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Cardiovascular Research 2001 51(1):108-121; doi:10.1016/S0008-6363(01)00249-8
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Post-ischemic PKC inhibition impairs myocardial calcium handling and increases contractile protein calcium sensitivity

Christof Stamma, Ingeborg Friehsa, Douglas B. Cowanb, Hung Cao-Danha, Sabrena Noriac, Mamoru Munakataa, Francis X. McGowan, Jr.b and Pedro J. del Nidoa,*

aDepartment of Cardiac Surgery, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
bDepartment of Anesthesia, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA
cDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada M5G 2C4

* Corresponding author. Tel.: +1-617-355-8290; fax: +1-617-232-2697 delnido{at}cardio.tch.harvard.edu

Received 23 August 2000; accepted 15 February 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
Objective: Protein kinase C (PKC) activation impairs contractility in the normal heart but is protective during myocardial ischemia. We hypothesized that PKC remains activated post-ischemia and modulates myocardial excitation–contraction coupling during early reperfusion. Methods: Langendorff-perfused rabbit hearts where subjected to 25 min unmodified ischemia and 30 min reperfusion. Total PKC activity was measured, and the intracellular translocation pattern of PKC-{alpha}, -{delta}, -{epsilon}, and -{eta} assessed by immunohistochemistry and fractionated Western immunoblotting. The PKC-inhibitors chelerythrine and GF109203X were added during reperfusion and also given to non-ischemic hearts. Measurements included left ventricular function, intracellular calcium handling measured by Rhod-2 spectrofluorometry, myofibrillar calcium responsiveness in beating and tetanized hearts, and metabolic parameters. Results: Total PKC activity was increased at end-ischemia and remained elevated after 30 min of reperfusion. The translocation pattern indicated PKC-{varepsilon} as the main active isoform during reperfusion. Post-ischemic PKC inhibition affected mainly diastolic relaxation, with lesser effect on contractility. Both PKC inhibitors increased the Ca2+ responsiveness of the myofilaments as indicated by a leftward shift of the calcium-to-force relationship and increased maximum calcium activated tetanic pressure. Diastolic Ca2+ removal was delayed and the post-ischemic [Ca2+]i overload further exacerbated. Depressed systolic function was associated with a lower amplitude of [Ca2+]i transients. Conclusion: PKC is activated during ischemia and remains activated during early reperfusion. Inhibition of PKC activity post-ischemia impairs functional recovery, delays diastolic [Ca2+]i removal, and increases Ca2+ sensitivity of the contractile apparatus, resulting in impaired diastolic relaxation. Thus, post-ischemic PKC activity may serve to restore post-ischemic Ca2+ homeostasis and attenuate contractile protein calcium sensitivity during the period of post-ischemic [Ca2+]i overload.

KEYWORDS Protein kinases; Calcium (cellular); Contractile function; e-c coupling; Ischemia; Reperfusion


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
Myocardial stunning is defined as reversible contractile dysfunction after a period of ischemia, but the cellular mechanisms underlying the observed phenomena are not fully understood [1]. High-energy phosphates and intracellular pH normalize after several minutes, but intracellular calcium [Ca2+]i and sodium homeostasis remain disturbed during early reperfusion. There are alterations of cellular and subcellular ion currents, and myofibrillar responsiveness to Ca2+ is depressed [2–4]. In addition to the deleterious effects of ischemia itself, reperfusion injury adds to the development of post-ischemic dysfunction. Both oxygen free radical production and calcium influx contribute to reperfusion injury, but the role of regulatory enzymes in contributing to or protecting from reperfusion injury is less clearly defined.

Protein kinase C (PKC) is expressed in cardiomyocytes and its various isoforms have been implicated in the regulation of intracellular ion concentrations and contractile protein calcium sensitivity. In the non-ischemic heart, PKC activation has been shown to impair contractile function by decreasing cytosolic [Ca2+]i and myofibrillar responsiveness to calcium [5,6]. Activated PKC is able to regulate the activity of actomyosin MgATPase by phosphorylating Troponin I (TnI) both in vitro and in vivo [7–10]. There is accumulating evidence that PKC plays a crucial role during myocardial ischemia and reperfusion. PKC is activated during ischemia and phosphorylates contractile and regulatory proteins as well as Ca2+ channels and proteins of the sarcoplasmic reticulum that are important for maintaining intracellular Ca2+ homeostasis and contractile efficiency [11]. Pre-ischemic activation of PKC improves post-ischemic myocardial function [12–14], and it has been shown that the beneficial effects of myocardial preconditioning are mediated by PKC [15–17]. Recently, it has been demonstrated that hearts from transgenic mice chronically expressing activated PKC-β are less susceptible to ischemia/reperfusion injury [18]. However, it remains unclear whether PKC activation exerts its beneficial effects during the ischemic period or rather during post-ischemic reperfusion by improving energy metabolism, restoring calcium homeostasis, or altering contractile protein response to cytosolic calcium. Studies of the role of PKC in the ischemic myocardium usually activate or inhibit PKC prior to the onset of ischemia, so that the effects of PKC during ischemia and during reperfusion myocardium cannot be distinguished. We hypothesized that PKC remains activated during reperfusion and regulates myocardial excitation–contraction coupling by modulating [Ca2+]i handling and/or calcium sensitivity of contractile proteins. To test this hypothesis, we chose an experimental protocol using pharmacologic inhibitors to suppress PKC activity specifically during reperfusion following a period of unmodified ischemia.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
2.1 Animal care
All 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 National Academy of Sciences and published by the National Institutes of Health (NIH Publication No 85-23, revised 1996). The protocol was reviewed and approved by the animal care committee at Children's Hospital, Boston.

