© 1996 American Heart Association, Inc.
Articles |
Thyroid Hormone Represses Protein Kinase C Isoform Expression and Activity in Rat Cardiac Myocytes
the Departments of Medicine (S.F.S.) and Pharmacology (V.R., S.F.S.), Columbia University, New York, NY.
Correspondence to Susan F. Steinberg, MD, Associate Professor of Medicine and Pharmacology, Department of Pharmacology, Columbia University, College of Physicians and Surgeons, 630 West 168 St, New York, NY 10032.
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Abstract |
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We have previously demonstrated that at least four isoforms of protein kinase C (PKC;
, 
, 
, and 
) are expressed in neonatal rat ventricular myocytes and that development is associated with a decline in their expression. The mechanism(s) regulating PKC isoform expression in ventricular myocytes is completely unknown. The developmental decline in PKC expression occurs, in large part, during the first 2 weeks of postnatal life, while thyroid hormone levels are known to be progressively increasing. Accordingly, this study examined the influence of thyroid hormone on PKC isoform expression to determine whether thyroid hormone can be implicated as a potential physiological regulator of PKC gene expression during normal cardiac development. Hypothyroidism was induced in adult rats by surgical thyroidectomy; thyroid status was manipulated in cultured neonatal ventricular myocytes by growth in serum-free medium with varying triiodothyronine (T3) levels. In each case, hypothyroidism was verified by a 10- to 50-fold increase in steady state mRNA for ß-myosin heavy chain. In hypothyroid adult ventricular myocardium, there was a selective 60% increase in the expression of PKC protein that corresponded to an increase in maximally stimulated PKC enzyme activity with PKC substrate peptide (pep) but not with histone as substrate. Northern blot analysis revealed a 70% increase in PKC mRNA, indicating that the regulatory effects of thyroid hormone are mediated, at least in part, at the message level. In neonatal ventricular myocytes, there was a T3-dependent reduction in immunoreactivity for both PKC and PKC that was associated with significant reductions in both histone- and pep-kinase activities. The concentration of T3 that half-maximally repressed PKC and PKC expression was 
0.5 nmol/L. Thyroid hormone had no effect on PKC and PKC expression in neonatal or adult ventricular myocytes. PKC isoform expression in cardiac fibroblasts was not influenced by variations in the thyroid hormone concentration during culture. These results provide evidence that thyroid hormone specifically represses PKC and PKC in the neonatal heart and PKC in the adult heart. Thyroid hormone–induced changes in PKC may play an important permissive role in the modulation of autonomic responsiveness in ventricular cardiomyocytes.
Key Words: thyroid hormone • protein kinase C • cardiac myocytes
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Introduction |
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Protein kinase C comprises a family of serine-threonine kinases that play a fundamental role in a wide variety of cellular functions.1 PKC becomes activated in response to receptor-dependent stimulation of membrane phospholipid hydrolysis and the resultant rise in DAG; activation of PKC involves redistribution to the particulate fraction of the cell, followed by downregulation due to proteolytic degradation. Molecular cloning and biochemical studies have revealed a family of structurally related PKC enzymes that can be grouped broadly into three closely related classes (reviewed in Reference 1). The cPKC isoforms (
, ß, and 
) are known as calcium dependent, as their activation by PS and DAG/phorbol esters is enhanced in the presence of calcium. cPKC isoforms contain a calcium-binding domain, a conserved pseudosubstrate sequence, which is presumed to maintain the enzyme in an inactive form in the absence of activator,2 and two adjacent cysteine-rich zinc finger–like motifs, which confer DAG/phorbol ester binding. The nPKC isoforms (, , 
/L, and 
) lack the putative calcium-binding domain and, as a result, do not require calcium for maximal enzymatic activation. aPKC isoforms (, 
/
, and µ) possess only a single copy or excessively widely spaced copies of the cysteine-rich zinc finger–like motif in the phorbol ester–binding/regulatory domain and are not activated by DAG or phorbol esters.3 4 5 6 7 8 9
In cardiac tissue, activation of PKC has been linked to the modulation of various steps in the excitation-contraction coupling process, thereby influencing cardiac rhythm and contractile performance.10 PKC also has been implicated in the regulation of gene expression and the induction of the hypertrophic response in these cells.10 Accordingly, there has been considerable interest in identifying the PKC isoform(s) expressed by cardiac myocytes. Results of original studies characterizing PKC isoform expression in the heart were inconsistent. However, recent studies provide evidence that age-dependent differences in PKC isoform expression may account for at least some of the earlier discrepancies.11 12 For example, several laboratories have presented evidence that calcium-sensitive ( and perhaps ß), novel ( and ), and atypical () PKC isoforms are coexpressed in neonatal rat ventricular myocytes.11 12 13 14 In contrast, nPKC isoforms (PKC and minor amounts of PKC) are detectable in adult rat ventricular myocytes; cPKC and aPKC isoforms generally are not detected in adult myocytes,11 15 although some laboratories have detected PKC in adult cardiac myocytes.12 16 These results have led to the intriguing speculation that individual PKC isoforms may subserve distinct roles in cardiac cell signaling and that developmental changes in PKC isoform expression may underlie age-dependent differences in hormonal modulation of myocyte function.
