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(Circulation Research. 1999;85:77-87.)
© 1999 American Heart Association, Inc.

Original Contribution

Two Distinct Mechanisms Mediate a Differential Regulation of Protein Kinase C Isozymes in Acute and Prolonged Myocardial Ischemia

Ruth H. Strasser, Gregor Simonis, Steffen P. Schön, Martin U. Braun, Renate Ihl-Vahl, Christof Weinbrenner, Rainer Marquetant, Wolfgang Kübler

From the Department of Cardiology, Angiology, and Pulmology, University of Heidelberg, Medical Center, Heidelberg, Germany.

Correspondence to Ruth H. Strasser, Department of Cardiology, Angiology, and Pulmology, University of Heidelberg, Bergheimer Str 58, 69115 Heidelberg, Germany. E-mail ruth.strasser{at}med.uni-heidelberg.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—An activation of protein kinase C (PKC) in acute myocardial ischemia has been shown previously using its translocation to the plasma membrane as an indirect parameter. However, whether PKC remains activated or whether other mechanisms such as altered gene expression may mediate an isozyme-specific regulation in prolonged ischemia have not been investigated. In isolated perfused rat hearts, PKC activity and the expression of PKC cardiac isozymes were determined on the protein level using enzyme activities and Western blot analyses and on the mRNA level using reverse transcriptase–polymerase chain reaction after various periods of global ischemia (1 to 60 minutes). As early as 1 minute after the onset of ischemia, PKC activity is translocated from the cytosol to the particulate fraction without change in total cardiac enzyme activity. This translocation involves all major cardiac isozymes of PKC (ie, PKC, PKC, PKC, and PKC). This rapid, nonselective activation of PKCs is only transient. In contrast, prolonged ischemia (15 minutes) leads to an increased cardiac PKC activity (119±7 versus 190±8 pmol/min per mg protein) residing in the cytosol. This is associated with an augmented, subtype-selective isozyme expression of PKC and PKC (163% and 199%, respectively). The specific mRNAs for PKC (948±83 versus 1501±138 ag/ng total RNA, 30 minutes of ischemia) and PKC (1597±166 versus 2611±252 ag/ng total RNA) are selectively increased. PKC and PKC remain unaltered. In conclusion, two distinct activation and regulation processes of PKC are characterized in acute myocardial ischemia. The early, but transient, translocation involves all constitutively expressed cardiac isozymes of PKC, whereas in prolonged ischemia an increased total PKC activity is associated with an isozyme-selective induction of PKC and PKC. Whether these fundamentally different activation processes interact remains to be elucidated.

Key Words: acute myocardial ischemia • protein kinase C • heart • signal transduction

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein kinase C (PKC) plays a central role in the regulation of signal transduction and cell proliferation. Activation of PKC has been reported to mediate inotropic responses,^{1 2} to induce apoptosis,³ and to promote myocardial hypertrophy.^{4 5} It also has been suggested to mediate cardiac protection in ischemic preconditioning.^{6 7 8} All of these processes may be important in acute myocardial ischemia and in the response of the myocardial tissue to chronic infarction.^{9 10}

As a classical hallmark for the activation of PKC, the translocation of the enzyme from the soluble to the particulate fraction has been used.¹¹ This translocation, which represents only an indirect parameter for the activation, has been observed in many model systems and organs.¹² It also has been demonstrated in the early phase of acute myocardial ischemia¹³ and after repetitive episodes of brief ischemia during the ischemic preconditioning and during reperfusion.^{7 8 9} The molecular signal mediating this activation process in short-term myocardial ischemia has not been characterized so far. An activation of ₁-adrenergic receptors mediated by the increased release of endogenous catecholamines in ischemia as the responsible mechanism for the translocation of PKC in the ischemic heart could be excluded.^{13 14} This contrasts with the regulation in the normoxic heart, in which activation of ₁-adrenergic receptors has been shown to translocate PKC.^{15 16} Recent data suggested an increased inositol phosphate release and metabolism during ischemia,¹⁷ which, in addition to the intracellular calcium release, may contribute to the ischemia-promoted activation of PKC. This activation of PKC in the ischemic or reperfused heart may contribute to the genesis of arrhythmias,¹⁸ to a reduced contractile force,¹⁹ or, as shown previously, to the sensitization of the adenylyl cyclase system.¹³ Whether activation of PKC especially in prolonged ischemia may modulate gene expression, as it has been shown in isolated cell systems,²⁰ has not been determined so far.

Molecular cloning and biochemical analysis revealed that PKC represents a family of at least 11 isozymes of closely related serine and threonine protein kinases, which can be classified into 3 major subclasses.²¹ The classical isozymes , ß, and require calcium for activation, whereas the newly characterized isozymes , , , and may be activated independently of calcium. A third group, the so-called atypical PKC isozymes such as , , , and µ, have been characterized not to be activated by calcium, by 1,2-sn-diacylglycerol (DAG), or by phorbol esters. The pattern of expressed isozymes of PKC may be distinct in different organs.^{20 21} Recently, it has been shown that the predominant isozymes in rat heart are the , , , and subtypes, depending on the developmental stage.^{22 23} Similar results have been described on the mRNA level using PCR methodology.²⁴ The functional significance of these distinct isozymes of PKC in the heart has not been characterized so far. In the heart, activated PKC may phosphorylate many distinct substrates, including contractile proteins such as troponin I or T, and thus interfere with myocardial contractility.¹⁹ However, phosphorylation of distinct substrates, which may mediate the specific cellular responses, could not be attributed to single isozymes.

In myocardial ischemia, the earliest time point of this translocation and activation of PKC has not been determined. More importantly, it has not been addressed whether in continuous ischemia this translocation process persists or whether alternative activation processes of PKC may be operative in prolonged ischemia.