2.2 Perfusion protocol
New Zealand white rabbits (2–2.5 kg) were euthanized by intravenous injection of ketamine (100 mg/kg), and heparin (500 U/kg). The hearts were rapidly excised and placed in 4°C cold buffer solution. After cannulation of the aorta, the hearts were perfused retrograde in the Langendorff mode at 80 mmHg constant perfusion pressure with modified Krebs–Henseleit (KH) buffer (115 mmol/l NaCl, 26 mmol/l NaHCO3, 11 mmol/l glucose, 1.8 mmol/l MgSO4, 1.8 mmol/l KH2PO4, 4.7 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 µm nylon filter. The final buffer pH was 7.35–7.45, PO2 was 550–600 mmHg, and PCO2 was 30–40 mmHg. The temperature of the hearts was maintained at 37°C and monitored with a thermistor probe placed in the right ventricle. The cava and pulmonary veins were sutured closed and the pulmonary artery cannulated in order to obtain samples of venous effluent under anerobic conditions. A fluid-filled latex balloon connected to a micromanometry catheter (Millar Instruments, Houston, TX) was placed in the left ventricle via the left atrium. The catheter was sutured to the mitral valve annulus and the balloon kept in place by a thread attached to the tip, guided through the apex of the left ventricle and secured with an external stopper. All hearts were paced at 180 beats/min via epicardial electrodes sutured to the right ventricle. To facilitate pacing, the AV-node was crushed with a ligature placed at the apex of the triangle of Koch.

After 30 min stabilization, the hearts were subjected to 25 min normothermic global ischemia in a heated chamber filled with humidified room air. At the onset of reperfusion, two specific but structurally different PKC inhibitors (chelerythrine 2 µmol/l and 5 µmol/l, and GF 109203X 0.2 µmol/l and 1.3 µmol/l [BIOMOL Research Laboratories, Plymouth Meeting, PA] in dimethylsulfoxide, DMSO) were added to the perfusate. Control hearts received vehicle only (250 µl DMSO/4 l of buffer). Hearts that fibrillated during early reperfusion and did not spontaneously convert to sinus rhythm within 2 min were discarded. After 3 min and 30 min of reperfusion function measurements were performed. Left ventricular developed pressure was measured pre-ischemia at a diastolic pressure of 6 mmHg and the same balloon volume was then used to measure contractile function during reperfusion. The pressure data were digitalized with a time resolution of 2 ms and + and –dP/dT derived electronically. Coronary flow was measured by timed collection of the coronary effluent. Myocardial oxygen consumption (MVO2) was derived from the arteriovenous difference in O2 tension (Stat Profile Plus 9, Nova Biochemical, Waltham, MA), multiplied by coronary flow and divided by dry heart weight.

2.3 Metabolic measurements
After 30 min of reperfusion with or without PKC inhibitors, the hearts were snap-frozen in liquid nitrogen. Left ventricular tissue samples were homogenized in 6% perchloric acid solution. Lactate levels were obtained using an enzymatic assay (Sigma Chemical Company, St Louis, MO) and expressed as mmol lactate/g dry weight. Myocardial ATP content was measured at end-ischemia by HPLC as described by Bernt et al. [19].

2.4 Calcium measurements
Measurement of beat-to-beat intracellular calcium transients was performed in intact perfused rabbit hearts as we have previously described and validated in detail [20]. After 15 min of post-ischemic reperfusion, the hearts were loaded with the Ca2+-sensitive dye Rhod-2-AM (Molecular Probes, Eugene, OR) by perfusion with the cell-permeable acetoxymethylester (Rhod-2-AM; 0.5 mg/0.25 ml DMSO infused over 2 min at 37°C without recirculation). Dye-loading was followed by a 15 min washout to remove any extracellular or unhydrolyzed dye, and all Ca2+ measurements were performed after 30 min reperfusion. A modified spectrofluorometer (SLM-Aminco, Springfield, IL) provided excitation light at 524 nm and recorded emission light at 589 nm. Recordings were performed with a time-resolution of 2 ms for analysis of single Ca2+ transients and 40 ms for observance of changes in [Ca2+]i levels over longer time-periods. Since Rhod-2 has no spectral shift after Ca2+ binding, it is necessary to account for differences in dye loading or changes in tissue dye concentration over time (leakage or photobleaching). Therefore, tissue absorbance was quantified using the ratio of scattered excitation light at 524 nm (peak Rhod-2 absorbance in myocardial tissue) and 589 nm (isosbestic point for myocardium). The change in absorbance over time was then used to normalize emission light intensity by calculating fluorescence/absorbance (F/A) for each time-point. In order to quantify [Ca2+]i in the tetanized heart study, at the end the respective experiment 2,2'-dithiodipyridine (100 µM) was infused over a period of 2 min to induce calcium release from the sarcoplasmic reticulum. This was immediately followed by bolus injection of calcium ionophore A23187 [GenBank] (calcimycin) in 10 ml 10% calcium solution to maximize calcium entry from the extracellular space. Fluorescence was recorded with a time resolution of 40 ms during the infusion, and maximum fluorescence (Fmax) was determined to calculate systolic and diastolic calcium concentration using the following equation:

Formula
where Cai is the free intracellular calcium concentration, Kd is the dissociation constant for rhod-2 with calcium (710 mmol/l in the presence of 0.5 mM myoglobin), Ft is fluorescence at a specific time-point, Fo is autofluorescence measured before dye loading, At and Amax are tissue light absorbance at the specific time-point or at the end of the experiment, respectively. Interference of chelerythrine with rhod-2 emission and absorption spectra was excluded in preliminary experiments (data not shown). The rate of diastolic Ca2+ removal from the cytosol was assessed by the time-period from systolic peak to half-systolic [Ca2+]i level (t1/2). To assess myofibrillar responsiveness to Ca2+ in the beating heart, the Ca2+ concentration in the perfusate was increased stepwise from 0.5 to 4.0 µmol/l.

2.5 Tetanized heart preparation
To assess myofibrillar responsiveness to Ca2+ more directly, a separate group of control and inhibitor-treated hearts loaded with rhod-2 were evaluated using steady-state pressure-[Ca2+]i curves produced by tetanus. After 30 min of post-ischemic reperfusion with buffer containing 0.1 µmol/l ryanodine (Calbiochem, San Diego, CA), tetani were produced by rapid electrical stimulation at 8 Hz and 40 ms pulse width, delivered approximately 25% above the threshold required for capture. Fusion of tetani was elicited at varying perfusate Ca2+ concentrations while fluorescence and left ventricular pressure were recorded simultaneously. Three tetanic signals were averaged at each Ca2+ concentration. Peak tetanic pressure was plotted against [Ca2+]i during the tetanic plateau phase. As described in the literature, the [Ca2+]i–pressure relation was fitted to the Hill equation using nonlinear regression analysis to derive [Ca2+]i 50n, Pmax and the Hill coefficient:

Formula
where P is tetanic pressure, Pmax is maximal tetanic pressure, n is the Hill coefficient, and [Ca2+]i 50n is [Ca2+]i required for 50% activation [21]. Myofibrillar responsiveness to calcium was characterized by the slope of the tetanic [Ca2+]i/LV pressure curve (Hill coefficient, n), maximal calcium activated tetanic pressure (Pmax) and by [Ca2+]i 50n.

2.6 Western immunoblotting
At the end of the respective perfusion protocol, the hearts were snap frozen in liquid nitrogen and stored at –80°C. Left ventricular tissue was homogenized in ice-cold buffer containing 20 mM Tris–HCl, 2 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 25 µg/ml leupeptin, 0.3 mM sucrose at pH 7.4 and centrifuged at 1000xg for 5 min (supernatant=unfractionated tissue extract). Cytosolic and particulate fraction were then separated by ultracentrifugation at 100,000xg for 60 min. The resulting supernatant (cytosolic fraction) was collected, and the pellet (particulate fraction) was resuspended in sucrose-free buffer containing 1% Triton X, incubated for 60 min at 4°C and centrifuged at 100,000xg for 15 min. Tissue extracts were stored at –80°C for later analysis. Protein samples of 30 µg each were separated by SDS–PAGE gel electrophoresis and transferred to nitrocellulose membranes. Coomassie Brilliant Blue R-250 staining of the gels confirmed equal protein loading. The membranes were incubated with the following primary antibodies: anti-mouse PKC-{varepsilon}, polyclonal (Upstate Biotechnology, Lake Placid, NY), anti-rabbit PKC-{alpha}, monoclonal (Upstate), anti-human PKC-{eta}, polyclonal (Calbiochem, San Diego, CA), and anti-human PKC-{delta}, monoclonal (Gibco, Gaithersburg, MD), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody and detection using enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). These four isoforms were chosen because they have previously been implicated in the regulation of calcium handling and/or contractility in rabbit hearts.

2.7 Immunohistochemistry and confocal microscopy
Myocardial tissue samples were excised prior to ischemia, at end-ischemia, and following 30 min reperfusion. Tissue was fixed in 2.5% paraformaldehyde overnight then paraffin-embedded and cut into 5 µm sections. Following de-paraffinization in xylene, slides containing tissue sections were rehydrated through a graded ethanol series and used immediately for immunostaining. The sections were incubated with PKC antibodies (see above) diluted 1:100 in 5% fetal bovine serum in PBS pH 7.4. Primary antibodies were detected with an anti-mouse Alexa 568TM-conjugated secondary antibody (Molecular Probes). Coverslips were applied to the sections with fluorescent mounting medium (Dako). Slides were visualized using a BioRad MRC 1024 Scanning Laser Confocal Microscope with a Nikon 63x oil immersion objective, NA=160/0.17. Alexa 568TM was excited at 568 nm and fluorescence was detected between 589 and 621 nm. Optical sectioning was performed at 0.5 µm increments and images were processed using the accompanying BioRad software.