The molecular mechanism(s) governing PKC isoform expression has received relatively little attention.17 18 19 20 21 Since sympathetic innervation has been implicated in the developmental maturation of certain aspects of myocardial cell function,22 23 24 25 and since the age-dependent decline in PKC isoform expression coincides with the maturation of sympathetic innervation in rat ventricular myocardium, we previously considered a role for sympathetic innervation as a candidate regulator of PKC expression. However, experiments using in vivo and in vitro paradigms to manipulate sympathetic innervation provided evidence that sympathetic innervation of the ventricle is not the dominant influence regulating PKC isoform expression in the heart.10 11 Another obvious important physiological regulator of gene expression in the perinatal period is thyroid hormone. The surge in thyroid hormone levels shortly after birth is believed to play a role in postnatal MHC26 27 28 and Na+,K+-ATPase29 isoform switching. The goal of the present study was to determine whether thyroid hormone also might be implicated in the developmental changes in PKC isoform expression in rat ventricular myocardium.
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Materials
Polyclonal antibodies against PKC
, PKC, and PKC were purchased from GIBCO-BRL, and monoclonal antibodies against PKC were purchased from Seikagaku and Upstate Biotechnology Inc. Polyclonal anti-PKC was the generous gift of Dr Doriano Fabbro (CIBA Geigy, Basel, Switzerland).30 125I-labeled goat anti-rabbit IgG F(ab')2 fragment, 125I-labeled sheep anti-mouse IgG F(ab')2 fragment, and [-32P]ATP (3000 Ci/mmol) were purchased from Du Pont NEN. pep was purchased from Upstate Biotechnology Inc. PMA was purchased from Sigma Chemical Co. All other chemicals were reagent grade.
Tissue and Cell Culture Preparations
Only Wistar rats were used in the present study. Thyroidectomized adult male rats were obtained from Charles River Breeding Laboratories, Inc and were used 4 weeks after surgery. At this time, they showed a profound increase in ß-MHC expression by Northern blot analysis (see Fig 1
). Hearts from anesthetized thyroidectomized rats and control rats were excised quickly and either rinsed in cold physiological saline solution, blotted with filter paper, weighed, and frozen rapidly in liquid nitrogen for subsequent preparation of protein extracts and RNA or used to prepare isolated ventricular myocytes according to methods published previously.31 Nine control and nine thyroidectomized adult rats were used in the present study. Three control and three thyroidectomized adult rats were used for comparisons of PKC isoform protein and mRNA expression in ventricular myocardial tissue. Because of limitations in tissue availability, a separate set of three control and three thyroidectomized adult rats was used for comparisons of PKC enzyme activity in ventricular myocardial tissue. Finally, a third set of three control and three thyroidectomized adult rats was used for comparisons of PKC isoform protein in myocytes isolated from the intact ventricle. However, immunoblot analyses verified comparable effects of thyroid hormone in each set of experimental animals (see Figs 1, 3, and 8).
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Cardiac myocytes were isolated from hearts of 2-day-old rats by a trypsin dispersion procedure according to a protocol that incorporates a differential attachment procedure to enrich for cardiac myocytes as described previously.24 Cells were suspended in MEM containing 5x10-6 mol/L hypoxanthine, 12 mmol/L NaHCO3, and 10% FCS, then preplated onto 100-mm culture dishes, and incubated at 37°C in a humidified atmosphere (5% CO2/95% air) for 1 hour. During this interval, the majority of the myocytes remain in suspension while nonmyocyte fibroblast-like cells preferentially attach to the surface of the culture dish. Subsequently, unattached cells were pelleted and resuspended in serum-free medium (1:1 DMEM/F-12 medium, GIBCO-BRL) supplemented as described by Mohamed et al32 with 4 µL/mL MEM amino acids, 2 µL/mL MEM vitamin solution (100x), 0.025% fetuin, 2.45 mg/mL NaHCO3, 0.1 mg/mL glucose, 20 µg/mL gentamicin, 1% BSA, 0.02 mg/mL ascorbic acid, 0.08 mg/mL calcium chloride, 50 µU/mL insulin, 5 µg/mL transferrin, 10 ng/mL sodium selenate, 0.012% BSA-palmitate complex, 1 ng/mL epidermal growth factor, 50 ng/mL hydrocortisone, and T3, which was added from a 1 mmol/L stock solution in 10 mmol/L NaOH to achieve a final concentration of 10-12 or 10-8 mol/L. The final yield of cells was 2.5 to 3x106 per neonatal heart. Cells were plated at a density of 0.5x106 cells per milliliter (10 mL per dish) onto 100-mm culture dishes that had been coated with fibronectin. The myocytes were used for experiments after 6 days in culture. Nonmyocyte fibroblast-like cells, derived from the cells that adhered to the plastic culture dishes during the preplating step, also were studied in some experiments. In this case, the cells were grown on 100-mm culture dishes for 6 days in the presence of serum-free medium containing 10-12 or 10-8 mol/L T3.
