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Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Caryn J McGill , Gavin Brooks
DOI: http://dx.doi.org/10.1016/S0008-6363(97)00041-2 368-376 First published online: 1 May 1997

Abstract

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Keywords
  • 80K/MARCKS
  • Cardiac myocyte
  • Development
  • Expression
  • Protein kinase C
  • Rat

Time for primary review 31 days.

1 Introduction

The protein kinase C (PKC) superfamily comprises a group of serine/threonine kinases which are able to phosphorylate a myriad of physiological substrates and are thought to play a pivotal role in the regulation of cell growth and differentiation in many cell types [1, 2]. The PKC family consists of at least 12 distinct isoforms, which can be sub-divided further into 3 groups according to their co-factor requirements for activation: (1) calcium/phospholipid-dependent or conventional PKC's (α, βI, βII and γ); (2) calcium-independent or novel PKC's (δ, ϵ, η, θ); and (3) atypical PKC's (ζ, λ, μ and ι) [3, 4]. Recently, a number of groups have reported the expression of PKC α, δ, ϵ and ζ isoforms in cardiac tissue [5–8]. Furthermore, Kohout and Rogers [9]have detected PKC α, δ, ϵ, η and ζ transcripts in both neonatal and adult cultured cardiac myocytes using reverse transcriptase-polymerase chain reaction (RT-PCR) amplification. In addition to having a central role in cellular growth control, activation of PKC has been shown to mediate the induction of hypertrophy in neonatal cardiac myocytes, through an up-regulation of immediate early genes such as c-fos, c-myc, Egr-1 and the induction of a specific cardiac gene programme producing, among others, myosin light chain, atrial natriuretic factor and β-myosin heavy chain [10]. However, the intracellular events which occur immediately downstream of PKC activation in cardiac tissue (e.g., specific substrate phosphorylation) have not been well characterised in the heart, although it has been proposed that PKC may activate the mitogen-activated protein kinase cascade in response to hypertrophic stimuli in cardiac myocytes [7].

The myristoylated alanine-rich C-kinase substrate (80K/MARCKS), which migrates with an apparent Mr of 80 kDa by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE), is a specific in vivo and in vitro substrate of PKC and recently all conventional and novel PKC isoforms have been shown to phosphorylate this protein in vitro [11]. Accordingly, phosphorylation of 80K/MARCKS by PKC has been used as a useful marker of PKC activation [12, 13]. A physiological role for this ubiquitous protein remains to be determined although we have shown recently that it may play a role in cellular proliferation and tumorigenesis [14, 15].

In the present study, we have determined the expression of 80K/MARCKS mRNA and protein during development of the rat myocardium, using both whole ventricular tissue and isolated cardiac myocytes. In addition, we have studied the possible regulation of 80K/MARCKS protein expression by PKC activation during development.

2 Methods

2.1 Materials

α-32P-dCTP (3000 Ci/mmol), γ-32P-ATP (3000 Ci/mmol), 125I-protein A (703 kBq/μg), donkey anti-rabbit Ig, horseradish peroxidase-conjugated antibody, ECL Western Blotting reagents and the PKC assay kit were purchased from Amersham International PLC (UK). Dulbecco's Modified Eagle's Medium (DMEM) with Glutamax, M199 medium, pancreatin, penicillin/streptomycin (1000 units/ml) and fetal calf serum (FCS) were obtained from Gibco (Paisley, Scotland). Murine 80K/MARCKS anti-peptide antibody was raised in rabbits against the C-terminal 11 amino acids of the murine 80K/MARCKS protein according to the method of Brooks et al. [16]. Oligo d(T)15 was purchased from Boehringer Mannheim (UK). Oligo d(T) cellulose, type 3, was supplied by Stratech (UK). DEAE-Cellulose (DE-52) was purchased from Fisons (UK). Collagenase CLS1 was obtained from Worthington (USA). Percoll and bovine serum albumin (BSA) type V were purchased from Sigma (UK). All other chemicals used were of the highest grade available commercially.

