inhibition had marked effects on the subcellular translocation pattern of PKC isoforms. In nonischemic hearts PKC- was mainly found in the cytosolic fraction, and translocated to the particulate fraction by end-ischemia. Pretreatment with the PI-PLC inhibitor U73122 effectively prevented translocation of PKC- during ischemia (Fig 2B). PKC- translocation to the membranous fraction during ischemia was restored by addition of the direct PKC activator PMA in U73122 hearts. Preischemia PKC- content was slightly higher in the membranous fraction and this isoform translocated to the cytosolic fraction during ischemia. U73122 prevented PKC- translocation during ischemia and concomitant administration of PMA restored the ischemic translocation pattern. PKC- translocated from the cytosolic to the membranous fraction during ischemia and this translocation was again prevented by U73122. However, simultaneous administration of PMA did not restore the translocation pattern seen in unmodified ischemia. Finally, PKC- content in both fractions did not significantly change during ischemia and neither U73122 alone nor U73122 plus PMA had any effect on its subcellular distribution.
PI-PLC inhibition and postischemic LV function
Administration of the PI-PLC inhibitor U73122 before the onset of ischemia led to impaired recovery of both developed pressure and diastolic pressure after reperfusion in a dose-dependent manner (Fig 3).
A highly significant inverse correlation was found between U73122 dose and developed pressure (r = -0.80, p < 0.001). Linear regression indicated a good linear fit, with 65% of the variation in developed pressure accounted for by dose (R2 = 0.65, F(1, 17) = 27.3; p < 0.001; Fig 3A). Also, a highly significant positive correlation was found between U73122 dose and diastolic pressure after reperfusion (r = 0.85; p < 0.001). Linear regression revealed an excellent fit with 72% of the variation in diastolic pressure accounted for by dose (R2 = 0.72, F(1, 17) = 44.7; p < 0.001; Fig 3B). A dose greater than 5 µmol/L U73122 resulted in rigor formation by 30 minutes of reperfusion (percent recovery of developed pressure < 30%, n = 3). In contrast, administration of U73343 (0.5 µmol/L), the inactive analogue of U73122, had no effect on cardiac function after reperfusion as compared with untreated controls (recovery of developed pressure 80.1% ± 4.5%, diastolic pressure 5 ± 1.4 mm Hg; Fig 4A,B).
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Fig 4. Postischemic recovery of contractile function. (A) Left ventricular (LV) developed pressure (expressed as percent of the preischemic value) after 20 minutes of ischemia at 37°C, and 30 minutes of reperfusion. (B) Left ventricular diastolic pressure measured at the same balloon volume as preischemia. (C) Rate-pressure product (RPP) LV developed pressure x heart rate, expressed as percent recovery of preischemic RPP). Vehicle or treatment was administered just before the onset of ischemia: control, vehicle (DMSO 0.05%, n = 4); U73343, 0.5 µmol/L U73343 (n = 4); U73122, 0.5 µmol/L U73122 (n = 8); chelerythrine, 2 µmol/L chelerythrine (n = 6); U73122 + PMA, 0.5 µmol/L U73122 plus 10 pmol/L PMA (n = 8). Data are mean ± SD. Groups are compared using ANOVA followed by the Dunnett procedure for multiple comparisons.