Studies of PKC Expression
Total cell extracts from ventricular myocardial tissue, isolated adult ventricular myocytes, and cultured neonatal rat ventricular myocytes were prepared as described previously.11 Briefly, ventricular myocyte cultures were washed with PBS and then immediately lysed in preheated (95°C) homogenization buffer (20 mmol/L Tris-HCl, pH 7.5, 2 mmol/L EDTA, 2 mmol/L EGTA, 6 mmol/L ß-mercaptoethanol, 50 µg/mL aprotinin, 48 µg/mL leupeptin, 5 µmol/L pepstatin A, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L sodium vanadate, and 50 mmol/L NaF) containing 1% SDS and homogenized by sonication. Similarly, total protein extracts were prepared from intact cardiac tissues by adding preheated homogenization buffer containing 1% SDS to minced myocardium (12 mL/g tissue), followed by homogenization with a Polytron tissue homogenizer. Protein content was determined according to a modified Lowry assay.
Immunoblot Analysis
Samples were electrophoresed on an 8% SDS-polyacrylamide gel and transferred to nitrocellulose. Prestained molecular weight markers were electrophoresed in parallel. After an incubation in 5% dry milk, 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, and 0.1% Triton-X 100 (blocking buffer I) for 1 hour at room temperature to block nonspecific binding, the nitrocellulose was probed with a 1:500 dilution of primary PKC isoform–specific antisera in 3% BSA, 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 0.1% Triton-X 100, and 0.02% NaN3 overnight at 4°C. Four PKC isoform–specific antisera were used. These antisera were generated against synthetic peptides corresponding to amino acids 313 to 326 for PKC or unique sequences in the carboxy-terminal variable region of PKC, PKC, and PKC. However, it should be noted that the anti-PKC has been shown to also recognize PKC.7 33 Moreover, additional atypical isoforms of PKC, which are structurally highly homologous to PKC in the carboxy-terminal end of the molecule have been identified recently (PKC/5 34 ). Given that the anti-PKC antiserum has been reported to cross-react with these other aPKC isoforms,5 34 the identification of the protein detected with this antibody as PKC is tentative at this time. The nitrocellulose was washed five times, 5 minutes each, with 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, and 2% Nonidet P-40 and incubated in the same buffer containing 5% dry milk (blocking buffer II) for 30 minutes at room temperature. To detect bound primary antibody, blots were incubated for 1 hour at room temperature with 125I-labeled goat anti-rabbit IgG F(ab')2 fragment at a final dilution of 0.25 µCi/mL in blocking buffer II. The nitrocellulose was washed seven times as described above, dried, and autoradiographed with Kodak XAR film with intensifying screens at -70°C. The specificity of all immunoreactive proteins was established previously by immunoblot analysis in the presence and absence of competing immunizing peptide.11 Preliminary experiments also established that staining intensity is linear with protein loading for each PKC isoform, validating the comparison of PKC isoform expression in the present study. PKC was clearly resolved as a protein doublet in some experiments (eg, see Fig 5). This is known to represent variability in the extent of PKC phosphorylation35 ; both molecular weight species of the enzyme were combined for the analysis of the relative abundance of PKC. For autoradiograms in which the densities of the bands linearly increased with loading of increased amounts of protein, the relative abundance of individual proteins identified was quantified by scanning densitometry.
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PKC Enzyme Assay
PKC was partially purified from the soluble extract of myocardial cells by DEAE-Sephacel column chromatography. All steps were carried out at 4°C. Myocytes grown in 100-mm tissue culture dishes were washed two times with PBS, scraped into 0.4 mL per dish homogenization buffer (2 mmol/L EGTA, 2 mmol/L EDTA, 5 µg/mL leupeptin, 1 µg/mL aprotinin, 0.25 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L pepstatin A, 1 mmol/L dithiothreitol, 0.1 mmol/L sodium vanadate, and 20 mmol/L Tris, pH 7.5), and sonicated. Intact ventricular myocardial tissue was homogenized with a Polytron in the same buffer (100 mg/mL). Homogenates were centrifuged at 100 000g for 1 hour at 4°C, and supernatants (soluble fractions) were subjected to chromatography on a DEAE-Sephacel (l-mL column bed). Samples (1 mg) were applied to a column that had been equilibrated in homogenization buffer. After sample application, the column was washed with 10 mL buffer A (2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L dithiothreitol, and 20 mmol/L Tris, pH 7.5), and PKC was eluted with 2 mL buffer A containing 250 mmol/L NaCl. The first 0.5 mL of the eluate was discarded, and the subsequent 1.5 mL was stored at -70°C in the presence of 10% glycerol.