2.8 PKC activity
Total PKC-activity was measured in unfractionated tissue extracts (see above) using the SignaTECT Protein Kinase C assay kit (Promega, Madison, WI). After purification of the enzyme by chromatography (DEAE cellulose column), the samples were incubated in the presence of 1.2 mM calcium, 0.5 mM ({gamma}32P)ATP, 1.6 mg/ml phosphatidylserine and 0.16 mg/ml diacylglycerol. Control reactions were run in the absence of phospholipids. The ({gamma}32P)-ATP transfer to the PKC-specific biotinylated peptide substrate AAKIQAS*FRGHMARKK was then quantified by absorption to a streptavidin matrix capture membrane. PKC activity is expressed as pmol ATP transfer/min/µg protein.

2.9 Infarct size
Three separate sets of hearts (n=4) were subjected to 25 min normothermic ischemia and reperfused with or without chelerythrine (5 µmol/l) or GF 109203X (1.3 µmol/l). Coronary effluent was collected before the onset of ischemia, after 30 s reperfusion, and then in 5-min intervals. Creatine kinase (CK) release from the heart was quantified using Oliver and Rosalki's enzymatic assay (Sigma). After 60 min reperfusion 2-mm-thick slices of left ventricular myocardium were prepared and incubated in 1% triphenyltetrazolium chloride (TTC) in phosphate-buffered saline at 37°C for 20 min. The stained slices were placed on a flatbed scanner and electronic images obtained. The area of TTC negative-stained infarct regions was measured using Scion image analysis software (Scion Corp.). Infarct size was quantified for each slide using the formula negative stained area/positive stained areax100, and the mean value of all slices obtained from one ventricle calculated.

2.10 Statistical analysis
Analysis of calcium recordings was performed using Sigma Plot software (version 4.0, SPSS Inc., Chicago, IL). Data are expressed as the mean±S.E.M. and statistical analysis performed using the SPSS software package (version 9.0, SPSS Inc., Chicago, IL). Differences between the groups were tested for significance by one-way ANOVA using the Bonferroni correction for multiple comparisons. If normal distribution and equal variance testing was passed, Student's t-test was used to compare individual data sets. A two-tailed P value of less than 0.05 was considered statistically significant throughout.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 Acknowledgments
 References
 
3.1 PKC activity and translocation during reperfusion
PKC activity in whole-cell extracts was significantly increased at the end of the 25 min ischemic period and remained elevated at 30 min reperfusion (Fig. 1A). Protein content and subcellular distribution of four PKC isoforms as determined by Western immunoblotting with subcellular fractionation of left ventricular tissue are shown in Fig. 1B. The distribution of potentially important PKC target proteins in the fractionated samples is shown in the insert. LDH activity was only detected in the soluble fraction, indicating that the particulate fraction contained no significant amount of soluble cytosolic proteins. Troponin I was detected only in the cytosolic fraction, and Na+/K+ ATPase protein as well as SERCA2 ATPase proteins were detected in the particulate fraction. Pre-ischemia, PKC-{varepsilon} was found mainly in the cytosolic fraction. At the end of the ischemic period, PKC-{varepsilon} translocated to the particulate fraction and remained there by 30 min reperfusion. PKC-{alpha} was found in both fractions pre-ischemia and at 30 min reperfusion. However, at end-ischemia PKC-{alpha} was only detected in the cytosolic fraction. Both PKC-{delta} and PKC-{eta} were mainly detected in the cytosolic fraction pre-ischemia, and translocated to the particulate fraction during the ischemic period. However, at 30 min reperfusion, protein content of both isoforms in the particulate fraction was markedly reduced.


Figure 1
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  		Fig. 1 (A) PKC activity in tissue extracts from control hearts and hearts subjected to 25 min ischemia (end-ischemia) followed by 30 min reperfusion (reperfusion). PKC activity is increased at end-ischemia and remains elevated during reperfusion. Both PKC inhibitors (chelerythrine and GF109203X) given during reperfusion effectively suppressed PKC activity. (B) Representative immunoblots of cytosolic and membranous fractions demonstrating the subcellular distribution of PKC-{alpha}, -{delta}, -{epsilon}, and -{eta} in control hearts (pre-ischemia), after 25 min ischemia (end-ischemia), and after 30 min reperfusion (reperfusion). The insert shows the distribution of troponin I, Na+/K+ ATPase, SERCA2 ATPase, and LDH activity in the fractionated samples. C, cytosolic fraction; P, particulate fraction.

 
Confocal microscopy of immunostained tissue sections confirmed the translocation pattern seen by Western immunoblotting. Fig. 2 demonstrates diffuse (cytosolic) staining for PKC-{varepsilon} pre-ischemia. At end-ischemia, the PKC-{varepsilon}-specific antibody localized predominantly in the plasmalemma, and at 30 min reperfusion there was some redistribution of PKC-{varepsilon} to intracellular organelles, resulting in a more patchy fluorescence pattern. The subcellular distribution of PKC-{alpha}, -{delta}, and -{eta} at 30 min reperfusion was not visibly different from pre-ischemia (images not shown).