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Simultaneous administration of PMA (10 pmol/L) to hearts receiving U73122 (0.5 µmol/L) restored recovery of developed pressure to near control levels (81.8% ± 8.3%, 65.6% ± 12.1%, and 76% ± 3.9% in control, U73122, and U73122 plus PMA, respectively; Fig 4A). This dose of PMA was chosen because our previous data indicated that PMA alone at this concentration had no effect on cardiac function [3]. Preischemic infusion of chelerythrine mimicked the effects of U73122 in our model. Recovery of developed pressure was significantly attenuated (83.8% ± 5.9% versus 69.1% ± 9.2% in control and chelerythrine, respectively), and diastolic pressure after reperfusion increased (4 ± 1 mm Hg versus 11 ± 4 mm Hg in control and chelerythrine, respectively; Fig 4). ANOVA indicated significant differences among the treatment groups shown in Figure 4A: F(4, 34) = 5.41, p = 0.002. Dunnett’s procedure revealed lower developed pressure in hearts treated with chelerythrine (p < 0.01) and U73122 (p < 0.01) compared with controls; no differences were observed between controls and U73343 (p = 0.98) or U73122 + PMA (p = 0.40). ANOVA also revealed significant treatment effects on diastolic pressure as seen in Figure 4B: F (4, 34) = 23.01, p < 0.001. Diastolic pressure was significantly elevated in the U73122 group (p < 0.001) and concomitant treatment with PMA ameliorated this rise (4 ± 1.4 mm Hg, 20.9 ± 7.6 mm Hg, and 10.8 ± 5 mm Hg in control, U73122, and U73122 plus PMA, respectively). The rate-pressure product at 30 minutes reperfusion also mirrored the detrimental effects of PLC/PKC inhibition on postischemic recovery and the beneficial effect of simultaneous PMA treatment: F(4, 33) = 13.59, p < 0.001 (Fig 4C). Furthermore, the incidence of ventricular fibrillation at the beginning of reperfusion was higher in the U73122 group than in the U73122 plus PMA group (Table 1).
PI-PLC inhibition and postischemic intracellular calcium
At 30 minutes reperfusion, diastolic cytosolic calcium levels were higher in U73122 treated hearts than in control hearts (4.1 ± 0.6 versus 6.1 ± 0.8 F/A arbitrary units; p = 0.001). Systolic cytosolic calcium levels were not significantly different between U73122 treated and control hearts (8.2 ± 1.3 versus 8.7 ± 1.9 F/A arbitrary units; p = 0.6). Consequently, the amplitude of the systolic calcium transient was lower in U73122 treated hearts than in control hearts (2.6 ± 1.2 versus 4.1 ± 1.3 F/A arbitrary units; p = 0.06). The time-to-half systolic calcium (t1/2) as an indicator of the diastolic decline of the calcium transient was longer in U73122 treated hearts than in control hearts (80.1 ± 10.6 ms versus 62.1 ± 10.3 ms; p = 0.01), suggesting delayed diastolic calcium removal from the cytosol after U73122 treatment.
Coronary flow, oxygen consumption, lactate, and ATP content
There were no significant differences before or after ischemia with respect to coronary flow, heart rate, MVO2, lactate, or ATP content of left ventricular tissue at the end of ischemia between control, U73122, and U73122 plus PMA groups, indicating that the observed differences in myocardial contractility and calcium handling postischemia were not due to altered oxidative metabolism (Table 1).
PI-PLC/PKC-inhibition in nonischemic hearts
Inhibition of PI-PLC by U73122 (0.5 µmol/L for 2.5 minutes) in nonischemic control hearts had no significant effect on developed pressure or diastolic pressure. Similarly, chelerythrine (2 µmol/L for 15 minutes) given to nonischemic hearts had no significant effect on developed pressure or diastolic pressure (Fig 5).
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Fig 5. Effects of U73122 or chelerythrine on contractile function in nonischemic hearts. Left ventricular developed pressure (A) and diastolic pressure (B) after administration of Krebs buffer (control), U73122 (0.5 µmol/L for 2.5 minutes), or chelerythrine (2 µmol/L for 15 minutes). There were no significant differences between control hearts and hearts treated with chelerythrine or U73122 (p > 0.1 each by ANOVA followed by the Dunnett procedure for multiple comparisons).