PKC activity was measured with substrate (0.5 mg/mL histone or 50 µmol/L pep) and 1.5 µg sample protein in a reaction mixture containing 27 mmol/L Tris, pH 7.5, 5 mmol/L MgCl2, 0.67 mmol/L EGTA, 0.67 mmol/L EDTA, 0.5 µmol/L PKI, and 0.4 mmol/L dithiothreitol in the absence and presence of 80 µg/mL PS, 100 ng/mL PMA, and excess calcium as indicated in individual experiments. pep is a synthetic peptide that corresponds to the pseudosubstrate site of PKC, but with a phosphorylatable serine for alanine substitution (ERMRPRKRQGSVRRRV). Preliminary experiments established that the reactions were linear with time and enzyme concentration and identified calcium-to-EGTA ratios optimal for maximal calcium-dependent activation of histone and pep phosphorylation (in the presence and absence of PMA). Calcium-sensitive histone-kinase activity was assayed in the presence of 3.33 mmol/L calcium. However, this concentration was found to exert an inhibitory effect on PKC enzyme activity when pep was used as the substrate, consistent with previous evidence for substrate-dependent inhibitory effects of calcium on PKC enzyme activity.36 Therefore, calcium-stimulated pep-kinase activity was measured at a lower concentration of calcium (0.5 mmol/L). Reactions were initiated by the addition of [-32P]ATP (4 µCi, 66 µmol/L) and were performed in triplicate at 30°C for 10 minutes. Assays were terminated by spotting 40 µL of the reaction mixture onto phosphocellulose filter papers (P-81), which were immediately dropped into water. After four 5-minute washes with water, the filters were counted for radioactivity.
Preparation and Electrophoresis of RNA
Total cellular RNA was isolated from intact adult ventricular myocardium or cultured neonatal myocytes by the method of Chomczynski and Sacchi.37 Total RNA yields were 826±47 and 852±40 µg per adult rat heart for euthyroid and hypothyroid preparations, respectively. Poly(A+)-enriched RNA was isolated by passing 380 µg of total RNA over oligo(dT) cellulose (GIBCO-BRL). The yield of poly(A+)-enriched RNA recovered from euthyroid and hypothyroid preparations was similar. Total or poly(A+)-enriched RNA was separated by electrophoresis on 1% agarose-formaldehyde gels. RNA was transferred to a nylon membrane and covalently attached using UV cross-linking. Two oligonucleotide probes were used in the present study. Levels of ß-MHC mRNA were measured using a synthetic 30-base oligonucleotide probe complementary to a unique sequence in the 3'-untranslated region of ß-MHC,38 which was kindly provided by Dr Richard Kitsis (Albert Einstein College of Medicine, Bronx, NY). Hybridization to total RNA was carried out in 50 mmol/L NaH2PO4, pH 7.4, 5x SSC, 1x Denhardt's solution, 2% SDS, and 0.2 mg/mL denatured salmon sperm DNA overnight at 55°C. The final wash stringency was at 55°C with 1x SSC and 0.1% SDS, five times for 10 minutes. Levels of PKC mRNA were measured using a 35-base oligonucleotide probe complementary to a portion of the V3 region of PKC essentially according to manufacturer's instructions (GIBCO-BRL). Hybridization was to poly(A+)-enriched RNA and was carried out in 50% formamide, 5x SSPE, 5x Denhardt's solution, 0.1% SDS, and 0.2 mg/mL denatured salmon sperm DNA overnight at 37°C. To correct for minor differences in loading and/or transfer, the blot was stripped and rehybridized with a GAPDH cDNA probe (provided by Dr Paul Rothman, Columbia University, New York, NY). After autoradiography at -70°C with Kodak XAR-5 film backed by intensifying screens, quantification was performed by proportional counting using a Betascope model 603 blot analyzer (Betagen Corp).
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Results |
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Effects of Thyroid Hormone on PKC Isoform Expression in Cardiac Myocytes
Initial experiments relied on the well-known ability of thyroid hormone to modulate ß-MHC gene expression to verify the hypothyroid state of the surgically thyroidectomized rats. Fig 1
(bottom) illustrates that ß-MHC mRNA abundance is markedly increased in the hypothyroid rats (50-fold compared with the euthyroid controls). Accordingly, we determined whether thyroid hormone also modulates PKC isoform expression in these rat ventricular myocardial preparations. Our previous studies established that neonatal rat ventricular myocytes coexpress PKC, PKC, PKC, and PKC but that PKC is the predominant isoform of PKC detected in adult rat ventricular myocardial tissue. These results are confirmed by the immunoblot illustrated in Fig 1 (top). The neonatal preparation contained immunoreactivity for each of the PKC isoforms tested (, , , and ). In contrast, there was abundant PKC immunoreactivity but only trace amounts of PKC, PKC, and PKC immunoreactivity in whole extracts from adult ventricular myocardial tissue. For each PKC isoform, immunoreactivity in the neonatal preparation exceeded that detected in the adult preparation.11 Levels of PKC, PKC, and PKC in adult ventricular myocardial tissue were not influenced by thyroid hormone (Figs 1 and 2). In contrast, PKC immunoreactivity was 60% higher in the extracts from hypothyroid rats compared with the euthyroid controls (Figs 1 and 2).
