Cardiovascular Research

Cardiovascular Research 2000 48(2):285-299; doi:10.1016/S0008-6363(00)00185-1
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Molecular heterogeneity of protein kinase C expression in human ventricle

Hyeon-Gyu Shin^*, Joey V. Barnett, Paul Chang, Seenu Reddy, Davis C. Drinkwater, Richard N. Pierson, Ronald G. Wiley and Katherine T. Murray

Departments of Medicine, Pharmacology and Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA

^* Corresponding author. Tel.: +1-615-936-1514; fax: +1-615-322-4707 hyeon-gyu.shin{at}mcmail.vanderbilt.edu

Received 19 April 2000; accepted 27 June 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Although activation of protein kinase C (PKC) modulates the function of normal cardiac myocytes and likely plays a role in the pathogenesis of cardiomyopathic disease states, the molecular basis of PKC expression in human ventricle has not been examined in detail. Methods: We have performed Western analysis and immunohistochemistry on explanted human cardiac tissue from nondiseased and diseased specimens using isoform-specific antibodies directed against all known PKC isozymes. Results: In homogenates from left and right ventricle, all isoforms except PKC- and were detected by immunoblotting, with confirmation using a second antibody directed against a different epitope when possible. For PKC-βII, , and , data indicated that these isoforms were variably phosphorylated in vivo, resulting in multiple bands during immunoblotting. Because of potential antibody cross-reactivity, reverse transcriptase polymerase chain reaction (RT-PCR) was performed which confirmed expression of PKC-, βI, and . Immunohistochemistry demonstrated that all isoforms detected in ventricular homogenate by Western analysis could be localized to cardiac myocytes. From a methodologic standpoint, significant degradation of PKC isoforms could be demonstrated when samples were either frozen or allowed to remain at room temperature, compared to immediate subcellular fractionation. Conclusions: These findings indicate that the PKC expression in human ventricular myocytes is remarkably diverse, with multiple conventional, novel, and atypical isoforms present, and highlight the importance of sample preparation in comparative studies of PKC isoform expression.

KEYWORDS Signal transduction; Heart failure; Cardiomyopathy; Protein phosphorylation; Protein kinases; Myocytes

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Protein kinase C (PKC) is a family of serine/threonine kinases which play a major role in signal transduction in the heart. In response to circulating hormones such as norepinephrine, endothelin, and angiotensin II, activation of PKC triggers a multitude of intracellular events, including phosphorylation of cardiac structural elements, stimulation of other proteins such as kinases, and altered gene expression [1–4]. These events can influence multiple physiologic processes in the heart, including contraction, relaxation, and chronotropy [3,5,6]. PKC activation has also been implicated in the pathophysiology of a number of cardiovascular disease states [1,3], including hypertrophy [3,5,6], ischemia and ischemic preconditioning [7,8], and heart failure [9,10], based on numerous studies using animal models.

It is now recognized that PKC comprises a group of at least 11 different isoforms which can generally be divided into three categories [2,4,11]: calcium-dependent or conventional (c) PKCs (, βI, βII, and ); calcium-independent or novel (n) PKCs (, , , and ); and atypical (a) PKCs ( and /) which are unresponsive to phorbol esters. The most recently-described isoform, PKC-µ, is structurally distinct with homology to the protein kinase D family. Substantial experimental evidence indicates that individual PKC isoforms differ with respect to tissue distribution and subcellular localization, as well as substrate specificity [3,12–15]. Such data has led to speculation of isoform-specific functions in vivo [14–18]. The recent development of isoform-specific PKC inhibitors should delineate the role of individual isozymes in physiologic and disease processes [17,19,20]. As an example, the spontaneous rate of contraction of neonatal rat cardiac myocytes can be inhibited by activation of PKC- but not , β, , or [17]. The potential therapeutic use of selective PKC inhibitors is currently under active investigation [19,20].

Although previous studies have investigated PKC expression in cardiac tissue from various mammalian species [1,9,10,13,21–30], the isoforms which reside in human heart have not been determined in detail. In rat heart, several reports have suggested that a limited repertoire of PKC isoforms are present in enzymatically isolated adult rat ventricular myocytes, and that detection of additional isoforms in tissue homogenates may result from contamination by non-myocyte elements [21,22,24]. However, recent results examining a limited number of isoforms in human heart suggest that expression in this species may be more diverse [31]. In this study, we have undertaken a comprehensive investigation of PKC expression in explanted human ventricle using Western analysis and immunohistochemistry with multiple isoform-specific antibodies directed against each of the PKC isoforms identified to date. Our results indicate that the molecular basis of PKC expression in human heart is complex, with identification of nearly all isoforms in ventricular myocytes.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Patient population
Tissue was obtained from five nondiseased human hearts, four of which were rejected as donor hearts for cardiac transplantation, as well as one autopsy specimen, in accordance with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3). The subjects were 14–67 years of age (all females), and the causes of death included trauma (1), intracranial hemorrhage (2), smoke inhalation (1), and bowel infarction in one. Four subjects were receiving dopamine at the time of organ harvest; additional intravenous agents included nitroprusside in two, phenylephrine in one, and norepinephrine in one. In all cases, the left and right ventricles were normal by visual inspection and the coronary arteries were widely patent. There was no history of cardiac disease in any individual. Tissue from abnormal cardiac specimens was obtained following explant for cardiac transplantation (n = 7). All patients had class III–IV congestive heart failure due to ischemic cardiomyopathy (ages 43–61, all male). Six patients were hospitalized in the cardiac intensive care unit at the time of transplantation. Medications prior to transplant included dopamine/dobutamine (6), phosphodiesterase inhibitors (3), angiotensin converting enzyme inhibitors (4), digoxin (7), and diuretics (7). Following explantation, specimens were quickly immersed into ice-cold cardioplegic solution and transported immediately for subcellular fractionation.