Figure 2
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  		Fig. 2 Confocal photomicrographs of rabbit ventricular myocardium stained for PKC-{varepsilon}. Tissue was paraffin-embedded, sectioned, and used for immunohistochemistry with an anti-mouse PKC-{varepsilon} polyclonal antibody. Primary antibody was detected with an Alexa568-conjugated secondary antibody. (A) End-ischemic control stained with a non-specific primary antibody; (B–D) representative PKC-{varepsilon} staining in hearts subjected to no ischemia (B), at end-ischemia (C), and following 30 min reperfusion (D), respectively. The micrographs represent 0.5 µm optical sections of tissue that was aligned in the longitudinal orientation. Scale bars=10 µm.

 
3.2 PKC inhibition in non-ischemic hearts
Neither treatment with chelerythrine nor with GF109203X significantly altered left ventricular contractility, relaxation, coronary flow, or myocardial oxygen consumption in non-ischemic hearts (Table 1). Furthermore, no significant changes in [Ca2+]i cycling were observed after treatment with PKC inhibitors. Mean [Ca2+]i levels, transient size, and the rate of diastolic Ca2+ removal were unchanged after treatment with both lower and higher concentrations of chelerythrine and GF109203X (Table 2). The steady-state responsiveness of myofibrils to calcium as assessed in tetanized hearts were also not affected by inhibition of PKC in non-ischemic hearts (Table 2).


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  		Table 1 Measurements of contractile function, coronary flow, and oxygen consumption in non-ischemic and post-ischemic hearts treated with different concentrations of chelerythrine and GF 109203X (mean values±S.E.M.)

 

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  		Table 2 Intracellular calcium measurements in beating hearts and during and tetanic stimulation after 30 min reperfusion

 
3.3 PKC inhibition in post-ischemic hearts
Both inhibitors significantly decreased PKC activity during reperfusion compared to untreated controls (Fig. 1A). Pre-ischemic contractile function measurements were not different from control (diastolic pressure=6±0.3 mmHg; developed pressure=112±4 mmHg; coronary flow=57±4 ml/min; MvO2=0.84±0.05 ml/min/g dry weight). Post-ischemic control hearts showed the features of myocardial dysfunction with elevated diastolic pressure, lower developed pressure (recovery of developed pressure in post-ischemic control hearts was 74±4% at 30 min reperfusion), reduced +dp/dt and –dp/dt, lower coronary flow, but unchanged or only mildly reduced MvO2 indicating energetic inefficiency (‘oxygen wasting’). Data after treatment with PKC inhibitors of non-ischemic and post-ischemic hearts are summarized in Table 1. Three minutes after the onset of reperfusion, there was no difference between the groups in terms of systolic and diastolic pressure. However, after 30 min reperfusion with chelerythrine or GF 109203X, relaxation was significantly impaired as indicated by increased diastolic pressures and reduced –dp/dt. Systolic LV pressure was elevated in hearts treated with 0.2 µmol/l GF109203X but unchanged in all other groups, as was +dp/dt. Developed pressure (LV systolic–LV diastolic pressure) was consistently lower in post-ischemic hearts treated with either of the two PKC inhibitors (Fig. 3). PKC inhibition in post-ischemic hearts did not affect coronary flow or myocardial oxygen consumption compared to untreated controls.


Figure 3
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  		Fig. 3 Amplitude of intracellular Ca2+ transients and LV developed pressure in post-ischemic hearts treated with: (A) chelerythrine 2 µmol/l and 5 µmol/l; (B) GF109203X 0.2 µmol/l and 1.3 µmol/l. [Ca2+]i amplitude is calculated as systolic [Ca2+]i–diastolic [Ca2+]i, both expressed as fluorescence corrected for absorption (F/A). *P<0.05 vs. control.

 
ATP content of non-ischemic control hearts was 18±3 µmol/g dry weight, and 15±5 µmol/g in post-ischemic control hearts, respectively. Treatment with PKC inhibitors did not significantly influence post-ischemic ATP content (14±1, 14±1, 13±2 and 13±2 µmol/g dry weight, with chelerythrine 2 and 5 µmol/l, and GF109203X 0.2 and 1.3 µmol/l, respectively). Myocardial tissue lactate levels at 30 min reperfusion were not significantly different from non-ischemic control hearts (72±4 vs. 85±7 mmol/g dry weight). Neither treatment with chelerythrine nor with GF109203X changed myocardial tissue lactate levels significantly (8.2±4, 7.1±5, 6.9±4 and 7.8±7 µmol/g dry weight, with chelerythrine 2 and 5 µmol/l, and GF109203X 0.2 and 1.3 µmol/l, respectively).

Cytosolic calcium levels in beating hearts and tetanized preparations are summarized in Table 2. Compared with non-ischemic hearts, mean intracellular free Ca2+ was elevated in post-ischemic hearts. The amplitude of the calcium transient was depressed and the rate of diastolic calcium removal from the cytosol delayed. In post-ischemic hearts treated with PKC inhibitors, mean [Ca2+]i, levels were further elevated in a dose-dependent manner. The rate of diastolic calcium removal, expressed as the time to half-systolic [Ca2+]i, was significantly delayed and the amplitude of the calcium transient further reduced (Fig. 3). Representative calcium transients are shown in Fig. 4. The diastolic [Ca2+]i-to-resting pressure relationship in post-ischemic beating hearts was shifted to the left in hearts treated with PKC inhibitors, indicating increased myofibrillar responsiveness to cytosolic calcium (Fig. 5A). However, the relationship between systolic calcium levels and developed pressure was essentially unchanged during reperfusion in the PKC inhibitor treated hearts (Fig. 5B).