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To verify that thyroid hormone represses PKC in cardiac myocytes (rather than other contaminating cellular elements in the ventricle), we performed immunoblot analyses on extracts prepared from myocytes isolated from euthyroid and hypothyroid ventricles. Fig 3 illustrates that hypothyroidism is associated with a 2.3-fold increase in PKC in this preparation. Levels of PKC were not influenced by thyroid hormone (data not shown). Using a polyclonal anti-PKC antiserum from GIBCO-BRL, PKC immunoreactivity was not convincingly detectable in euthyroid adult ventricular myocytes and did not become detectable in the hypothyroid preparation. However, in view of recent reports from other investigators that PKC immunoreactivity is present in extracts from isolated adult rat ventricular myocytes,12 we also used two other anti-PKC antisera to optimize our ability to detect an effect of thyroid hormone to influence PKC expression. Using monoclonal antibodies to PKC from Seikagaku and Upstate Biotechnology Inc, prominent immunoreactive bands that were similar in size to PKC were detected in similar abundance in extracts from euthyroid and hypothyroid ventricular myocytes. However, in each case, the subcellular fractionation of the immunoreactivity was not influenced by phorbol esters (under conditions in which phorbol ester–induced translocation of PKC from the soluble to the particulate fraction was evident; data not shown). The absence of the requisite phorbol ester sensitivity constitutes evidence that the prominent immunoreactive species identified by the Seikagaku and Upstate Biotechnology Inc antisera are nonspecific and not PKC. Thus, although it still is possible that thyroid hormone induces changes in PKC that are beneath the limits of detection with three commercially available antisera, these results are most consistent with the conclusion that (in the context of the four isoforms examined in this study) the effects of thyroid hormone to influence PKC in adult ventricular myocytes are confined to the isoform.
To determine whether changes in the abundance of PKC protein are associated with changes in steady state levels of PKC mRNA, Northern blot hybridization analysis was carried out. An oligonucleotide probe complementary to PKC identified a 7.5-kb transcript in euthyroid and hypothyroid adult rat ventricular myocardial preparations.39 The hybridization signal for PKC obtained with this probe was 70% higher in hypothyroid than in euthyroid rat ventricular myocardial preparations (Fig 4). These results indicate that thyroid hormone acts, at least in part, at the message level to regulate PKC.
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The next series of experiments examined whether thyroid hormone regulates PKC isoform expression in primary cultures of neonatal rat ventricular myocytes. Studies in this in vitro system were considered critical in eliminating the influence of secondary changes induced by hypothyroidism in the intact animal (such as changes in the secretion of other hormones and neurotransmitters as well as changes in cardiovascular hemodynamics) and isolating the direct regulatory effects of thyroid hormone on PKC isoform expression in cardiac myocytes. For these experiments, cells were maintained in a serum-free chemically defined medium with either 10-8 or 10-12 mol/L T3. These concentrations of T3 were chosen to maximally stimulate (10-8 mol/L) or not influence (10-12 mol/L) thyroid hormone-responsive gene expression28 29 and straddle the reported value for plasma-free T3 in the euthyroid rat (1.5 nmol/L40 ). Initial experiments verified the thyroid hormone responsiveness of these cultures, since ß-MHC mRNA was markedly repressed in cultures grown for 6 days in the presence of 10-8 mol/L T3 (10-fold compared with cultures grown in the presence of 10-12 mol/L T3, Fig 5B). Having established that myocyte cultures are amenable to studies of the regulatory actions of thyroid hormone, we immunoblotted whole protein extracts from these preparations with PKC isoform–specific antisera (Fig 5A). The levels of PKC and PKC were 73±7% and 68±14% higher in myocytes maintained for 6 days in serum-free medium with 10-12 mol/L T3 than in cultures maintained for a similar time interval with 10-8 mol/L T3 (n=10, P<.05). In contrast, levels of immunodetectable PKC and PKC did not differ between these preparations (ie, 10-12 mol/L T3 was associated with trivial increases in the level of expression of PKC [9±8%] and PKC [8±8%], n=10, P=NS).
The time course for the T3-dependent changes in PKC and PKC expression in cultured neonatal ventricular myocytes was examined. To the best of our knowledge, the effects of culturing cardiac myocytes on PKC isoform expression have not been examined previously. Therefore, it was necessary to consider the possibility that PKC isoform expression might not be stable during the first few days of culture, particularly since culture-dependent changes in the expression of other signaling proteins (ie, Go) have been described previously.41 Apart from actual culture-dependent changes in protein expression, there is a rather substantial increase in cell size (signaled by the 2- to 2.5-fold increase in protein recovery observed in Fig 6, bottom) that occurs during the first few days of culture that must also be considered. These factors introduce an element of uncertainty regarding the optimal denominator to use to track the time course for T3-dependent changes in PKC expression in culture. Rather than attempt to correct for culture-dependent changes in protein recovery that may influence the measurements, we elected to assay PKC isoform immunoreactivity in a constant amount of protein extract from each sample. Accordingly, PKC isoform immunoreactivity is expressed in Fig 6 according to the standard denominator used in the literature (ie, PKC per milligram protein). However, it is clear that the changes in PKC isoform expression must be interpreted in the context of the progressive time-dependent increase in protein recovery that, importantly, is not influenced by T3 (Fig 6). Immunoreactivity for PKC (and to a lesser extent PKC) increased during the first 4 to 5 days of culture in the presence of 10-12 mol/L T3 (Fig 6, top and middle). Immunoreactivity for PKC and PKC also increased modestly (23% and 52%, respectively) during the first 4 days of culture in the presence of 10-12 mol/L T3 (data not shown). Whether these increases in PKC isoform immunoreactivity actually underestimate true culture-dependent increases in PKC isoform expression per myocyte (in view of the time-dependent increase in protein recovery) requires further study. Nevertheless, Fig 6 illustrates that the culture-dependent increase in PKC immunoreactivity was attenuated in the presence of 10-8 mol/L T3 and that PKC immunoreactivity actually decreased in the presence of 10-8 mol/L T3. PKC and PKC immunoreactivity were not influenced by T3 at any time point (data not shown). The effect of T3 to reduce PKC and PKC expression was not evident on day 1 but became detectable after 2 days of culture. T3-dependent reductions in PKC and PKC expression were maximal by 4 to 5 days of culture and persisted for at least an additional 4 days. The time course for T3-dependent changes in PKC and PKC appeared to be quite similar.