2.2 Preparation of subcellular fractions
Portions of the left and right ventricular free walls were dissected and minced into small pieces of approximately 2x2 mm. Subcellular fractionation was performed at 4°C using approximately 2–3 g of tissue, while the remainder was immediately frozen in liquid nitrogen until processed. Tissue was homogenized in a buffer (mM: Tris–HCl 25, EDTA 2, EGTA 0.5, PMSF 1, DTT 1, pefebloc 0.5, benzamide 1, iodoacetamide 1, 1,10-phenanthrolene 1, aprotinin 0.004, leupeptin 0.2, pepstatin A 0.0015, and bacitracin 0.7). The homogenate was separated into soluble and particulate fractions by centrifugation at 50 000xg for 10 min, subjected to a protein assay (BCA Protein Assay Reagent, Pierce, Rockford, IL), diluted in 2x sodium-dodecyl-sulfate (SDS) sample buffer, aliquoted, and stored at –80°C until use.

Rat brain cellular extracts were prepared for use as a positive control during Western blot analysis for PKC isoforms , βI, βII, , , , and . The use of rats in this investigation was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult Sprague–Dawley rats were anesthetized by intraperitoneal injection of ketamine (80 mg/kg), decapitated, and the entire brain rapidly harvested. Tissue was rinsed with phosphate-buffered saline (PBS), minced, and subjected to subcellular fractionation as described above for human heart.

2.3 Selection of antibodies for Western blot analysis
Because antibodies from different companies demonstrated substantial differences in sensitivity to detect a given PKC isoform, a systematic comparison was undertaken initially between polyclonal antibodies from multiple sources (typically GibcoBRL, Santa Cruz, Research & Diagnostics, and Calbiochem) using recombinant PKC isoforms or other positive controls, in order to identify the most sensitive and specific antibodies for Western analysis. The initial antibody dilutions tested were based on the manufacturer's recommendations and previous reports in the literature, with final concentrations determined by testing a range of dilutions with appropriate positive controls. Based on these results, two antibodies were chosen, directed against unique, non-overlapping epitopes when possible, for detection of each PKC isoform in tissue (Table 1). Antibodies against the most recently described PKC isoforms (PKC-, and µ) were available from a more limited number of commercial sources. In some cases, monoclonal antibodies were used when polyclonal antibodies were either inadequate or unavailable for Western analysis. A PKC isoform was scored as present if both antibodies recognized bands of similar molecular mass, with loss of immunoreactivity following peptide block for each polyclonal antibody.

View this table: [in this window] [in a new window]   Table 1 Epitope site used for antibody generation based on inferred human sequence

 
2.4 Western blot analysis
Fifty µg of human heart protein was diluted with SDS sample buffer, boiled for 2 min, and immediately loaded into lanes of a 6–10% SDS–polyacrylamide gel. A positive control was also loaded for each blot; as noted above for most isoforms, this was rat brain soluble protein (0.5–1.5 µg); for PKC-, and µ, 50–100 µg of particulate protein from EL4 cells, whole cell lysate from a lymphoma cell line (Jurkat cells), and mouse kidney homogenate were used, respectively. Proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane by a semi-dry transfer cell apparatus (Bio-Rad, Hercules, CA). Membranes were incubated in 5% nonfat dry milk in TBST solution (mM: Tris-base 10, NaCl 150, Tween-20 0.1%, pH 7.6) for at least 2 h at room temperature, followed by incubation with primary antibody in TBST at 4°C overnight. Most antibodies selected for Western analysis were polyclonal, affinity-purified rabbit IgG from either Santa Cruz (Santa Cruz, CA) or GibcoBRL (Grand Island, NY), as shown in Table 1. For Santa Cruz antibodies, the following dilutions were used: PKC- 1:5000; PKC-βI, , and 1:1000; PKC-βII 1:2000; and PKC-, , and µ 1:500. For GibcoBRL antibodies, the dilutions used were: PKC-, and 1:500; PKC-βI, βII, , and 1:250. Additional antibodies used included: Research & Diagnostics (Berkeley, CA) for PKC- (1:500); Santa Cruz polyclonal, affinity-purified goat antibody for PKC- (1:500); monoclonal antibodies from Transduction Laboratory (Lexington, KY) for PKC-, , , and (1:250); and Calbiochem for PKC-µ (1:400).

Following removal of the primary antibody, horseradish peroxidase-conjugated secondary antibody at an appropriate dilution (1:15 000 for polyclonal antibodies, 1:7500 for monoclonal antibodies) was added for 1 h at room temperature. An enhanced chemiluminescence kit (ECL, Amersham, Arlington Heights, IL) was used for detection of protein bands. The specificity of polyclonal antibody detection was determined by performing Western blot analysis in the absence and presence of a 20-min pre-incubation of antibody with a 10-fold excess of the peptide used for antibody generation. As noted above, detection of a given PKC isoform by Western blot analysis was confirmed not only by the peptide block experiments, but also by detection of a protein having the same molecular mass by two isoform-specific antibodies.