Using the well-characterized model of isolated perfused hearts, the present study demonstrates that 2 distinct activation mechanisms of PKC occur in sequence in myocardial infarction.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
[-³²P]ATP, [-³²P]ATP, and ¹²⁵I-labeled protein A were purchased from New England Nuclear. Reagents for the Bradford protein assay were from Bio-Rad. Phosphocellulose P81 was purchased from Whatman, and polyclonal peptide antibodies against the isozymes of PKC (PKC, PKC, PKC, PKC, PKC, PKCß, PKC, and PKC) and their respective peptides were purchased from GIBCO/BRL. Reagents for the PKC ELISA were from Calbiochem. Hexanucleotides were from Boehringer Mannheim, and Taq polymerase was from Perkin Elmer. All other reagents were bought from Sigma. Male Wistar rats (200 g) were purchased from Thomae (Biberach, Germany).

Perfusion of Isolated Rat Hearts
Male Wistar rats were anesthetized with thiopentolbarbital (50 mg/kg IP). After heparinization (1000 IU heparin IV), the hearts were rapidly removed and perfused at 37°C according to the method of Langendorff²⁵ at a constant flow of 4.5 mL/min per gram wet weight tissue, using a modified Krebs-Henseleit solution (Tyrode) containing, in mmol/L, NaCl 125, MgC1₂ 1, CaCl₂ 1.85, KCl 4, glucose 11, sodium EDTA 0.027, NaHCO₃ 17, and NaH₂PO₄ 0.2). The Krebs-Henseleit solution (Tyrode) was oxygenated using 95% O₂/5% CO₂, and the pH was maintained at 7.4. After preperfusion for 10 minutes for equilibration, global ischemia was induced by stopping perfusion. The hearts were kept at constant humidity and temperature (37°C). The incubation chamber was simultaneously gassed with nitrogen to prevent oxygen uptake at the surface. In each experiment, controls and treatment groups were perfused in parallel to avoid interassay variations. At the end of the perfusion experiments, the hearts were freeze clamped and stored at -80°C.

Partial Purification of PKC Isozymes
Hearts were homogenized in buffer A (containing [in mmol/L] Tris-HCl 20, sucrose 250, EDTA 5, EGTA 5, PMSF 1, ß-mercaptoethanol 10, and benzamidine 1) using a Polytron (LS10-35, Kinematica; 3 times for 6 seconds, 10 000 rpm). The homogenate was centrifuged (360g, 10 minutes, 4°C). The resulting supernatant was centrifuged at 100 000g (60 minutes, 4°C) to separate the soluble fraction from the particulate fraction. The pellet corresponding to the membrane fraction was solubilized in buffer A containing Triton X-100 at a final concentration of 0.1% by stirring on ice for 45 minutes at 4°C. Insoluble membrane particles were sedimented by centrifugation at 100 000g (60 minutes, 4°C). Triton X-100 was added as concentrated stock to the cytosolic fraction to give the same final concentration of 0.1% as was present in the solubilized membrane fraction. Cytosolic and solubilized membrane fractions were applied to DEAE-cellulose columns (1 mL bed volume), which had been equilibrated before with buffer A including 0.1% Triton X-100. The DEAE columns were washed with 5 mL of buffer A including 0.1% Triton X-100 and 5 mL of buffer B, containing (in mmol/L) Tris-HCl 20, sucrose 250, EDTA 1, EGTA 1, PMSF 1, ß-mercaptoethanol 10, and benzamidine 1, and 0.1% Triton X-100. PKC was eluted with 2 mL of buffer B including 400 mmol/L NaCl. The recovery of PKC activity in the purified cytosolic and particulate fraction amounted to 68% of the total, stimulated cardiac activity as determined in the crude homogenate. The yield was identical in the controls and in the ischemic hearts. Since basal kinase activity cannot be determined in the nonpurified fractions because of residual calcium, the preparations were purified according to the method of Takai et al,²⁶ so that we would be able to exclude a proteolytic activation with certainty.

Determination of PKC Activity
PKC activity was determined in the cytosol and the soluble particulate fraction using 2 different substrates in the assay (ie, with histone III-S, according to the method of Takai et al,²⁶ and with a substrate peptide derived from the pseudosubstrate region of the enzyme).²⁷ Basal activity was determined in the presence of 10 mmol/L EDTA and 10 mmol/L EGTA. Maximally stimulated PKC activity was measured in the presence of 1.25 mmol/L CaCl₂, 100 µg/mL phosphatidylserine, and 20 µg/mL DAG.

To avoid isozyme selectivity of the substrate, the activity of PKC was also determined by using a PKC ELISA kit with a pseudosubstrate peptide from the C1 region common to all isozymes present in rat heart.^{28 29} For background determination, the reaction mixture was incubated in the absence of protein and amounted to <5% of stimulated activity. Purified PKC from rat brain (Calbiochem) with known specific activity was used as a standard to calculate specific activities.

Immunoblot Analysis
Proteins were separated on 8% SDS-polyacrylamide gels according to the method of Laemmli³⁰ and transferred to nitrocellulose using the method of Towbin et al.³¹ Prestained molecular weight standards were electrophoresed and transferred in parallel.

Autoradiograms, in which densities of the specific bands correlated linearly with the amount of protein loaded, were evaluated by laser densitometry using the LKB laser densitometer (Ultroscan XL, LKB).

As a control to identify the specific bands with certainty, parallel aliquots were analyzed in the presence of the antigenic peptide. To quantify the relative expression of PKC isozymes, increasing amounts of the individual antigenic peptides of the different isozymes of PKC were spotted on nitrocellulose and detected as described above to create peptide standard curves.

RNA Preparation
The extraction of total cardiac RNA was carried out according to a modification of the guanidine isothiocyanate ethanol precipitation method of Chirgwin et al.³² Two hundred to five hundred milligrams of the pulverized rat left ventricles were used. The purity of the RNA probes was determined by UV absorption at 260 and 280 nm with a 260/280 yield >1.7 in all samples. RNA concentrations were determined by UV absorption at 260 nm. RNA samples were stored in H₂O/ethanol (1:1) at -80°C.

Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)
Total cardiac RNA, which represents the RNA not only of cardiomyocytes, was reverse transcribed into cDNA using a modified protocol of RT-PCR as described by Ponzoni et al.³³ Total RNA (300 and 600 ng) was heated with 50 mmol hexanucleotide mix (Boehringer Mannheim) in a final volume of 12 µL for 10 minutes at 95°C and chilled on ice. After centrifugation for 30 seconds at 10 000g, 2 µL 10x PCR buffer (500 mmol/L KCl, 100 mmol/L Tris-HCl [pH 9.0], and 1% Triton X-100), 2 µL 10 mmol/L dNTPs, 20 units RNase inhibitor, 2 µL 0.1 mol/L DTT, and 1 unit RT were added to a final volume of 20 µL. Reverse transcription was performed at 42°C for 30 minutes and stopped by heating at 95°C for 10 minutes. In each experiment, 2 concentrations of template RNA were chosen to confirm the linearity of reverse transcription and amplification.

cDNA amplification was performed by adding 2 µL of the RT reaction (equivalent to one tenth of the starting template, ie, 30 or 60 ng, respectively) into 50 µL PCR buffer containing isozyme-specific sense and antisense primers (0.5 mmol/L), 0.55 mmol/L MgCl₂, 60 µmol/L dNTP, and 1 unit Taq DNA polymerase (Perkin Elmer) using the following protocol: denaturation at 95°C for 45 seconds, primer annealing at 55°C for 45 seconds, and primer extension at 74°C for 45 seconds. For the PCR, sense primers and antisense primers specific for each PKC isozyme were selected according to Ponzoni et al³³ (see Table).

View this table: [in this window] [in a new window]   Table 1. Specific PKC Primer Sequences

The lengths of amplification products were 180 bp for PKC, 237 bp for PKC, 280 bp for PKC, and 247 bp for PKC. Depending on the linearity tests (Figure 6), the number of the amplification cycles used was 28 cycles for all PKC isoforms to assure amplification in the linear range. After the last cycle, the samples were incubated for 5 minutes at 74°C to extend incomplete products. To verify the specificity of the amplification products, restriction digests (1 hour, 37°C) were performed using MscI, Eco47III, HindIII, and XbaI, resulting in a specific restriction pattern for each PKC isozyme (Figure 5).

View larger version (25K): [in this window] [in a new window]   Figure 6. Exponential range for RT-PCR of PKC isoforms. Shown are the densities of the amplification product labeled with individual internal oligonucleotides (inset toward bottom of each panel) depending on number of PCR cycles. For all isoforms, the amplification is linear up to 28 cycles.

View larger version (38K): [in this window] [in a new window]   Figure 5. A, RT-PCR products after subtype-specific amplification of mRNA for cardiac desmin, PKC, PKC, PKC, and PKC. Total RNAs (600 ng) extracted from rat heart were reverse transcribed with hexanucleotide primers, and cDNA (60 ng) was amplified with specific primers. The resulting PCR products had the expected sizes (desmin, 289 bp; PKC, 180 bp; PKC, 237 bp; PKC, 280 bp; and PKC, 247 bp). In the negative control in the absence of RNA (), no product emerged. B, Restriction digest of the amplification product for PKC. Sample restriction digest for PKC using 2 external restriction enzymes (HindIII and XbaI) and 2 internal restriction enzymes (MscI and Eco47III) is shown. In the upper part of the panel, a scheme of the amplification product of PKC indicating the specific restriction sites is shown. The resulting restriction fragments shown in the lower panel for MscI and Eco47III have the expected sizes, whereas no restriction fragments were observed in the absence of restriction enzymes (no enzyme) or in the presence of HindIII or XbaI.

As an internal standard for amplification, the expression of desmin was quantified in parallel. As external standard, PKC isozyme–specific synthetic RNAs were used in each amplification procedure in increasing concentrations from 5 to 40 fg RNA. All PCR amplification products were separated on a 2% agarose gel containing 0.01% ethidium bromide and were visualized by UV radiation. As molecular size standard, a 100-bp DNA ladder (GIBCO/BRL) was used. The electrophoresed PCR products were vacuum blotted on a NY-13 nitrocellulose membrane using the following buffers (with components in mol/L): denaturation buffer for 12 minutes (NaCl 1.5 and NaOH 0.5), neutralization buffer for 8 minutes (Tris [pH 5.0] 1 and NaCl 2), and SSC buffer (20x SSC) for 40 minutes (NaCl 3 and sodium citrate 0.3). Fixation of DNA on the nitrocellulose membrane was performed by UV radiation (1200 J/cm², 254 nm). The blots were hybridized with ³²P-labeled internal oligonucleotides (1 µg) to further confirm the specificity of the amplified PCR products and to allow quantification. As internal oligonucleotides, the following base sequences were used:

PKC: d(ATT GAA GTC CGT GAG TTT CAC)

PKC: d(AGC CAG AGA CAC CAG AGA CTG)

PKC: d(GAA GTT GAA CTC ATC CAG GCC)

PKC: d(CAG TAG ATG GAC AAG AAC GAT G)

Southern blot hybridization was performed overnight at 42°C in 4x SSC, 0.5% polyvinylpyrrilidone (PVP), 0.5% Ficoll, 0.5% BSA, 0.5 mg/mL herring sperm DNA, and 0.6 mg/mL yeast RNA. The autoradiograms (Kodak Xomat AR 5) were analyzed with a laser densitometer (Ultrascan XL, Pharmacia).