Figure 4
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  		Fig. 4 Representative recordings of intracellular Ca2+ transients in non-ischemic, post-ischemic, and post-ischemic hearts treated with PKC-inhibitors (chelerythrine 2 and 5 µmol/l). Time resolution is 2 ms.

 

Figure 5
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  		Fig. 5 Pressure-to-calcium relationship in postischemic beating hearts treated with chelerythrine 5 µmol/l, and in postischemic control hearts, perfused with extracellular Ca2+ concentrations of 1.0 to 5.0 µmol/l. (A) diastolic pressure and (B) developed pressure is plotted against diastolic/systolic intracellular Ca2+ level, expressed as fluorescence corrected for absorption (F/A). Linear regression, R2-value displayed on plot.

 
The steady-state [Ca2+]i–force relationship in the tetanized whole heart preparation is demonstrated in Figs. 6 and 7Go. The marked rightward and downward shift in reperfused hearts indicates reduced myofibrillar calcium responsiveness post-ischemia. Inhibition of PKC during reperfusion partially reversed this effect, indicating an increase in myofibrillar calcium sensitivity compared with untreated post-ischemic hearts. Maximum calcium activated pressure (Pmax) and [Ca2+]i at half-maximal tetanic pressure ([Ca2+]i 50n), both assessed using the modified Hill-equation, are shown in Table 2 for the respective groups. Again, calcium activated force was significantly depressed post-ischemia, but inhibition of PKC resulted in an increase in maximal calcium activated LV pressure (Pmax) with corresponding decrease in calculated [Ca2+]i 50n, both indicating an increase in Ca2+ sensitivity of the contractile apparatus due to inhibition of post-ischemic PKC activity. The calculated values for the Hill coefficient, also shown in Table 2, were 3.6 for post-ischemic untreated hearts, and 4.24 or 4.84 in chelerythrine or GF109203X treated hearts.


Figure 6
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  		Fig. 6 Representative recordings of left ventricular pressure (LVP) and intracellular free calcium [Ca2+]i during tetanization for 2 s in the presence of ryanodine. Depicted are recordings in a post-ischemic control heart and a heart treated with the PKC-inhibitor chelerythrine (5 µmol/l). Hearts were tetanized at different extracellular Ca2+ concentrations (here: 3 mmol/l). Time resolution of the recording is 2 ms, the signal then filtered with a lowpass filter (sampling interval 2 ms, half power point of filter 5 Hz).

 

Figure 7
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  		Fig. 7 (A) Steady-state pressure–calcium relationship in non-ischemic hearts (control), hearts subjected to 25 min ischemia and 30 min reperfusion (IR), and post-ischemic hearts treated with chelerythrine 5 µM during reperfusion (IR+chel). Peak tetanic pressure is plotted against [Ca2+]i. Measurements were performed at increasing perfusate Ca2+ concentrations. The data were then fitted to the modified Hill equation (R2>0.9 for each group). (B) illustrates the differences in Pmax by plotting maximal tetanic pressure as a function of [Ca2+]i. (C) tetanic pressure is expressed as percent of maximal pressure to demonstrate the shift in (iCa2+)n50, the EC50 of the tetanic calcium–force relationship.

 
3.4 Myocyte necrosis
The average infarct size in control hearts subjected to ischemia and reperfusion was 8.6±1.8%. Post-ischemic treatment with 5 µmol/l chelerythrine or 1.3 µmol/l GF109203X had no significant impact on myocardial infarct size (9.5±2.1%, or 7.9±1.1%, respectively) (Fig. 8). Furthermore, CK release during reperfusion was not different between the groups (Fig. 9).


Figure 8
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  		Fig. 8 TTC-stained slices of left ventricular myocardium of hearts subjected to 25 min ischemia followed by 60 min reperfusion (Control) and hearts treated with 5 µmol/l chelerythrine (Chelerythrine) or 1.3 µmol/l GF109203X (GF109203X). The average infarct size was not different between the groups.

 

Figure 9
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  		Fig. 9 Creatine kinase (CK) activity measured in coronary effluent from of hearts subjected to 25 min ischemia followed by 60 min reperfusion (Control) and hearts treated with 5 µmol/l chelerythrine (Chelerythrine) or 1.3 µmol/l GF109203X (GF109203X).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 Acknowledgments
 References
 
The main findings of our study are that PKC remains activated throughout early reperfusion and its inhibition aggravates myocardial reperfusion injury without affecting the extend of cardiomyocyte necrosis. PKC inhibition by both chelerythrine and GF109203X given only during reperfusion, not during ischemia, resulted in significant impairment of myocardial relaxation and systolic contractile function. The changes in contractile function were associated with changes in [Ca2+]i homeostasis: post-ischemic calcium overload was further aggravated, diastolic calcium removal was delayed, and the amplitude of calcium transients was smaller. In post-ischemic control hearts, responsiveness of the contractile apparatus to calcium was reduced, and this effect was partially reversed by PKC inhibition. In the concentrations used, none of the PKC inhibitors had a significant effect on contractile function, calcium handling, or metabolic parameters in non-ischemic hearts.