2.5 Detection of PKC isoform mRNA by RT-PCR
Approximately 1–2 mg of left ventricle was used to extract total RNA using a modified single step extraction procedure [32]. The sample was incubated with RNAse-free DNase, and cDNA was reverse transcribed (RT) according to the manufacture's instruction (First-strand cDNA synthesis Kit, Pharmacia, Piscataway, NJ). PKC sequences were amplified by using the polymerase chain reaction (PCR) and isoform-specific primers: for PKC-: forward 5' CGA CTG TCT GTA GAA ATC TGG 3' (nt 742–762), reverse 5' CAC CAT GGT GCA CTC CAC GTC 3' (nt 1185–1165); for PKC-βI: forward 5' CTG TGG AAC TGA CTC CCA CTG 3' (nt 2134–2154), reverse 5' ATA CTG AAG CAT TTT GGT ATC 3' (nt 2538–2518); for PKC-: forward 5' AGG AAG CTG TAC CGT GCC AAC 3' (nt 349–369), reverse 5' CTT TAA TGC TGT CAT GCT TCC GG 3' (nt 659–637). Primers for the human cardiac Na⁺ channel, hH1, were used as a positive control for each PCR reaction. DEPC water was used in place of primers as a negative control. PCR amplification was performed with 35 cycles and a denaturing temperature of 94°C for 30 s, annealing temperature of 58°C for 30 s (for PKC-βI, 60°C), and extension temperature of 72°C for 1 min. PCR products (10 µl) were loaded onto 1.5% agarose gel, stained with ethidium bromide and visualized under UV light.

2.6 Dephosphorylation of PKC isoforms
Dephosphorylation of PKC isoforms was stimulated by warming 50 µg of soluble or particulate protein to 30°C for 60 min. This permitted activation of endogenous phosphatases present in human heart, and dephosphorylation achieved in this manner was as efficient as the addition of exogenous phosphatases (PP) including PP1 or PP2A (data not shown). [³²P]glycogen phosphorylase a was used as a positive control, with a reduction in radioactivity indicative of dephosphorylation (with conversion to glycogen phosphorylase b). To terminate the reaction, 2x SDS sample buffer was added. In some reactions, dephosphorylation was inhibited using a combination of phosphatase inhibitors, including (mM): NaF 10, β-glycerophosphate 10, Na pyrophosphate 100, Na orthovanadate 2.5, and microcystine 1. All samples were boiled 2 min prior to loading onto a 6% gel for Western analysis.

2.7 Preparation of tissue sections
For human heart, a small piece of left ventricle (3x3 mm) was dissected from the heart, immersed in 4% paraformaldehyde solution at 4°C overnight, dehydrated with 50–100% ethanol, and embedded into paraffin wax. The tissue was sliced into 7-µm thick sections using a microtome and baked at 55°C overnight. Slides were stored at 4°C until processed for immunohistochemistry.

Rat brain was used as a positive control for immunohistochemistry for most PKC isoforms [33–35]. Rats were anesthetized with intraperitoneal pentobarbital and transcardially perfused with 1 l of 4% paraformaldehyde. The brain was removed and immersed in cold 30% sucrose overnight at 4°C. The cerebellum was dissected, frozen to a microtome stage with dry ice and sectioned at 40 µm thickness. Sections were collected in 0.05 M Tris–saline solution (pH 7.6) and stored either in Tris–saline at 4°C or in antifreeze solution (30% ethylene glycol, 30% glycerol in sodium phosphate buffer) at –20°C until processed for immunohistochemistry. As noted below, Jurkat cells were also used as positive control for some PKC isoforms. Cells were collected onto a slide by cytocentrifugation, air-dried, and fixed in 4% paraformaldehyde immediately prior to immunohistochemistry.

2.8 Immunohistochemistry
Based on the results of Western blot analysis, primary antibodies from Santa Cruz were used in these experiments except for PKC-, , , (Transduction), and µ (Calbiochem). Initially, the sensitivity of each of these antibodies was determined with positive controls using a range of concentrations, with peptide block to assess the specificity of immunodetection. While rat brain cerebellar sections were used as a positive control for most antibodies, Jurkat cells concentrated by cytocentrifugation on a microscope slide served this purpose for PKC-, , and µ. Immunohistochemistry of rat brain cerebellum was performed using methods described previously [36]. Dilution of primary antibody solutions ranged from 1:1000 to 1:2500. The eight PKC isoforms tested were detected in this preparation (data not shown) with specific patterns of cellular distribution as previously reported [33–35]. Jurkat cells, which were used as a positive control for the remaining three isoforms, underwent immunostaining using the method described below for human heart sections.