Synthetic RNA Standards
To allow an absolute quantification of specific mRNA levels, synthetic external RNA standards with increasing concentrations were analyzed in parallel in all experiments.³⁴ For synthesis of the RNA standards, the PCR amplification products of each PKC isozyme, using the cDNA clones as templates, were separated in 2% agarose gels. Specific bands were cut out and electroeluted for 1 hour at 100 mA (GE 200 Sixpac gel eluter, Hoefer) and subcloned into a pcDNA I vector carrying the promoter for SP6 and T7 RNA polymerases using the Sure clone ligation kit (Pharmacia). The inserts were in vitro transcribed on the basis of their orientation with T7 RNA polymerase or with SP6 RNA polymerase (Boehringer Mannheim) to obtain RNA transcripts. After DNase I digestion, aliquots of the synthetic RNAs were analyzed for length in 5% acrylamide-urea gels. To eliminate DNA fragments as well as the enzymes, the samples were layered onto 5.7 mol/L CsCl₂ cushions (2 mL) and centrifuged (20°C, 180 000g, 21 hours, Beckmann SW 40 T-Rotor). The pellets were resolved in 0.3 mol/L sodium acetate (pH 6.0) and precipitated with 100% ethanol (3:1 vol/vol) at -20°C overnight. The RNAs were analyzed for purity in 5% acrylamide urea gels. To verify the absence of plasmid DNA in the RNA preparations, aliquots of synthetic RNAs were digested by 1 unit of RNase A (Boehringer Mannheim) before RT-PCR. To determine synthetic RNA concentrations, the UV absorption at 260 nm was measured (Ultraspec III, Pharmacia). Increasing concentrations of the subtype-specific synthetic RNA standards (5 to 40 fg RNA) were used as external standard templates both for reverse transcription and for amplification to create standard curves in each amplification experiment. With the use of these external and internal standards and 2 concentrations of starting template for each individual sample to be analyzed, the intra-assay and interassay variation was cut down to 6% to 10%.

Protein Determination
Protein determination was performed according to the method of Bradford³⁵ using BSA as standard.

Statistical Analysis
Statistical analysis was performed using ANOVA and the Student-Newman-Keuls test for significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PKC Activity and Subcellular Distribution During Short-Term and Prolonged Myocardial Ischemia
As shown in Figure 1A, periods of ischemia as brief as 1 minute led to an increase of PKC activity in the particulate fraction with a concomitant decrease of enzyme activity in the cytosolic fraction. This ischemia-induced translocation of PKC as early as 1 minute after the onset of global ischemia resulted in an almost doubling of the enzyme activity in the particulate fraction (37±3 versus 63±2 pmol/min per mg protein, n=14, *P0.05). The activity reached a maximum after only 2.5 minutes of ischemia (65±8 pmol/min per mg protein, n=14). This rapid increase of PKC activity in the particulate fraction (Figure 1A, middle panel) was accompanied by a decrease of the total enzyme activity in the cytosolic fraction (119±7 versus 87±2 pmol/min per mg protein, n=14, *P0.05, Figure 1A, left panel). However, total cardiac PKC activity in the ischemic hearts remained unaltered at up to 10 minutes of ischemia (Figure 1A, right panel). Using the pseudosubstrate for the determination of PKC activity to exclude an isozyme selectivity (see Materials and Methods), a similar translocation of the enzyme after 2.5 minutes of ischemia was observed with an increase in the particulate fraction by 94% and a decrease in the cytosolic fraction by 38% (Figure 1B). These data show that very brief periods of ischemia led to a rapid translocation of PKC from the cytosol to the membrane fraction, which indicates its very rapid activation. As shown here for the first time in intact hearts, the activation occurs as early as 1 minute after the onset of ischemia. Periods shorter than 1 minute after the onset of ischemia could not be evaluated for technical reasons. As additional controls, the relative distribution of PKC activities in nonperfused hearts, which were frozen directly ex vivo, were compared. Normoxic perfusion had no influence on the activity or distribution of PKC activities (data not shown). These data confirmed that the mounting of the isolated hearts to the perfusion apparatus, which was performed in <20 seconds, followed by a 10-minute preperfusion, did not result in a redistribution or an activation of PKC.

View larger version (41K): [in this window] [in a new window]   Figure 1. Activity of PKC and intracellular distribution after various periods of global ischemia. A, PKC activity was determined using the histone phosphorylation assay (see Materials and Methods) in the cytosolic and particulate fractions derived from isolated perfused rat hearts after normoxic perfusion (control) and after various periods of global ischemia (1 to 60 minutes). The total amount of PKC activity in the cytosolic fraction and in the particulate fraction was summarized and expressed as PKC activity from the individual hearts (right panel). Left panel, Mean±SEM of 14 individual hearts at each time point in triplicate determinations. In the cytosol (left panel, left side), PKC activity is significantly reduced after 1 and 2.5 minutes of ischemia (*P0.05). In the particulate fraction (left panel, right side), PKC activity is significantly increased after 1, 2.5, and 10 minutes of ischemia without any significant change in the total cardiac activity (ie, the sum of cytosolic and particulate PKC activity in the whole heart [right panel]). Total cardiac PKC activity was significantly augmented after 15, 30, and 60 minutes of ischemia (right panel). This increase is attributed to a significant enhancement of cytosolic PKC activity (left panel), whereas in the particulate fraction, PKC tended to decrease after 30 minutes of ischemia without reaching significance. B, Cardiac PKC activity in the absence of activators (=basal activity) and in the presence of calcium and phosphatidylserine (see Materials and Methods) was determined in the cytosol of normoxic controls and of hearts after various periods of global ischemia using the pseudosubstrate assay. Shown are the mean±SEM (n=6) for each point. Early decrease of PKC activity in the cytosol after 2.5 minutes of ischemia is followed by an increase to 145% of control in prolonged ischemia without any change in basal PKC activity.