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) or 10-8 mol/L T3 (
) for the indicated time intervals. Whole-cell extracts were prepared and subjected to SDS-PAGE and immunoblot analysis with PKC isoform–specific antibodies as described in "Materials and Methods." Immunoreactivity for PKC
To determine the dose-response relationship for the effect of T3 on PKC isoform expression, myocytes were grown in the presence of a range of T3 concentrations. Fig 7 illustrates that PKC and PKC decrease as a function of increasing T3 concentration with similar dose-response characteristics. The concentration of T3 that half-maximally represses PKC and PKC expression is 0.5 nmol/L; this is similar to the value previously reported for T3 modulation of Na+,K+-ATPase29 42 and MHC28 gene expression in cultured rat ventricular myocytes. This concentration is well within the reported concentration range for plasma-free T3 levels in the euthyroid developing rat.40
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Myocyte cultures grown in serum-free chemically defined medium contain a minor population of contaminating nonmyocyte fibroblast-like cells. We considered the possibility that the measured changes in PKC isoform expression are attributable to a large increase in PKC isoform expression in this minor population of contaminating cells. Therefore, additional experiments were performed to determine whether thyroid hormone represses PKC isoform expression in cultures enriched for cardiac fibroblasts. In agreement with a previous study, these highly enriched cultures of cardiac fibroblasts expressed PKC, PKC, PKC, and PKC immunoreactivity.11 However, PKC isoform expression was not influenced by T3 added to the culture medium (Fig 8). These results argue that the differences in PKC and PKC immunoreactive protein detected in myocyte cultures must be attributed to an effect of thyroid hormone to repress PKC expression in cardiac myocytes.
Effects of Thyroid Hormone on PKC Enzyme Activity in Adult Ventricular Myocardium and Cultured Neonatal Rat Ventricular Myocytes
As a first approach to understand the functional significance of thyroid hormone–dependent changes in PKC expression in ventricular myocardium, PKC enzyme activity was measured. The soluble fraction from euthyroid and hypothyroid ventricular myocardium was used in these experiments. We reasoned that this preparation would optimize our ability to detect changes in PKC enzyme activity that arise as a result of changes in the abundance of only a subset of the PKC isoforms expressed in the tissue since (1) all of PKC and much of PKC are recovered as soluble proteins from unstimulated cells homogenized in the presence of divalent cation chelators (PKC, which is not influenced by thyroid hormone and is highly associated with the particulate fraction, is largely eliminated from the samples by the subfractionation) and (2) preliminary studies indicated that the thyroid hormone–dependent difference in PKC immunoreactivity is most pronounced in the soluble fraction prepared from adult rat ventricular myocardium (data not shown). Fig 9A establishes that the difference in PKC immunoreactivity induced by thyroid hormone is retained in samples subjected to DEAE chromatography to enrich for PKC. Accordingly, PKC activity was measured with histone and pep as substrates and a series of activation conditions. Given the distinct substrate specificities and cofactor requirements of individual PKC isoforms, this strategy was designed to optimally discriminate cPKC and nPKC enzyme activity (Fig 9B). We detected ample calcium-sensitive histone-kinase activity in the adult ventricular myocardial preparations. This was contrary to our expectation, since histone is a poor substrate for calcium-insensitive isoforms of PKC, such as PKC, the predominant isoform of PKC detected by immunoblot analysis in adult rat ventricular myocardium. Nevertheless, consistent with immunoblotting experiments demonstrating that in vivo hypothyroidism does not influence PKC immunoreactivity in adult rat ventricular myocardium, PKC enzyme activity with histone as substrate was similar in preparations from the euthyroid and hypothyroid ventricle.
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In contrast, a different result was observed when pep was included as substrate in the assay (Fig 9B). This peptide is the preferred substrate for calcium-insensitive PKC isoforms but also can be phosphorylated in a calcium-dependent fashion by cPKC isoforms. Thus, it is not entirely surprising that both calcium-sensitive and calcium-insensitive PKC enzyme activity was detected with pep as substrate. PKC activity with pep as substrate was significantly reduced in the euthyroid preparation relative to the hypothyroid preparation when measured with PS alone or when maximally stimulated with PS/calcium/PMA. Although pep activity with PS/PMA also tended to be lower in the euthyroid preparation relative to the hypothyroid preparation, this difference was not statistically significant. Given that differences in nPKC isoform expression would be predicted to be accompanied by differences in pep-kinase activity with both PS/calcium/PMA and PS/PMA, the explanation for the absence of a statistically significant difference between euthyroid and hypothyroid preparations in pep-kinase activity with PS/PMA is not obvious. Nevertheless, the effect of thyroid hormone to modulate maximally stimulated pep-kinase, but not histone-kinase, activity is most consistent with an effect of thyroid hormone to modulate a nPKC isoform and agrees with the results of immunoblotting experiments.