To determine PKC isoform expression in human left ventricle, sections mounted on slides were allowed to re-hydrate by extensive washing with 50–100% ethanol and xylene solution. Sections were further washed and incubated with a blocking solution (e.g. 10% goat serum in 1% bovine-serum albumin (BSA)/PBS) for at least 1 h at room temperature followed by the addition of primary antibody (e.g. diluted in 1% BSA/PBS with 1% goat serum) and incubation overnight at 4°C in a humidified chamber. Dilution of the antibodies for PKC-, βI, βII, , , and was 1:100; for PKC-, , , , and µ, 1:50. Pre-immune rabbit serum at 1 mg/ml was used as a negative control. Sections were washed three times with PBS and incubated with alkaline phosphatase-conjugated secondary antibody for 1 h at room temperature followed by three washes with PBS. Detection was achieved using fast naphthol-red as the enzyme substrate and was terminated by washing with PBS. Sections were counter-stained with hematoxylin (Anatech, Battle Creek, MI) for 60 s, washed, air dried, and covered with Aquamount (Lerner Laboratories, Pittsburgh, PA) and a coverslip. The specificity of polyclonal antibody detection for each isoform was determined using pre-absorption of the antibody by incubation with a 10-fold excess of specific epitope peptide.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 PKC isoforms in normal human ventricle: Western analysis
Initial studies were conducted to determine which PKC isoforms were expressed in nondiseased human left ventricle using immunoblotting. Total ventricular homogenate was subjected to subcellular fractionation to obtain soluble (S) and particulate (P) fractions (n = 5 and n = 7 for nondiseased and diseased hearts, respectively). These fractions were then analyzed by Western blotting using the antibodies in the first column of Table 1. The results for two nondiseased specimens (ND1, ND2) are shown for the conventional PKC isoforms in Fig. 1, the novel PKCs in Fig. 2, and atypical isoforms plus PKC-µ in Fig. 3 (left side of each figure). For all isoforms except PKC- and , one or more bands was detected having a molecular mass that correlated with previously-published values for individual PKC isoforms [1,22,24,37–39]. Most isoforms were present as single bands, although for PKC-βII, , and , two or three bands were detected on most blots. The identity of these bands with respect to the positive control in each blot is described in more detail below. PKC expression was predominantly in the soluble fraction for some isoforms (PKC- and ), the particulate fraction for others (PKC- and µ), or both, with some variability between specimens. A detailed analysis of isoform location was not undertaken, given the fact that specimens were largely explanted from potential organ donors who were maintained on life support and intravenous inotropes prior to donation. Under such conditions, it is likely that hearts were subjected to a significant degree of neuro-hormonal stimulation which could activate or down-regulate PKC prior to explant. The bands shown in Figs. 1–3 were eliminated by preincubation of primary antibody with the epitope peptide (peptide block) where appropriate. In addition, Western analysis with a second panel of isoform-specific antibodies (column 2 of Table 1) detected bands of similar size, which were also removed by peptide block. Taken together, these data indicate that the bands shown in Figs. 1–3 represent isoforms of PKC, with the exception of PKC-βII as described below. PKC- and were not detected when a twofold higher concentration of either antibody or 100 µg of protein was used.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Western blot analysis of conventional PKC isoforms in nondiseased and cardiomyopathic human heart. Western blot analysis was performed on left ventricular tissue from nondiseased subjects (ND) as well as patients with ischemic cardiomyopathy (ICM). Soluble (S) and particulate (P) fractions were obtained for each sample following acute or immediate subcellular fractionation after tissue procurement. Fifty µg of protein was loaded in each sample lane, with rat brain (RB) as a positive control (). On the right side of the figure, the arrows indicate the molecular size of bands that represent PKC isoforms (except for possibly PKC-βII; see text).

 

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Western blot analysis of novel PKC isoforms in human left ventricle. Results for novel PKC isoforms are shown, using the same format and sample preparation as for Fig. 1. The positive controls for PKC- and were EL4 (EL4) and Jurkat (JRT) cells, respectively.

 

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Western blot analysis of atypical PKC isoforms and PKC-µ. Results are shown for atypical PKC isoforms and PKC-µ, using the same format and sample preparation as for Fig. 1. The positive control for PKC-µ was mouse kidney (KID).

 
In order to compare PKC isoform expression between left and right ventricle, samples from both regions containing identical amounts of protein (50 µg) were run together on the same gel (experiments replicated using four separate gels for each isoform). The results of these studies demonstrated that there was no obvious difference in expression between the left and right ventricle for any of the PKC isoforms detected (data not shown).

3.2 Phosphorylation of PKC isoforms
It is known that PKC isoforms, including PKC-βII, are substrates for phosphorylation in vivo [40,41]. To determine whether the multiple bands seen for PKC-βII, , and during Western analysis represented differentially phosphorylated isoforms, experiments were performed to dephosphorylate PKC in vivo (n = 3 and n = 6 for nondiseased and diseased hearts, respectively). Pilot experiments demonstrated that dephosphorylation of a radiolabelled substrate, [³²P]glycogen phosphorylase a, was readily accomplished by incubation with human left ventricular homogenate at 30°C for 1 h, which activates endogenous phosphatases in the heart extract (Fig. 4A). This method was comparable to the addition of exogenous phosphatases such as PP1 or PP2A (data not shown). Samples of human left ventricle before and following incubation at 30°C were subjected to Western blotting using antibodies directed against PKC-βII, , and , with the results shown in Fig. 4B–D. In all three cases, phosphatase activation led to an obvious enhancement of the lowest molecular weight band for each isoform (indicated by the arrowheads at 78, 70, and 90 kDa for PKC-βII, , and , respectively), particularly in the membrane fraction. This enhancement could be inhibited by concomitant incubation with a combination of phosphatase inhibitors (lanes 6 and 7), further confirming that it resulted from dephosphorylation of PKC rather than degradation. As illustrated in Figs. 1–3 as well as Fig. 4, these lower molecular weight bands were also present, and usually dominant, in the rat brain positive control, further confirming their identity as dephosphorylated species of the PKC isoforms. For PKC-, the upper band at 74 kDa diminished in intensity with dephosphorylation. For PKC-, dephosphorylation led to the virtual disappearance of the upper band at 96 kDa, as well as the emergence of the lower molecular weight band at 90 kDa (arrowhead), which is also present in the rat brain positive control (also in Fig. 2). For PKC-βII, obvious change in the prominent band at 83 kDa with dephosphorylation was not apparent, suggesting that it may not represent a form of PKC-βII. Thus with the possible exception of PKC-βII, the multiple bands detected by immunoblotting represent differentially phosphorylated species of PKC-βII, , and , with phosphorylated forms predominating in vivo under these conditions.