With continued ischemia for longer periods of time, the maximally stimulatable activity of PKC gradually, but not significantly, decreased in the particulate fraction (Figure 1A, middle panel), thus returning to control values after 15 minutes of ongoing ischemia. In the cytosolic fraction, PKC activity first returned to control values after 10 minutes and then gradually increased during ongoing ischemia for >20 minutes (Figure 1A, left panel). After 30 minutes of ischemia, PKC in the cytosolic fraction was significantly above that of normoxic controls, reaching an almost doubling of total PKC activity in the ischemic hearts after 60 minutes of ischemia. In contrast, in the particulate fractions, PKC activity tended to decrease in prolonged ischemia. This change, however, did not reach statistical significance. During the complete time course, the yield of total protein in both fractions, the particulate and the cytosolic fraction, remained unaltered.

These data demonstrate that in contrast to the early ischemia-induced translocation, the activation process of PKC in prolonged myocardial ischemia includes an increase of total PKC activity, which does not involve a translocation of the enzyme. The maximally stimulatable enzyme activity is increased in the cytosolic fraction after prolonged ischemia without significant loss of enzyme activity from the membrane fraction. Consequently, total PKC activity in the infarcted hearts increased from 1820±150 to 3620±140 pmol/min per heart after 60 minutes of global ischemia. These data suggest that distinct processes for the regulation of PKC may be operative in short-term and prolonged myocardial ischemia.

To exclude that this novel, unexpected increase of PKC activity was not overestimated by the histone assay known to be more specific for the conventional isozymes, additional measurements were performed using the pseudosubstrate peptide as substrate (Figure 1B). Also in these experiments, PKC activity significantly increased by 70% after 45 minutes of global ischemia, thus confirming the data obtained with the histone assay. We emphasize that these measurements were done in fully independent experiments. Moreover, to exclude a proteolytic activation of PKC in prolonged ischemia, basal enzyme activity (ie, in the absence of any stimulators [calcium and phosphatidylserine]) was included. As shown in Figure 1B, basal PKC activity remained unaltered even after 45 minutes of ischemia, thus excluding with certainty a proteolytic activation of the enzyme. Only the stimulated enzyme activity was significantly increased, which suggests an increased expression of PKC isozymes in cardiac tissue.

To address the questions of which of the dominant isozymes of PKC in rat heart may be involved in these regulation processes and whether the regulation of the individual isozymes of PKC may allow us to further differentiate distinct regulation processes, PKC isozymes were studied by isozyme-specific Western blot analyses using subtype-specific polyclonal peptide antibodies in the cytosolic and particulate fractions after various periods of ischemia.

The expression of PKC isozymes in adult rat heart is somewhat controversial. In the present study the dominant isozymes of PKC were , , and , accompanied by a weaker expression of the PKC. These results are in good agreement with data of other groups.^{22 23} An absolute quantification, however, is not possible using Western blot analysis because of the different affinities of the specific antibodies to their respective antigen. The relative expression of the single isozymes was quantified using the individual immunogenic peptides of the antibodies as an external standard (data not shown). Using this assay, PKC (equivalent to 1.25±0.2 pmol peptide), PKC (equivalent to 1.27±0.1 pmol peptide), and PKC (equivalent to 0.77±0.05 pmol peptide) were the most abundant isozymes expressed in the adult rat heart. The expression of PKC was much lower (equivalent to 0.22±0.01 pmol peptide). The ß-subtype of PKC, which has been shown to be expressed in nonmyocardial cells of the heart at extremely low levels and in fetal cardiomyocytes,²³ could not consistently be detected in these experiments using adult rat heart (data not shown). Similarly, the isozymes , µ, and could not be revealed in either fraction using Western blot analysis (data not shown). Thus, in adolescent rat hearts, only one of the calcium-dependent isozymes, PKC, and the calcium-independent isozymes , , and are expressed.

Subcellular Distribution and Translocation of PKC Isozymes in Short-Term Myocardial Ischemia
As shown in the sample Western blots in Figure 2, brief periods of ischemia led to a significant increase of all major cardiac isozymes of PKC in the particulate fraction. This increase of all PKC subtypes was observed as early as 2.5 minutes after the onset of ischemia, which is in good accordance with the translocation of enzyme activity, which was detected as early as 1 minute after the onset of ischemia (see Figure 1). This increase of the dominant cardiac isozymes of PKC in the particulate fraction was accompanied by the reduction of these isozymes in the cytosolic fraction (Figure 2), suggesting a translocation of these isozymes from the cytosol to the particulate fraction. A comparable translocation of these isozymes could be demonstrated up to 10 minutes after the onset of ischemia (data not shown). The quantitative analysis of 5 independent sets of experiments, which included the analysis of all 4 isozymes both in the cytosolic and the particulate fractions, always directly comparing the normoxic controls and the ischemic hearts, is shown in the graphs of Figure 2. In the particulate fraction, all 4 isozymes of PKC were concomitantly increased, whereas in the cytosolic fraction, the immunodetectable enzymes significantly decreased after 2.5 minutes of ischemia. The relative loss of enzyme in the cytosolic fraction and the relative increase in the particulate fraction was comparable for each of the 4 cardiac isozymes. These data show that the early ischemia-induced activation process nonspecifically translocates all 4 major cardiac isozymes of PKC from the cytosolic fraction to the particulate fraction of rat heart irrespective of their calcium sensitivity.

View larger version (47K): [in this window] [in a new window]   Figure 2. Western blot analyses of cardiac PKC isozymes in particulate and cytosolic fractions after 2.5 minutes of ischemia. In the upper part of each panel, representative Western blots of the cytosolic fractions (lanes 1 and 2) and of the particulate fractions (lanes 3 and 4) in normoxic controls (lanes 1 and 3) or after 2.5 minutes of global ischemia (lanes 2 and 4) are shown. Samples from controls and ischemic hearts were always analyzed in parallel. In the lower part of each panel are mean±SEM of the densitometric analysis of 5 sets of experiments from independent hearts with duplicate determinations for each point. For all isozymes of PKC, short-term ischemia induced a significant reduction in the cytosol and a concomitant, significant increase in the particulate fraction (*P0.05), ie, translocation of the isozymes, indicating their activation. Control and ischemic samples were analyzed in parallel and related to the controls. AUC indicates area under the curve.