The established mechanism of PKC activation in the heart involves receptor-mediated activation of phosphatidyl–inositol specific phospholipase C (PI–PLC), resulting in phosphoinositide turnover with production of diacylglycerol (DAG) and inositol triphosphate. DAG in turn activates PKC, requiring Ca2+ as a cofactor for activation of Ca2+-dependent isoforms. Activation of PKC, as indicated by translocation from the cytosolic to the particulate fraction, has been shown to occur during ischemia [22–25]. The mechanism and time-course of receptor-independent myocardial PKC activation during ischemia are less clearly defined [11,26]. Several studies have demonstrated that an increase of extracellular or intracellular calcium induces PKC-mediated cardioadaptation to ischemic injury [15,16,27], and we have recently shown that PI–PLC activation is necessary for translocation of PKC-{alpha} and {epsilon} during myocardial ischemia [12]. In the present study we assessed PKC activity in non-fractionated tissue extracts as well as the translocation pattern of several isoforms in fractionated tissue and using immunohistochemistry. Increased expression of PKC-{delta} and -{epsilon} has been shown during prolonged periods of of ischemia [26], but less is known about the reperfusion period. Kawamura et al. previously demonstrated transient translocation of PKC-{alpha} and -{zeta} following ischemic preconditioning, and only the {epsilon}- and {delta}-isoforms remained translocated after 35 min reperfusion [25]. Of the four isoforms we studied, PKC-{varepsilon} was the only one that consistently remained translocated to the particulate fraction after 30 min reperfusion. This finding is consistent with other studies which have identified PKC-{varepsilon} as the isoform mediating ischemic myocardial preconditioning [28,29], a phenomenon that requires some form of sustained activation or translocation to bridge the gap between the preconditioning stimulus and the onset of the actual ischemia.

The role of PKC in the non-ischemic heart remains controversial. Capogrossi et al. demonstrated a negative inotropic effect of PKC activation in non-ischemic myocytes, associated with smaller Ca2+ transients and lower mean [Ca2+]i [30]. Our laboratory previously confirmed these findings for the isolated perfused rabbit heart [5], and Gwathmey and Hajjar described impaired sarcoplasmic Ca2+ release after PKC activation in human myocardial trabeculae [31]. PKC inhibition in non-ischemic myocytes was shown to increase the Ca2+ transient amplitude [6]. Conversely, several groups have reported an increase in contractility following PKC activation and/or decrease in contractility following PKC inhibition in different non-ischemic myocardial preparations [32,33]. In our experiments we chose inhibitor concentrations that had no significant effect on myocardial function or metabolism in non-ischemic hearts, but did have an effect in post-ischemic hearts. We based this on our hypothesis that activated PKC has a greater regulatory impact on excitation–contraction coupling in the reperfused myocardium than in perfused non-ischemic heart. There is accumulating evidence that PKC activation before or during ischemia is protective for the ischemic heart and that inhibition of PKC results in impaired myocardial recovery [12–14]. On the other hand, Lasley et al. observed a reduced infarct size and better functional recovery following pre-ischemic application of low-dose chelerythrine in an in situ model, suggesting a beneficial effect of PKC inhibitors during prolonged ischemia [34]. Furthermore, several groups have found that chelerythrine given before the onset of a period of unmodified ischemia had no significant impact on post-ischemic recovery [35–37]. However, the aforementioned experiments have in common that PKC was manipulated before the onset of ischemia, so that it is not possible to distinguish effects occurring during ischemia from those during post-ischemic reperfusion. Similarly, experiments using transgenic mice with chronically activated PKC or mutant PKC-specific phosphorylation sites are not able to differentiate pre-ischemic from post-ischemic effects of PKC activation. We found that inhibition of PKC during reperfusion results in deterioration of cardiac function, particularly diastolic relaxation, and conclude that PKC is important for limiting the reperfusion injury after unmodified ischemia. Therefore, the apparent discrepancies regarding the effects of PKC inhibition before unmodified ischemia may be due to variations of the inhibitory effect during reperfusion. When the inhibitor is given pre-ischemia but exerts most of its effects during reperfusion, factors such as the dose and mode of application, as well as duration of ischemia are likely to influence the net effect on post-ischemic recovery.

Due to the importance of intracellular free Ca2+ as the mediator of contractile protein interaction, the relation between PKC and [Ca2+]i has been of great interest. In vitro experiments have shown that PKC activation inhibits sarcolemmal L-type Ca2+ channels and increases Ca2+ uptake by the sarcoplasmic reticulum by phosphorylating phospholamban [38,39]. Hence, activation of PKC during ischemia may serve as a mechanism to reduce Ca2+ accumulation in the cytosol. This is indirectly supported by our finding that inhibition of PKC during reperfusion results in even higher [Ca2+]i, exacerbating post-ischemic [Ca2+]i overload. The delayed diastolic Ca2+ removal points to decreased activity of SERCA-2, rather than to ‘leaking’ sarcolemmal Ca2+ channels. Additional evidence supporting the theory of impaired SR Ca2+ re-uptake is the observation that the amplitude of Ca2+ transients decreased following PKC inhibition, indicating progressive SR Ca2+ depletion. Considering the fact that an increase of [Ca2+]i activates PKC, it is intriguing to speculate that PKC plays an important role in a feedback mechanism to limit myocyte Ca2+ overload and preventing excessive rigor bond formation, energy wasting and activation of proteolytic enzymes.