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Dephosphorylation of PKC-βII, , and in human left ventricle. (A) [32P]Glycogen phosphorylase a was incubated in water at 30°C in the presence and absence of phosphatase inhibitors (lanes 5 and 6). Phosphatase inhibitors had no effect on the basal degree of radioactivity in the absence of phosphatase. The radiolabelled protein was then incubated in soluble (S) or particulate (P) fractions of human left ventricle at 30°C to activate endogenous phosphatase (lanes 1 and 2), with a reduction in radioactivity. The reduction was inhibited by co-incubation at 30°C with phosphatase inhibitors (lanes 3 and 4). (B–D) Subcellular fractions of human left ventricle after routine sample preparation (lanes 1 and 2), following incubation at 30°C to activate endogenous phosphatases (lanes 4 and 5), and following incubation at 30°C in the presence of multiple phosphatase inhibitors (lanes 6 and 7), as well as a positive control (rat brain, RB; lane 3). Samples were subjected to SDS–PAGE followed by Western analysis using the antibodies indicated underneath the figure. Enhancement of the lowest molecular weight band in lane 5 of each panel (indicated by the arrowhead) indicates dephosphorylated PKC, which is reduced in the presence of phosphatase inhibitors (lane 7). Samples shown are from hearts with ischemic cardiomyopathy.

 
3.3 Antibody cross-reactivity and confirmation of PKC isoform expression
The experimental results using Western analysis are summarized in Table 2, indicating molecular mass, predominant site of expression, and the number of bands detected for each isoform. Because of amino acid sequence homology between PKC isoforms, antibodies directed against one isoform might cross-react with another, as previously reported [27,37]. In such cases, the pattern of detection by immunoblotting should be identical for the two isoforms. With this concept in mind, the data presented in Table 2 eliminate the possibility of antibody cross-reactivity except for two cases: PKC- and βI; and PKC- and . To explore this possibility further, experiments were performed to determine the sensitivity of the PKC antibodies in question to detect a range of concentrations of purified recombinant PKC isoforms. As shown in panel A of Fig. 5, antibodies directed against PKC- could easily detect purified PKC-βI in similar quantities, while the sensitivity of the PKC-βI antibody was much greater to detect PKC-βI than PKC-. In panel B, recombinant PKC- was easily detected by the antibody for PKC- but not by the PKC- antibody, which nonetheless detected PKC- in human heart and rat brain samples (recombinant PKC- is not commercially available). These results indicated that the antibodies for PKC- might be detecting PKC-βI, while the PKC- antibody may be recognizing PKC-. Such cross-activity is less likely to occur with human heart samples due to the reduced quantity of PKC present, compared with the amounts of recombinant enzymes used in the experiments shown in Fig. 5. Nevertheless, to confirm expression of PKC-, βI and in human heart, reverse transcriptase-polymerase chain reaction (RT-PCR) using isoform-specific primers was performed using total RNA from left ventricle (n = 3 for diseased hearts). The results are shown in Fig. 6 for three individual specimens (ICM1, 2, and 3). In each case, RT-PCR was performed in the presence (A) and absence (B) of DNase treatment, while RNA samples in the absence (C) and presence (D) of DNase which were not subjected to RT-PCR were run as controls. In addition, RNase-treated water (E) and samples probed with primers for the human heart Na⁺ channel (F) were analyzed by RT-PCR as negative and positive controls, respectively, in each experiment. Bands of the appropriate size were detected in all three specimens for PKC-, βI, and , consistent with expression of these PKC isoforms in human left ventricle.

View this table: [in this window] [in a new window]   Table 2 Characterization of PKC isoforms in human left ventricle by Western analysis

 

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Cross-reactivity of PKC antibodies. (A) Cross-reactivity between antibodies for PKC- and βI was examined by loading increasing concentrations of human LV soluble (S) and particulate (P) fractions (lanes 1–4), purified recombinant PKC- (lanes 5–7), purified recombinant PKC-βI (lanes 8–10) and rat brain (RB, lanes 11 and 12) onto a gel. SDS–PAGE was performed followed by Western analysis with antibodies for either PKC- (left panel) or PKC-βI (right panel). (B) Results are shown for antibodies directed against PKC- and using a similar format as in panel A, except that purified recombinant PKC- was not available for use.

 

View larger version (55K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Detection of mRNA for PKC-, βI, and in human left ventricle using RT-PCR. RT-PCR was performed on total RNA isolated from the left ventricle of three patients (ICM 1, 2, and 3) using isoform-specific primers for PKC-, βI, and . Samples in each lane were processed as follows: A: (+) DNase, RT-PCR; B: (–) DNase, RT-PTR; C: (–) DNase, no RT-PCR; D: (+) DNase, no RT-PCR; E: RNase-treated water, PT-PCR, as a negative control; and F: (+) DNase, RT-PCR using primers for the human heart sodium channel, hH1, as a positive control. A DNA ladder was run in the lanes at both ends of the gel. The size of the predicted base pair (bp) fragment is shown on the right of the figure for the PKC isoform tested.

 
3.4 PKC expression in cardiomyopathic states and the effect of sample preparation
To determine whether PKC isoform expression was altered in the presence of disease states, immunoblotting was also performed on samples from patients with ischemic cardiomyopathy (n = 7). The results are shown for four patients with ischemic cardiomyopathy (ICM 1, 2, 3, and 4) in Figs. 1–3. Our results indicate that the same isoforms which are expressed in nondiseased heart are also present in this pathologic state, with no evidence of an isoform switch (i.e. pathologic expression of an isoform that was not detected in nondiseased tissue, and vice versa).