Using a similar strategy, soluble fractions from neonatal rat ventricular myocytes cultured in serum-free chemically defined medium with 10-8 or 10-12 mol/L T3 were partially purified by DEAE chromatography, and PKC immunoreactivity and enzyme activity were assessed. The goal was to determine whether the T3-dependent changes in PKC and PKC immunoreactivity correlate with changes in histone- and pep-kinase activities. Fig 10A illustrates that T3-dependent differences in PKC and PKC immunoreactivity were retained during partial purification of PKC. Fig 10B reports histone- and pep-kinase activities in these preparations. Substantial calcium-dependent PKC activity with histone as substrate was detected. Consistent with the observation that growth in the presence of 10-8 mol/L T3 reduces the level of immunodetectable PKC, calcium-sensitive histone-kinase activity was significantly reduced in myocytes cultured for 6 days with 10-8 mol/L T3.
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Neonatal ventricular myocytes are enriched in PKC, PKC, and PKC relative to adult ventricular myocytes. Thus, the striking disparity between the maximal pep-kinase activity in neonatal and adult preparations was not surprising (compare Figs 9 and 10). Maximal pep-kinase activity markedly exceeded maximal histone-kinase activity in the neonatal preparation. Under all assay conditions, PKC enzyme activity with pep as substrate was lower in preparations from myocytes grown in the presence of the higher concentration of T3 compared with myocytes grown in the presence of 10-12 mol/L T3. These results constitute additional evidence that thyroid hormone acts as a negative modulator of both calcium-sensitive PKC and calcium-insensitive PKC in neonatal ventricular myocardial tissue. Moreover, despite rather high background levels of PKC enzyme activity due to the copresence of other PKC isoforms (PKC and PKC), which contribute to total PKC enzyme activity and are not influenced by T3, thyroid hormone–dependent changes in PKC and PKC significantly modulate total PKC enzyme activity in neonatal ventricular myocardial tissue.
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Discussion |
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Recent studies provide evidence that multiple isoforms of PKC are expressed simultaneously in cardiac myocytes in an age-dependent fashion; cPKC isoforms (
and perhaps ß) and the aPKC are confined to fetal and neonatal myocytes, whereas the nPKC isoforms predominate in adult myocytes.11 15 Initial studies demonstrating that the developmental decline in PKC expression (around the time of birth) precedes the decline in PKC and PKC expression (during early postnatal life) provided the first evidence that multiple factors must regulate PKC isoform expression in ventricular myocytes. The observation that the decline in PKC precedes the postnatal surge in thyroid hormone secretion made thyroid hormone an unlikely candidate as a physiologic modulator of PKC expression. Accordingly, the studies reported herein were designed to test the hypothesis that thyroid hormone acts as a negative modulator of PKC, PKC, and/or PKC expression in ventricular myocytes. The most important finding of the present study is that certain PKC isoforms are influenced by thyroid hormone. Thyroid hormone represses PKC and PKC expression in neonatal ventricular myocytes, suggesting that the postnatal changes in thyroid status may play a role in the developmental decline in PKC and/or PKC expression in ventricular myocytes.
PKC was the only isoform that was highly sensitive to thyroid hormone manipulations in both neonatal and adult ventricular myocytes. The coordinate increase in PKC protein and mRNA in the hypothyroid adult ventricle is consistent with the notion that thyroid hormone acts at a transcriptional level to influence PKC. This result could suggest that the promoter region of the PKC gene contains a thyroid hormone–responsive element(s), although thyroid hormone–dependent translational and/or posttranslational events also have been described and must be considered in future studies.29 43 Thyroid hormone acts as a negative modulator of PKC expression and enzyme activity in vivo in the intact adult ventricle as well as in primary cultures of neonatal ventricular myocytes grown in serum-free chemically defined medium. The results in cultured myocytes are particularly important as they provide evidence that thyroid hormone directly regulates PKC expression; these results rule out the need to postulate a significant indirect effect of thyroid hormone that might be mediated by changes in hemodynamics, metabolism, or other mechanisms in the intact animal model.
Thyroid hormone also regulates PKC expression but apparently in a developmental stage–specific fashion. Although thyroid hormone modulates PKC immunoreactivity and enzyme activity in neonatal myocytes, there was no evidence that PKC is influenced by thyroid hormone in the intact adult ventricle, where PKC appears to reside in a non–cardiomyocyte-contaminating cell population(s).11 We would postulate that there is stable repression of PKC in adult ventricular myocytes in a manner that makes it unresponsive to changes in thyroid status. In this regard, developmental stage–specific regulatory actions of thyroid hormone have been reported,44 although the molecular mechanisms that underpin this phenomenon currently are uncertain.