Selective Induction of PKC and PKC in Prolonged Ischemia
The significant increase of PKC activity in the cytosol after prolonged ischemia (>30 minutes), as determined by histone III-S phosphorylation and by phosphorylation of a pseudosubstrate peptide from the C₁ region (compare methods), could be due to a regulatory modulation of all constitutively expressed PKC isozymes or, alternatively, to an altered expression of selective PKC isozymes. Moreover, it is conceivable that this regulation may not involve all cardiac isozymes of PKC. To address these questions, immunoblot analyses of the cytosolic and particulate fractions of control and ischemic hearts (15 to 60 minutes of ischemia) were performed. As shown in representative Western blots for the dominant cardiac isozymes of PKC (Figure 3), prolonged periods of ischemia for >30 minutes induce an increase of PKC and PKC in the cytosolic fraction without any change of the isozymes PKC and PKC. In the immunoblots, no proteolytic breakdown products were observed. The quantitative analysis of several series of independent experiments (n=6, ie, 6 individual hearts at each time point) revealed that prolonged ischemia had no significant influence on the expression of PKC and PKC (Figure 4). In contrast, the amounts of PKC and PKC were selectively increased in prolonged ischemia (Figure 4). After 15 minutes of ischemia, the immunodetectable enzyme PKC was increased by 36%, followed by a further increase up to a doubling of the enzyme after 60 minutes of ischemia (up to 198% of control values). Similarly, the expression of PKC was significantly increased 45 and 60 minutes after the onset of global ischemia (Figure 4). In the particulate fraction, the densities of PKC isozymes , , and were not significantly changed after prolonged ischemia (data not shown).

View larger version (66K): [in this window] [in a new window]   Figure 3. Western blot analyses of PKC isozymes in the cytosolic fraction after 15 to 60 minutes of global ischemia. Shown are representative Western blots for all PKC isozymes detected in the cytosolic fraction of normoxic controls (Co) and after various periods of global ischemia (15 to 60 minutes) in isolated perfused rat hearts. To the right of each gel the relative mobility as related to prestained molecular mass standards (55 to 97 kDa) is indicated, and to the left, specific bands for PKC, PKC, PKC, and PKC. Each heart was analyzed for all 4 isozymes of PKC in sequence. Control and ischemic hearts of the complete time course were always analyzed in parallel to allow relative quantification. These experiments are quantified in Figure 4.

View larger version (60K): [in this window] [in a new window]   Figure 4. Quantitative Western blot analysis of the cardiac isozymes of PKC in the cytosolic fractions after prolonged ischemia. Shown are quantitative, laser densitometric analyses of Western blots for all PKC isozymes in the cytosolic fraction of normoxic controls (Co) and after various periods of global ischemia (15 to 60 minutes) in isolated perfused rat hearts. Indicated are mean±SEM of 6 sets of experiments with 6 individual hearts at each time point and duplicate determinations for each heart. All time points in the time course were analyzed in parallel (compare Figure 3, which shows representative Western blots for this series of experiments) and compared with the normoxic controls. No significant changes in the expression of PKC and PKC could be detected after 15 to 60 minutes of ischemia. PKC significantly increased after 15 to 60 minutes of global ischemia, whereas PKC revealed a significant increase after 45 to 60 minutes of ischemia (*P0.05).

These data indicate that prolonged myocardial ischemia promotes, distinct from the early isozyme-unspecific translocation process, a selective induction of PKC and PKC, but not of PKC and PKC. A relocation of PKC or PKC from the particulate fraction to the cytosolic fraction cannot account for the ischemia-induced increased levels of immunodetectable PKC and PKC nor for the increased total enzyme activity (Figure 1, right panel). These data support the notion that a distinct, isozyme-selective regulation process for PKC is operative in prolonged ischemia, which leads to an increased expression of PKC and PKC at the protein level.

Induction of mRNA Specific for PKC and PKC in Prolonged Ischemia
To address the question whether the increase of total PKC activity and of immunodetectable enzyme in the cytosolic fraction 30 minutes after the onset of ischemia may be due to an increased expression, the mRNA levels for the 4 isozymes of PKC were determined at various time points after the onset of ischemia. Because in our hands the abundance of mRNAs was too low to use Northern blot analysis, quantitative RT-PCRs specific for the PKC isozymes were developed. As shown in Figure 5A, for each isozyme single amplification products of the expected sizes were obtained using specific sense and antisense primers (compare methods). As internal control, the mRNA for desmin was amplified using specific primers (Figure 5A, lane 2). In the absence of RNA, no amplification product was observed irrespective of the primer pair used (negative control). To additionally test for specificity, internal oligonucleotides were used in Southern blot analyses. The specificity of the amplification products was assessed by specific restriction digests. As one example, the restriction digest for the amplification product for the mRNA specific for PKC is shown in Figure 5B. The resulting pattern of bands after digestion was as expected (see scheme in Figure 5B). For absolute quantification, linearity of amplification was verified (Figure 6). The synthetic RNA standards specific for each isozyme were reverse transcribed and amplified in parallel in each experiment (Figure 7). To verify for each sample that the analysis was performed in the linear range, 2 different concentrations of starting template (ie, of total RNA) were analyzed in parallel. As shown in Figure 7 for PKC and PKC, 300 and 600 ng of total RNA were reverse transcribed and 30 and 60 ng were used for PCR amplification. After 30 minutes of ischemia, the amplification products for PKC remained unaltered. In contrast, expression of PKC was increased after 30 minutes of ischemia, as shown in the lower part of Figure 7 (lanes 3 through 6). The concentration of specific mRNA for desmin, which was used as internal standard, remained unchanged. The quantitative analysis of all series of experiments, which covered the time course from 15 to 60 minutes after the onset of ischemia, revealed that the mRNA levels for PKC and PKC did not change (Figure 8). In contrast, the mRNA levels both for PKC and for PKC increased as early as 30 minutes after the onset of ischemia. These data suggest an isoform-specific induction of PKC and PKC at the mRNA level in prolonged ischemia, supporting the data at the protein level.