Because of the small number of samples which were not sex-matched (five nondiseased females and seven diseased males), we did not undertake a quantitative comparison to determine whether PKC isoform expression was either up- or down-regulated during cardiomyopathic states. However for a given specimen, variability in isoform expression was apparent when Western blot analysis was performed at multiple time points following sample collection. The source of this variability was ultimately identified and is illustrated in Fig. 7. Following explant, a portion of the left ventricle for all hearts was subjected to immediate or acute subcellular fractionation in the presence of protease inhibitors, while the remainder of the tissue was frozen in liquid nitrogen until processed in an identical manner at a later date. In Fig. 7A, samples prepared acutely were compared to samples from the same specimens which were initially frozen for a period of at least 12 months prior to subcellular fractionation. It is apparent that expression of PKC- is reduced in at least 2 of the specimens that were frozen initially, indicative of protein degradation, compared to tissue processed acutely, despite equal amounts of protein present (as shown in Fig. 7B, which represents ponceau staining of the same blot). This degradation was also apparent for PKC-βI, , , and in our samples (data not shown). In addition, evidence of PKC isoform degradation was also present in the normal specimen obtained at autopsy, when it was compared to acutely prepared, explanted normal hearts (data not shown). These findings highlight the critical importance of sample preparation when comparing PKC isoform expression under different conditions.

View larger version (49K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Degradation of PKC isoforms: Comparison of frozen and acutely-prepared samples. (A) Left ventricle obtained from one nondiseased heart (ND1) and two hearts with ischemic cardiomyopathy (ICM 1,2) was subjected to subcellular fractionation either immediately upon explant (acute) or following a period of storage in liquid nitrogen (frozen). Equal amounts (50 µg) of soluble (S) or particulate (P) protein were loaded onto a gel followed by SDS–PAGE and Western analysis for PCK-. (B) Ponceau staining of the same blot shown in panel A is demonstrated.

 
3.5 Immunohistochemistry
In previous studies of rat heart, PKC isoform expression in pure populations of acutely-isolated ventricular myocytes differed from isoform content of ventricular tissue homogenate, due to contamination of nonmyocyte elements in the latter preparation [1,13,21,22,24]. Similar studies are difficult with human heart due to the very small proportion of viable myocytes obtained using cell dispersion techniques [42,43]. Therefore in order to identify which PKC isoforms were present specifically in human ventricular myocytes, immunohistochemistry was performed on tissue sections of left ventricle obtained from three nondiseased and four diseased hearts as described above. As demonstrated in Fig. 8, all of the PKC isoforms detected by Western analysis were also localized to ventricular myocytes. In appropriate cases, incubation of the antibody with epitope peptide eliminated isoform detection (data not shown). On the other hand, no staining was observed for PKC- or , which were also absent by immunoblotting. While myocyte staining was present for the PKC-βII antibody, this result must be interpreted with caution, given that the most prominent band detected during Western analysis at 83 kDa may not be PKC-βII, as discussed above.

View larger version (149K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Immunohistochemistry of PKC isoforms in human left ventricle. Each panel is labelled according to the PKC isoform antibody (, βI, βII, , , , , , , , and µ) used for immunostaining in that section. The top left panel demonstrates results with pre-immune serum. All samples are from a heart with ischemic cardiomyopathy.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In this study, we have investigated the molecular basis for PKC isoform expression in nondiseased and diseased human ventricle. Using Western analysis, our results demonstrate a large number of isoforms normally present in left and right ventricle, including conventional, novel, and atypical types. To determine which PKC isoforms detected by Western blotting were present specifically in cardiac myocytes, immunohistochemistry was performed, with the localization of all nine isoforms to this cell type.

This work represents the first comprehensive study of PKC isoform expression in human ventricle, with utilization of antibodies directed against all known PKC isoforms. It is known that PKC antibodies from different commercial sources vary widely in their properties, which may account for discrepancies in previous reports of PKC expression in rat heart [27,30]. For this reason, considerable effort was undertaken initially to identify antibodies with optimal sensitivity and specificity, as described in the Methods. In addition, we established a rigorous set of criteria which had to be satisfied before an isoform was scored to be present, including detection of similar-sized bands by two different antibodies, ideally directed against different epitopes, with appropriate peptide block and localization to cardiomyocytes. Potential antibody cross-reactivity was identified, with confirmation of PKC isoform expression using RT-PCR. This exhaustive approach minimized errors in detection due to variability in antibody properties and allowed assignment of expression to cardiac myocytes.

Our experimental results suggest that human cardiac myocytes express a greater variety of PKC isoforms than usually seen in other mammalian species. Numerous investigations have focused on identification of the PKC isoforms in rat heart at various stages of development [13,21–24,26,27,30]. In studies of isolated, enriched populations of adult rat ventricular myocytes, there is general agreement that PKC- and are present [21], and probably PKC- and as well [27,30]. Some investigators have also identified the β isoforms [13,44]. Additional studies in rat and other mammalian species have primarily used total ventricular homogenate for Western analysis. While PKC isoform expression is more diverse under these circumstance, there is also a greater contamination with non-myocyte elements. Using this preparation for dog heart, the PKC- and isoforms have been detected, as well as possibly PKC- and β [1,29]. Similar investigations have identified PKC-, and in hamster [28] and 10 different isoforms in rabbit [8,10,25], while PKC-, , and were detected in guinea pig heart [9]. Although not entirely comprehensive, these studies demonstrate the diversity of PKC isoform expression in the heart.

For human ventricle, our findings are in general agreement with the limited data available from previous studies. Using Western blotting and immunohistochemistry, a recent report identified PKC-, βI, βII, and in nonfailing and failing human left ventricle, while other isoforms were not investigated [31]. Additional information has been derived from studies of tissue other than ventricle. In total homogenate from adult human right atrium [45], the , , and isoforms have been detected. On the other hand, Clerk et al. [23] identified PKC-, , and in total ventricular extract from human fetal (12–24 weeks) and neonatal heart.