Nonmyocyte fibroblast-like cells coexpress PKC, PKC, PKC, and PKC, but in contrast to cardiomyocytes, these were not regulated by thyroid hormone under the conditions used in the present study. Cardiac fibroblasts have been reported to contain high-affinity nuclear T3 receptors as well as mRNAs for c-erbA and c-erbAß, which encode T3-binding proteins and regulate the expression of T3-responsive genes.45 Thus, it is possible that thyroid hormone can modulate PKC isoform expression in fibroblasts, but our conditions were not optimal to discern such an effect. We are not aware of genes that could have been monitored as positive controls to verify that the cardiac fibroblasts retained thyroid hormone responsiveness in culture; in the absence of such markers for thyroid hormone action in fibroblasts, the conclusion that thyroid hormone does not influence PKC isoform expression in fibroblasts may be premature. However, this uncertainty does not undermine our conclusion that changes in PKC and PKC immunoreactivity observed in neonatal rat ventricular myocyte cultures cannot be attributed to a large effect occurring in a minor contaminating cell population.
Previous studies using immunoblot analysis to compare PKC isoform expression in fetal, neonatal, and adult ventricular myocardium reported a dramatic decline in PKC and PKC and a modest decline in PKC immunoreactivity during the first 2 weeks of postnatal life. Results reported herein constitute the first comparison of PKC enzyme activity in neonatal and adult ventricular myocytes. The markedly higher level of maximal pep-kinase activity in the neonatal than in the adult preparation provides independent confirmation that development is associated with a decline in PKC. Given that the enzymatic activity of purified PKC isoforms can be distinguished on the basis of their substrate specificities and cofactor requirements, we attempted to use this strategy to compare PKC immunoreactivity and enzyme activity in neonatal and adult preparations. However, the comparison was not entirely straightforward. For example, PKC immunoreactivity vastly exceeded PKC immunoreactivity in adult ventricular myocardium. Yet maximal pep-kinase activity was only slightly higher than maximal histone-kinase activity. We propose several mechanisms to reconcile the discrepancy between PKC immunoreactivity and enzyme activity. First, immunoblot analyses were performed on total protein extract, whereas enzyme activity was assayed on PKC that partitions to the soluble fraction. The soluble fraction of unstimulated cells is enriched in PKC (which resides exclusively in this fraction) relative to PKC, PKC, and PKC (which distribute between the soluble and particulate fractions of unstimulated cells). Second, recent studies indicate that the chromatographic fractions used for PKC enzyme assays may be further enriched in PKC relative to other isoforms of PKC (data not shown). Third, several laboratories have identified endogenous inhibitors of PKC enzyme activity that coelute with PKC during typical chromatographic procedures and interfere with the in vitro analysis of PKC activity.46 47 48 Although to the best of our knowledge isoform-specific inhibition has not been reported, it remains a possibility. Fourth, it is possible that PKC immunoreactivity in adult ventricular myocardial preparations is prominent compared with other isoforms, because the PKC antiserum is highly sensitive and can detect low levels of this protein. Studies that correct for potential differences in titer and hybridization efficiencies between the individual antisera will be required to address this issue. Finally, although assays of PKC enzyme activity with histone and pep as substrates provide a useful approach to distinguish the enzymatic activity of calcium-sensitive and -insensitive PKC isoforms, these cannot be quantitatively precise under conditions in which the enzymatic activities of multiple PKC isoforms are measured simultaneously. Nevertheless, we identified an increase in maximally stimulated pep-kinase, but not histone-kinase, activity that correlated with an increase in PKC immunoreactivity in hypothyroid adult ventricular myocardial preparations. Although the thyroid-dependent difference in maximally stimulated pep-kinase activity was relatively modest (certainly compared with the differences in -pep kinase activity between neonatal and adult preparations), pep-kinase activity measures all PKC isoforms in the preparation and will underestimate a change that is confined to a single isoform. Given the recent evidence that individual PKC isoforms are targeted to distinct intracellular sites in cardiac myocytes,13 the possibility that the reduction in PKC induced by thyroid hormone is substantial at a distinct intracellular site(s) must be considered in future studies. This may be particularly pertinent, given the recent evidence that PKC plays a role in the modulation of automaticity, the regulation of intracellular calcium homeostasis, and the activation of MAPK in cardiac myocytes.49 50 T3 also reduced PKC and PKC immunoreactivity (as well as histone- and pep-kinase activities) in neonatal myocytes. Thus, the results of the studies reported herein provide consistent evidence that thyroid hormone influences PKC expression in neonatal and adult rat ventricular myocardium but that the effects of thyroid hormone to influence PKC are confined to the neonatal ventricle.
In conclusion, results of the present study implicate thyroid hormone as a potentially important physiological modulator of PKC expression in ventricular myocytes. Given the recent evidence that stable alterations in the expression of individual PKC isoforms can lead to disturbances in the growth and hormone responsiveness of other noncardiac cell types51 52 53 and the integral role of PKC in the regulation of numerous cardiac cell functions, thyroid hormone–dependent changes in PKC are likely to be associated with important physiological ramifications. The challenge of future studies will be to assign to individual isoforms of PKC specific functions in intracellular signal transduction. This will facilitate efforts to define the physiological and/or pathological implications of thyroid hormone–induced changes in the expression of specific PKC isoforms in ventricular myocardial tissue.
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This study was supported by US Public Health Service, National Heart, Lung, and Blood Institute grant HL-28958.
Received October 25, 1995; accepted May 10, 1996.
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