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Comment
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In our experiments, PI-PLC activity increased during normothermic ischemia, and inhibition of PI-PLC during ischemia prevented translocation of PKC- in association with impaired postischemic recovery of LV contractility and, more pronounced, relaxation as well as intracellular calcium handling. Bypassing the PI-PLC signaling step by direct PKC activation with a phorbol ester restored postischemic cardiac recovery to control levels. Direct PKC inhibition with chelerythrine mimicked the effects of PLC-inhibition on postischemic recovery. The inactive analogue of U73122, U73343, did not impair cardiac recovery. Taken together these data indicate that protein kinase C activation during ischemia occurs through phosphatidyl-inositol specific phospholipase C activation and is protective for the heart.
To date, at least 10 isoforms in 3 subgroups of PI-PLC (PLC-ß1 to 4, PLC-
1 to 2, and PLC-1 to 4) have been identified in mammalian cells [14]. It is still unclear which isoform of PI-PLC is most prevalent in cardiac tissue. However, PI-PLC-1 seems to be the predominant isoform in adult rabbit ventricular myocytes [15]. PI-PLC activation through membrane receptor binding leads to phosphoinositide turnover resulting in production of diacylglycerol and activation of PKC. The effects of ischemia on PI-PLC activity appear to be time dependent. With very long ischemic times (hours), PI-PLC activity and PI-PLC protein content in the membrane have been reported to be decreased [6–8]. The effects of shorter periods of ischemia on activation of cardiac isoforms of PI-PLC are not completely understood. Takeishi and coworkers [16] have reported that PKC- but not PKC- translocation occurs during hypoxia through a PI-PLC-pathway in adult guinea pig heart. However, they used tricyclodecan-9-yl-xanthogenate (D609) as a PI-PLC-inhibitor, which also inhibits phosphatidylcholine-specific PLC (PC-PLC). Thus the role of PI-PLC activation in their study was not completely clear. Based on our results we postulate that PI-PLC activation is occurring during short periods of ischemia, presumably in response to membrane receptor activation by adenosine, endothelin, bradykinin, or rising cytosolic calcium [14–17].
Protein kinase C has also been shown to be activated in the heart during ischemia in a time-dependent manner as detected by membrane translocation and increased membrane activity [9–11]. However, isoform-specificity and species-specificity of PKC translocation are still controversial. We investigated the translocation of four PKC isoforms (two calcium dependent and two calcium independent) that are present in rabbit hearts and that have previously been implicated in ischemia-reperfusion injury [18]. Several investigators have reported ischemia- or hypoxia-induced translocation of PKC- and PKC- from cytosol to membrane in rat heart [9, 11, 19]. In the rabbit heart, Ping and associates [18] have demonstrated that PKC- but not PKC- translocated from cytosol to membrane during ischemia. In our study PKC- translocated to the membranous fraction and PKC- from the membranous fraction to the cytosolic fraction. This discrepancy may be explained by the period of ischemia, as only 4 minutes of ischemia was imposed in the Ping study as opposed to 20 minutes in ours. Because PKC- is a Ca2+-dependent isoform, prolonged ischemia with a significant rise in cytosolic calcium may be required for PKC- activation.
Recently PKC- activation during ischemia has been found to be cardioprotective. Chen and colleagues [20] and others [23] have reported an ethanol-induced cardioprotective effect related to PKC- activation in the ischemic rat cardiomyocyte. In the rabbit heart, Qiu and associates [21] and Liu and colleagues [22] have implicated PKC- activation in the ischemic preconditioning phenomenon. In our study the beneficial effect of infusing PMA to the U73122 treated hearts, "PMA rescue," indicates that PKC activation plays a central role in protection during longer periods of still reversible ischemia. Furthermore, PI-PLC activity is likely to be necessary for activation of both PKC- and PKC- during ischemia because diacylglycerol, a product of PI-PLC activation and PI turnover, activates both Ca2+-independent PKC- as well as the Ca2+-dependent PKC-. Direct PKC inhibition by administration of chelerythrine before the onset of ischemia significantly impaired postischemic diastolic function. This finding is consistent with the results found in U73122 treated hearts and underscores the important cardioprotective role for PKC during ischemia, but is at variance with other studies showing that chelerythrine had no effect on postischemic function in rat heart [24] or on infarct size in rabbit heart [25, 26]. Again, differences in species and experimental model likely explain the different findings.