Our experimental results demonstrate that in ischemic cardiomyopathy, there was no evidence of an isoform switch in PKC expression. In particular, isoforms which are normally not present, PKC- and , were not upregulated in the remaining viable tissue. Thus although it is well described that a number of fetal genes normally absent in adult tissue can be turned on during disease processes such as hypertrophy [46,47], PKC- and are not targets of this gene expression paradigm.

An important finding in this investigation is the sensitivity of PKC isoforms to degradation when tissue is either frozen or allowed to sit at room temperature (prior to autopsy). The time course of this phenomenon was not studied in detail but was clearly apparent within the duration of these experiments. These findings have major implications for studies in which isoform expression under one condition is compared to another: if all specimens are not processed in a similar manner, differential degradation may occur with confusing or erroneous results.

In summary, we have found that a large repertoire of PKC isoforms are present in both nondiseased and diseased human ventricle, with important methodologic caveats for future studies. The definition of the PKC isoforms present in individual human organs such as heart assumes increasing importance with the active development of isoform-specific inhibitors, which appear to offer promise thus far in animal models of human disease [19,20].

Time for primary review 23 days.

	Acknowledgements

 
This work was supported by a grant (R01 HL55665) from the National Institutes of Health. The immunohistochemistry pictures were processed with help from the Center for Molecular Neuroscience Histology/Imaging Facility.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Steinberg S.F., Goldberg M., Rybin V.O. Protein kinase C isoform diversity in the heart. J Mol Cell Cardiol (1995) 27:141–153.[Web of Science][Medline]
2. Liu J.P. Protein kinase C and its substrates. Mol Cell Endocrinol (1996) 116:1–29.[CrossRef][Web of Science][Medline]
3. Harrington E.O., Ware J.A. Diversity of the protein kinase C gene family: Implications for cardiovascular disease. Trends Cardiovasc Med (1995) 5:193–199.[CrossRef][Web of Science]
4. Mellor H., Parker P.J. The extended protein kinase C superfamily. Biochem J (1998) 332:281–292.[Web of Science][Medline]
5. Puceat M., Vassort G. Signalling by protein kinase C isoforms in the heart. Mol Cell Biochem (1996) 157:65–72.[Web of Science][Medline]
6. Sugden P.H., Bogoyevitch M.A. Intracellular signalling through protein kinases in the heart. Cardiovasc Res (1995) 30:478–492.[Abstract/Free Full Text]
7. Sandhu R., Diaz R.J., Mao G.D., Wilson G.J. Ischemic preconditioning: differences in protection and susceptibility to blockade with single-cycle versus multicycle transient ischemia. Circulation (1997) 96:984–995.[Abstract/Free Full Text]
8. Qiu Y., Ping P., Tang X.L., et al. Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved. J Clin Invest (1998) 101:2182–2198.[Web of Science][Medline]
9. Paul K., Ball N.A., Dorn G.W. II, Walsh R.A. Left ventricular stretch stimulates angiotensin II-mediated phosphatidylinositol hydrolysis and protein kinase C isoform translocation in adult guinea pig hearts. Circ Res (1997) 81:643–650.[Abstract/Free Full Text]
10. Rouet-Benzineb P., Mohammadi K., Perennec J., Poyard M., Bouanani N., Crozatier B. Protein kinase C isoform expression in normal and failing rabbit hearts. Circ Res (1996) 79:153–161.[Abstract/Free Full Text]
11. Newton A.C. Protein kinase C: structure, function, and regulation. J Biol Chem (1995) 270:28495–28498.[Free Full Text]
12. Bareggi R., Narducci P., Grill V., Lach S., Martelli A.M. Selective distribution of multiple protein kinase C isoforms in mouse cerebellar cortex. Biol Cell (1996) 87:55–63.[CrossRef][Web of Science][Medline]
13. Disatnik M.H., Buraggi G., Mochly-Rosen D. Localization of protein kinase C isozymes in cardiac myocytes. Exp Cell Res (1994) 210:287–327.[CrossRef][Web of Science][Medline]
14. Dekker L.V., Parker P.J. Protein kinase C — a question of specificity. Trends Biochem Sci (1994) 19:73–77.[CrossRef][Web of Science][Medline]
15. Jaken S. Protein kinase C isozymes and substrates. Curr Opin Cell Biol (1996) 8:168–173.[CrossRef][Web of Science][Medline]
16. Zhang Z.H., Johnson J.A., Chen L., El-Sherif N., Mochly-Rosen D., Boutjdir M. C2 region-derived peptides of beta-protein kinase C regulate cardiac Ca²⁺ channels. Circ Res (1997) 80:720–729.[Abstract/Free Full Text]
17. Johnson J.A., Mochly-Rosen D. Inhibition of the spontaneous rate of contraction of neonatal cardiac myocytes by protein kinase C isozymes. A putative role for the epsilon isozyme. Circ Res (1995) 76:654–663.[Abstract/Free Full Text]
18. Kiley S.C., Jaken S., Whelan R., Parker P.J. Intracellular targeting of protein kinase C isoenzymes: functional implications. Biochem Soc Trans (1995) 23:601–605.[Web of Science][Medline]
19. Simpson P.C. Beta-protein kinase C and hypertrophic signaling in human heart failure. Circulation (1999) 99:334–337.[Free Full Text]
20. Ishii H., Jirousek M.R., Koya D., et al. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science (1996) 272:728–731.[Abstract]