View larger version (45K): [in this window] [in a new window]   Figure 7. Ethidium bromide gels of the RT-PCR analyses for PKC and PKC. Two concentrations of total RNA (300 and 600 ng) from normoxic controls (co) or after 30 minutes of ischemia (isch) were reverse transcribed. A tenth of the reverse transcription volume (equivalent to 30 or 60 ng of total cardiac RNA as starting template, as indicated) was amplified with specific primers for PKC (upper 2 panels) or for PKC (lower 2 panels). As internal standard, desmin (lane 1 and 2, left panels) was analyzed. For absolute quantification, synthetic RNA standards were analyzed in increasing amounts (0.25 to 8 fg and 1 to 16 fg, respectively, right panels). Indicated to the left are the sizes of the amplification products (desmin, 289 bp; PKC, 180 bp; and PKC, 280 bp).

View larger version (48K): [in this window] [in a new window]   Figure 8. Quantitative analysis of the mRNA levels for the cardiac PKC isozymes after various periods of global ischemia. The specific mRNAs were quantified in normoxic controls (Co) and after 15 to 60 minutes of global ischemia using quantitative RT-PCR with external synthetic RNA standards for absolute quantification relative to total RNA. Shown are mean±SEM of 5 sets of experiments with 5 individual hearts for each time point and duplicate determinations. For PKC and PKC, the absolute concentrations of specific mRNA were in the low range (40 to 60 and 200 to 300 ag/ng total RNA, respectively). These mRNAs were without any significant changes during the time course of ischemia. The absolute mRNA levels for PKC and PKC were in a higher range and significantly increased after 30 and 60 minutes of global ischemia (*P0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
As a hallmark for the activation of PKC, the translocation of PKC from the cytosol to the particulate fraction has been characterized in many organs and tissues, including the heart. Such translocation of PKC activity has also been described in early myocardial ischemia.

The salient finding of the present study is that beyond the translocation of PKC in early myocardial infarction, a second activation mechanism of PKC occurs in persistent ischemia. This second activation process does not involve a translocation of the enzyme to the particulate fraction. Figure 9 illustrates these distinct activation processes schematically. Brief periods of ischemia (Figure 9, left panel) promote a translocation of the PKC activity from the cytosol to the particulate fraction, which suggests its activation and confirms previously published data.^{13 36} As demonstrated here for the first time, this translocation occurs as early as 1 minute after the onset of ischemia and is rapidly reversible in ongoing ischemia. Using Western blot analysis, it could be shown that all major isozymes of PKC are involved in this transient translocation, suggesting a non–subtype-selective activation of the dominant isozymes of PKC in the ischemic heart. Total cardiac enzyme activity and mRNA levels for these isozymes remain unaltered after this brief period of ischemia.

View larger version (33K): [in this window] [in a new window]   Figure 9. Scheme of the distinct regulation processes of PKC in early and prolonged myocardial ischemia.

A second activation mechanism was observed in persistent ischemia (15 minutes, Figure 9, right panel). This newly identified mechanism is characterized by a significant increase of total cardiac PKC activity residing in the cytosolic fraction and by an isozyme-selective augmented expression of the immunodetectable PKC and PKC. Furthermore, this regulation at the protein level is mirrored by a subtype-selective induction of specific mRNAs only for PKC and PKC.

This is the first characterization of 2 distinct regulation processes of PKC in the infarcted heart after short-term and prolonged ischemia. The molecular signals responsible for these 2 activation mechanisms are not known at this point. However, the different characteristics imply that 2 distinct regulation mechanisms may be operative.

The early translocation of PKC activity has been shown previously.^{13 16 36 37} To our knowledge, the earliest time point of translocation of PKC shown before was 2 minutes of ischemia in rabbit hearts.³⁶ Moreover, the reversibility of this process in myocardial ischemia has not been demonstrated before. In few other systems has translocation of PKC been shown to be transient.³⁸ So far, it has not been evaluated whether the activation of PKC in the ischemic heart may persist even after the relocation of the enzyme to the cytosol in prolonged ischemia.

Four PKC isozymes, PKC, PKC, PKC, and PKC, are the predominant isozymes detected in cardiac tissue of adolescent rats. Only minute levels of the ß-isozyme could inconsistently be found in unfrozen cardiac tissue (data not shown). Other isozymes of PKC, such as PKC or PKC, could not be detected (data not shown). These data are in good agreement with previously published data.^{22 23} Although the absolute quantification of the expression of these isozymes was not a focus of the present study, it could be shown using the immunogenic, specific peptides as external standards that the isozymes PKC, PKC, and PKC are expressed at similar quantities. Only PKC was expressed at a lower amount. Rybin and Steinberg,²³ also using Western blot analyses, found the highest abundance for PKCa, PKC, and PKC in the neonatal rat heart followed by a developmental decline of PKC and PKC.

The focus of the present study was the relative changes of the isozymes of PKC, which can reliably be determined using Western blot analyses. The relative distribution of PKC isozymes in the control hearts, however, is somewhat different in the present study compared with data in isolated cardiomyocytes.²³ Using hearts immediately frozen ex vivo or hearts frozen after normoxic perfusion, it could be demonstrated that the predominant amount of PKC resides in the cytosol and that perfusion of isolated hearts had no influence on the relative distribution of PKC isozymes. The isolation of ventricular myocytes,²³ which had been used to characterize the developmental changes of PKC isozymes in rat heart in other studies, may have induced a translocation of PKC to the particulate fraction. This may explain the relatively higher representation of PKC in the particulate fraction of isolated cardiomyocytes in those studies²³ compared with the particulate fraction of cardiac tissues used here. To avoid even brief periods of ischemia, no separation of atria and ventricles was performed in the present study. Thus, the relatively high expression of the -isozyme of PKC may additionally be due to atrial tissue, which has been shown to express this isozyme to a higher extent than the ventricles.