Several possibilities exist with respect to the mechanism by which PKC prevents diastolic dysfunction during the reperfusion period [27]. These include decreased cytosolic calcium accumulation during ischemia by inhibiting L-type calcium channel opening [28], decreased sensitivity of contractile proteins to calcium by thin filament protein phosphorylation [2, 5, 11, 29], and potentially, improved preservation of high energy phosphates required for breaking actomyosin rigor bonds. Tissue ATP and lactate levels at the end of ischemia were not different between the control untreated, U73122, or U73122 plus PMA treated hearts, suggesting that PI-PLC or PKC inhibition during ischemia did not affect energy metabolism (Table 1). Moreover, the ATP levels observed at end-ischemia were sufficiently high to prevent ischemic contracture in all groups. Also, MvO2 recovered to equal levels in all four groups (Table 1), indicating that oxidative metabolism during reperfusion was not significantly different between the groups. Cytosolic calcium rise during ischemia may also lead to contractile protein interaction and rigor bond formation [29, 30]. PKC activation has been found to inhibit sarcolemmal L-type Ca2+ channel opening and increase Ca2+ uptake into the sarcoplasmic reticulum in vitro [28]. Thus activation of PKC during ischemia may serve to prevent calcium accumulation in the cytosol, resulting in decreased rigor bond formation. On the other hand, activation of PI-PLC leads to IP3 release, which may further increase cytosolic calcium by promoting release from intracellular calcium stores [31]. As both PIP2 breakdown products, IP3, and diacylglycerol are produced it is unclear which product of PI-PLC activation predominates in myocardium during ischemia with respect to cytosolic calcium regulation. Our finding that diastolic cytosolic calcium levels were higher in U73122 treated hearts than in control hearts indicates that DAG-mediated PKC activation is the dominating effect of PLC activation during myocardial ischemia. PLC inhibition also led to delayed diastolic calcium removal from the cytosol and a reduced amplitude of the calcium transient, which is consistent with the hypothesis that PLC-mediated PKC-phosphorylation of sarcoplasmic calcium pumps (phospholamban or sarcoplasmic Ca2+ ATPase, or both) serves to protect the heart from cytosolic calcium accumulation during ischemia. The observed changes in intracellular calcium handling help explain the impaired diastolic relaxation associated with PLC/PKC inhibition. However, PKC has also been shown to modulate contractile protein sensitivity to calcium by phosphorylating troponin I (TnI) and troponin T (TnT) resulting in decreased Ca2+ sensitivity of the actomyosin ATPase and decreased actin-myosin interactions [4, 5, 32, 33]. Hence a PKC-mediated reduction of contractile protein calcium sensitivity may be an additional mechanism by which PLC/PKC activation preserves diastolic compliance during ischemia and reperfusion.
In conclusion, PI-PLC activation during myocardial ischemia appears to be an important step in the signaling cascade leading to the protective PKC effects on intracellular calcium regulation and the postischemic calcium-force relationship.
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Acknowledgments
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Supported by NIH grants HL-46207 (PJdN) and HL-52589 (FXM). Dr Christof Stamm was supported by a grant from the Deutsche Forschungsgemeinschaft (STA 497/2-1). We gratefully acknowledge Christine Rader and Dimitrios N. Poutias for their technical assistance.
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Abstract
Introduction
. (B) Diastolic pressure measured at a fixed balloon volume, after 20 minutes ischemia at 37°C, followed by 30 minutes reperfusion. (Each datum point represents one ischemia-reperfusion experiment.) The linear model for predicting diastolic pressure in mm Hg (y) from U73122 dose (x) can be described by the regression equation: 
.