21. Rybin V.O., Steinberg S.F. Protein kinase C isoform expression and regulation in the developing rat heart. Circ Res (1994) 74:299–309.[Abstract/Free Full Text]
22. Puceat M., Hilal-Dandan R., Strulovici B., Brunton L.L., Brown J.H. Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes. J Biol Chem (1994) 269:16938–16944.[Abstract/Free Full Text]
23. Clerk A., Bogoyevitch M.A., Fuller S.J., Lazou A., Parker P.J., Sugden P.H. Expression of protein kinase C isoforms during cardiac ventricular development. Am J Physiol (1995) 269:H1087–1097.[Web of Science][Medline]
24. Bogoyevitch M.A., Parker P.J., Sugden P.H. Characterization of protein kinase C isotype expression in adult rat heart. Protein kinase C-epsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin. Circ Res (1993) 72:757–767.[Abstract/Free Full Text]
25. Ping P., Zhang J., Qiu Y., et al. Ischemic preconditioning induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res (1997) 81:404–414.[Abstract/Free Full Text]
26. Kohout T.A., Rogers T.B. Use of a PCR-based method to characterize protein kinase C isoform expression in cardiac cells. Am J Physiol (1993) 264:C1350–1359.[Web of Science][Medline]
27. Rybin V.O., Steinberg S.F. Do adult rat ventricular myocytes express protein kinase C-? Am J Physiol (1997) 272:H2485–H2491.[Web of Science][Medline]
28. Cai J.J., Lee H.C. Protein kinase C isozyme-specific modulation of cyclic AMP-dependent phosphodiesterase in hypertrophic cardiomyopathic hamster hearts. Mol Pharmacol (1996) 1:81–88.
29. Domenech R.J., Macho P., Velez D., Sanchez G., Liu X., Dhalla N. Tachycardia preconditions infarct size in dogs: role of adenosine and protein kinase C. Circulation (1998) 97:786–794.[Abstract/Free Full Text]
30. Rybin V.O., Buttrick P.M., Steinberg S.F. PKC-lambda is the atypical protein kinase C isoform expressed by immature ventricle. Am J Physiol (1997) 272:H1636–1642.[Web of Science][Medline]
31. Bowling N., Walsh R.A., Song G., et al. Increased protein kinase C activity and expression of Ca²⁺-sensitive isoforms in the failing human heart. Circulation (1999) 99:384–391.[Abstract/Free Full Text]
32. Webb B.L., Lindsay M.A., Seybold J., et al. Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level. Biochem Pharmacol (1997) 54:199–205.[CrossRef][Web of Science][Medline]
33. Battaini F., Elkabes S., Bergamaschi S., et al. Protein kinase C activity, translocation, and conventional isoforms in aging rat brain. Neurobiol Aging (1995) 16:137–148.[CrossRef][Web of Science][Medline]
34. Wetsel W.C., Khan W.A., Merchenthaler I., et al. Tissue and cellular distribution of the extended family of protein kinase C isoenzymes. J Cell Biol (1992) 117:121–133.[Abstract/Free Full Text]
35. Kosaka Y., Ogita K., Ase K., Nomura H., Kikkawa U., Nishizuka Y. The heterogeneity of protein kinase C in various rat tissues. Biochem Biophys Res Commun (1988) 151:973–981.[CrossRef][Web of Science][Medline]
36. Wrenn C.C., Lappi D.A., Wiley R.G. Threshold relationship between lesion extent of the cholinergic basal forebrain in the rat and working memory impairment in the radial maze. Brain Res (1999) 847:284–298.[CrossRef][Web of Science][Medline]
37. Murray N.R., Fields A.P. Atypical protein kinase C iota protects human leukemia cells against drug-induced apoptosis. J Biol Chem (1997) 272:27521–27524.[Abstract/Free Full Text]
38. Johannes F.J., Prestle J., Eis S., Oberhagemann P., Pfizenmaier K. PKCµ is a novel, atypical member of the protein kinase C family. J Biol Chem (1994) 269:6140–6148.[Abstract/Free Full Text]
39. Kanashiro C.A., Khalil R.A. Signal transduction by protein kinase C in mammalian cells. Clin Exp Pharmacol Physiol (1998) 25:974–985.[Web of Science][Medline]
40. Dutil E.M., Keranen L.M., DePaoli-Roach A.A., Newton A.C. In vivo regulation of protein kinase C by transphosphorylation followed by autophosphorylation. J Biol Chem (1994) 269:29359–29362.[Abstract/Free Full Text]
41. Keranen L.M., Dutil E.M., Newton A.C. Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr Biol (1995) 5:1394–1403.[CrossRef][Web of Science][Medline]
42. Nabauer M., Beuckelmann D.J., Uberfuhr P., Steinbeck G. Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation (1996) 93:168–177.[Abstract/Free Full Text]
43. Wettwer E., Amos G.J., Posival H., Ravens U. Transient outward current in human ventricular myocytes of subepicardial and subendocardial origin. Circ Res (1994) 75:473–482.[Abstract/Free Full Text]
44. Gu X., Bishop S.P. Increased protein kinase C and isozyme redistribution in pressure-overload cardiac hypertrophy in the rat. Circ Res (1994) 75:926–931.[Abstract/Free Full Text]
45. Erdbrugger W., Keffel J., Knocks M., Otto T., Philipp T., Michel M.C. Protein kinase C isoenzymes in rat and human cardiovascular tissues. Br J Pharmacol (1997) 120:177–186.[CrossRef][Web of Science][Medline]
46. Chien K.R., Zhu H., Knowlton K.U., et al. Transcriptional regulation during cardiac growth and development. Annu Rev Physiol (1993) 55:77–95.[CrossRef][Web of Science][Medline]
47. Sugden P.H., Clerk A. Cellular mechanism of cardiac hypertrophy. J Mol Med (1998) 76:725–746.[CrossRef][Web of Science][Medline]

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