The early, ischemia-induced translocation was comparable for all 4 isozymes of PKC. This early activation involves both calcium-dependent (PKC) and calcium-independent (PKC, PKC, and PKC) isozymes of PKC, which suggests that intracellular calcium may not be responsible for this early activation process. Also, a receptor-mediated release of phosphoinositides, which has been shown to activate predominantly calcium-independent isozymes of PKC in cardiac tissue,³⁹ is unlikely to be involved in this ischemia-induced activation and translocation process. This concept is supported further by our previously published data, which demonstrated that the ischemia-induced translocation of PKC activity could not be prevented by blockade of ₁-adrenergic receptors using prazosin.¹³ Moreover, PKC is one of the atypical PKC isozymes, which, because of its characteristic sequence in its regulatory domain, is not activated by DAG.²⁰ Given that PKC is also subjected to the early activation and translocation in short-term ischemia, it may be concluded that DAG may not be responsible for this ischemia-induced early translocation of PKC. Other subcellular localizations of the PKC isozymes, such as a nuclear translocation or the translocation to caveolae,^{40 41} that may be important for the substrate specificity were not a focus of the present study. As shown here, this translocation of PKC is reversible on prolonged ischemia. Whether PKC after return to the cytosol remains active in the intact heart cannot be determined at this point.

The second process of ischemia-induced activation of PKC in prolonged ischemia resulted in an increased cytosolic enzyme activity, whereas the activity in the particulate fraction has returned to control values. This augmented activity is associated with an almost doubling of the immunodetectable enzymes PKC and PKC. The extent of the expressional increase of these isozymes closely matches the increase in enzyme activity as determined by histone III-S phosphorylation or the phosphorylation of a pseudosubstrate from the C₁ region.

The augmented expression at the protein level is mirrored by the enhanced levels of mRNAs specific for these isozymes of PKC. These data support the notion of an ischemia-induced, subtype-selective expressional regulation of these isozymes. To our knowledge, this is the first description of such a relatively rapid expressional regulation of PKC in cardiac tissue. Recently, a subtype-selective, slow, developmental regulation of the expression of PKC isozymes has been described in rat heart.²³ A rapid regulation of mRNAs specific for PKC has been shown for nonphysiological activation of PKC using phorbol esters. These lead to a downregulation of the expression of PKC both at the protein and at the mRNA level.⁴² Also, in isolated cells a pharmacological activation of PKC by phorbol esters was able to induce a selective downregulation of mRNA for PKC but a selective induction of mRNA for PKC.⁴³

Several studies have investigated the mRNA levels for PKC subtypes in cerebral tissue not during ischemia but during reperfusion after an ischemic insult.⁴⁴ These studies show that in brain of different species a rapid transcriptional, isotype-selective regulation of PKC isozymes may occur during reperfusion. However, none of these studies have characterized the regulation at the protein level or have focused on the regulation during ischemia.

In acute myocardial ischemia, a rapid induction of mRNAs followed by a rapid increase of the specific proteins has been observed for other components of signal transduction pathways such as for ß-adrenergic receptors,³⁴ for the uptake₁ carrier,⁴⁵ for the ß-adrenergic-receptor kinase,⁴⁶ and for heat shock proteins.⁴⁷ For some proteins, such as the heat shock protein SP71 and fibronectin, it has been shown that the ischemia-induced increase of mRNA is followed by an increased expression of the specific protein as determined by electrophoresis.⁴⁷ The coincidence of the increased immunodetectable amount of proteins and the increased levels of specific mRNAs for those proteins, as well as for PKC and PKC in the present study, suggests that the ischemic myocardium turns on the machinery for protein synthesis. However, a direct evaluation of translation in the ischemic heart has not been feasible for methodological reasons. Whether, additionally, a reduced degradation of specific proteins may contribute to the increased levels of these proteins cannot be decided at this point.

We would like to emphasize that this is the first coherent characterization of 2 distinct regulation processes of PKC in early and prolonged ischemia. It clearly distinguishes between the early, isozyme-unselective translocation process and the late, subtype-specific regulation process. In prolonged ischemia an augmented enzyme activity coincides with a subtype-selective induction of PKC and PKC at the protein level and at the expression of specific mRNAs. These 2 regulation processes were characterized in their temporal sequence in the widely accepted model of isolated rat hearts, such that species and model differences can be neglected.

Both the rapid, non–subtype-selective activation of PKC in very early ischemia and the subtype-selective increase in prolonged ischemia may have distinct functional consequences. Thus, the early activation of PKC may contribute to the genesis of arrhythmias possibly mediated by the activation of ATP-sensitive K⁺ channels or the delayed rectifier K⁺ current.⁴⁸

Activation of PKC in prolonged ischemia may promote myocardial hypertrophy⁴⁹ and the induction of immediate-early genes.⁴⁹ The activation and especially the newly characterized increased expression of PKC isozymes in the infarcted heart may contribute to the process of remodeling.¹⁰ So far it has not been clarified which isozyme of PKC may be responsible for such processes.

The clear distinction of the 2 regulation processes of PKC in short-term and in prolonged myocardial ischemia will contribute to a better understanding of the functional consequences of an activation of PKC.

	Acknowledgments

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB320). R.H.S. was supported by the Hermann and Lilly Schilling Foundation. We thank Ulrike Oehl and Annette Kempkes for expert technical assistance and Drs Carsten Schwencke and Mathias M. Borst for critical review of the manuscript.

	Footnotes

 
Parts of this study were presented at the 68th and 69th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13–16, 1995, and New Orleans, La, November 10–13, 1996, respectively.

Received September 16, 1998; accepted April 22, 1999.

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