Decreased calcium responsiveness of the contractile apparatus is commonly expected to exert a negative inotropic effect. However, there is accumulating evidence that attenuation of contractile protein calcium sensitivity has in fact beneficial effects on myocardial function, which may be of particular importance in the post-ischemic heart. Decreased Ca2+ responsiveness of the myofilaments contributes to the increased relaxation rate of the beating heart under the influence of β-adrenergic stimulation through PKA-mediated phosphorylation of the myofibrillar troponin I (TnI) subunit, resulting in reduced affinity of troponin C (TnC) for Ca2+. The role of PKC-mediated phosphorylation of contractile proteins is less clearly defined. PKC has been shown to phosphorylate TnI and troponin T in vitro, resulting in inhibition of actomyosin ATPase activity [7,8,10], and reducing Ca2+ sensitivity of the myofilaments [9]. However, in vivo, PKC phosphorylation of contractile proteins and its functional consequences have proven more difficult to demonstrate [40]. The Ca2+ responsiveness of myofibrils from stunned rabbit myocardium shifts to higher Ca2+ concentrations, indicating decreased Ca2+ sensitivity [41]. Bezstarosti et al. found no differences in the maximal activity of Ca2+ stimulated actomyosin MgATPase, the pCa2+ of myofibrils, or the degree of phosphorylation of myofibrils from stunned and non-stunned porcine myocardium [42]. Moreover, PKC-induced in vitro phosphorylation of myofibrils isolated from non-stunned myocardium resulted in a leftward shift of pCa2+50, indicating an increase in Ca2+ sensitivity. Following inhibition of PKC during post-ischemic reperfusion, we found a marked increase of Ca2+ sensitivity compared with post-ischemic controls, indicated by a leftward-shift of the [Ca2+]i–diastolic pressure relation in the beating heart. We also found an increase in myofibrillar Ca2+ responsiveness under the steady-state conditions of the tetanized heart. It has been suggested that activation of PKC in response to ischemia may be a signal for contractile quiescence and represents an energy-conserving defense mechanism against ischemia [13]. However, there is no difference in myocardial content of high-energy phosphates at end-ischemia following pre-ischemic activation or inhibition of PKC [12]. Instead, we found evidence that PKC exerts much of its effects during reperfusion, when contractility and relaxation have to be re-established in the presence of elevated [Ca2+]i. A recently evolved concept of myocardial stunning involves proteolytic breakdown of myofibrillar proteins. Breakdown products of TnI and TnT have been identified in stunned myocardium [43,44], and McDonough et al. linked the TnI phosphorylation state with the rate of proteolytic breakdown [45]. This hypothesis offers another explanation as to how post-ischemic PKC activation may affect myofibrillar integrity and function. PKC-mediated desensitization of myofibrils and improved diastolic Ca2+ removal in the post-ischemic myocardium may serve to protect the heart from excessive rigor bond formation in the setting of elevated [Ca2+]i post-ischemia. Additional evidence for the important role of PKC during reperfusion comes from a recent study using a novel pharmacologic PKC-activator (JTV519), which improved post-ischemic function even when given during reperfusion only [46].

4.1 Limitations of the study
An inhibitor-based approach to study the role of a particular enzyme is always open to criticism as inhibitors are rarely as selective as is claimed. We used two structurally different inhibitors, which produced similar results, but this does not exclude the possibility that inhibition of other kinases contributed to the observed effects. However, the IC50 values of both chelerythrine and GF109203X for other protein kinases are more than 100-fold higher than for PKC; thus we expect minimal effects on other signaling pathways at the low inhibitor concentrations used in the present study. The role of PKC in the excitation–contraction coupling can now be studied in various transgenic mouse models with high selectivity. However, we chose to use a pharmacologic inhibitor model in order to suppress PKC activity during a specific and limited time period following unmodified ischemia. We also confirmed the effectiveness of the inhibitors by measuring PKC activity.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
Acknowledgments
 References
 
Using an inhibitor-based model to suppress PKC activity only during reperfusion after unmodified ischemia, we observed impaired myocardial relaxation and contractility. This was associated with higher intracellular free calcium levels, delayed diastolic calcium removal, and increased myofibrillar responsiveness to calcium. In the post-ischemic heart, the increase in calcium responsiveness results in impaired relaxation rather than improved systolic function. We conclude that PKC plays an important role in the regulation of myocardial excitation–contraction coupling during reperfusion. Hence, physiologic activation of PKC during ischemia may serve a mechanism to restore and maintain cardiac function during reperfusion in the presence of post-ischemic intracellular calcium overload. Furthermore, strategies to facilitate PKC activity post-ischemia may serve to treat myocardial stunning.

Time for primary review 29 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
References
 
This work was supported in part by NIH Grants HL-52589 (Francis X. McGowan) and HL-46207 (Pedro J. del Nido). Christof Stamm was supported by a grant from the German Research Foundation (STA 497/2-1).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 Acknowledgments
 References
 

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