2.2 Animals

Wistar rats (mixed-sex litters) aged 2, 5, 6, 7, 8, 9, 10, 11, 14, 17, 21, 28 days and adult males (42 days, 200–250 g) were obtained from Binton and Kingman (Hull, UK). Animals were killed by an approved method, in accordance with the UK Home Office Animals (Scientific Procedures) Act, 1986.

2.3 Cell isolation

Adult and neonatal cardiac cells were isolated according to established methods as follows: Adult cardiac cells: Calcium-tolerant adult ventricular myocytes and non-myocytes (endothelial cells, fibroblasts, smooth muscle cells) were prepared from freshly excised hearts. Hearts were digested by Langendorff perfusion with 20 mg collagenase CLS1 by a modification of the method of Piper et al. [17]. Myocytes and non-myocytes were separated first by centrifugation (25×g), then by gravity, resulting in cultures that contained >95% pure myocytes. Non-myocytes were removed by pre-plating on Falcon tissue culture plates for 30 min at 37°C. Cells were either frozen in liquid nitrogen until required or used immediately. Neonatal cardiac cells: Neonatal myocytes were isolated from ten 2-day-old Wistar rats by a modification of the method of Gunn et al. [18]. Briefly, ventricles were dissociated by serial enzymatic digestion with 26 mg collagenase CLS1 and 30 mg pancreatin in a water-jacketed biological spinner flask (Wheaton, USA). Myocytes and non-myocytes were separated by differential centrifugation through a percoll gradient. Cells were resuspended in 68% DMEM-Glutamax, 17% M199, 15% FCS and 100 μg/ml penicillin/streptomycin and were either frozen in liquid nitrogen until required or used immediately. Both methods resulted in preparations containing >97–99% cardiac myocytes as determined by light microscopy and immunostaining with a cardiospecific troponin I antibody (data not shown).

2.4 Poly A+ RNA isolation

Poly A+ RNA was isolated as described previously [19]. Briefly, whole ventricular tissue (powdered in liquid N2) or cardiac myocytes were homogenised using a Polytron in a buffer (10 ml/500 mg tissue) containing 0.2 mol/l NaCl, 0.2 mol/l tris (hydroxymethyl) aminomethane buffer (Tris) HCl, 0.15 mmol/l MgCl2, 2% SDS and 200 μg/ml proteinase K (pH 7.5). The homogenate was passed through a 21 G needle to reduce viscosity and incubated overnight at 45°C. Poly A+ RNA was obtained by binding to oligo d(T) cellulose (20 mg/500 mg tissue) as follows: NaCl was added to the homogenate to a final concentration of 0.3 mol/l and the mixture incubated with gentle agitation (1 h at room temperature) with oligo d(T) cellulose. Following centrifugation, the pellet was washed 3 times in binding buffer (0.5 mol/l NaCl and 0.01 mol/l Tris HCl, pH 7.5). The poly A+ RNA was then eluted in 2×200 μl washes of elution buffer (0.01 mol/l Tris HCl, pH 7.5) and ethanol precipitated (0.1 vol of 3 mol/l NaOAc and 2 vol of absolute ethanol) overnight at −70°C. The quantity of RNA was determined by spectrophotometric measurement of absorbance at 260 nm. Following centrifugation, the poly A+ RNA pellet was resuspended in RNase-free water to a final concentration of 1 mg/ml.

2.5 Reverse transcription-polymerase chain reaction (RT-PCR)

Messenger RNA (1 μg) was reverse-transcribed to give a stable cDNA copy of the mRNA using the Invitrogen cDNA Cycle Kit, exactly as recommended by the manufacturer. Following ethanol precipitation, samples were dissolved in RNase-free water (20 μl) and a 1 μl aliquot taken and diluted to 50 μl with RNase-free water prior to carrying out the reaction. PCR was performed using 1 μl of diluted cDNA in a total volume of 50 μl containing HEPES (50 mmol/l; pH 7.9), MgCl2 (1.5 mmol/l), dNTPs (100 μmol/l each), 100 pmol each of forward (5′-GCC TTC CAA AGC AAA TGG GCA-3′) and reverse (5′-CTC TTC TTG AAG GAG AAG CCG-3′) primers (both synthesised by Pharmacia, UK), and 2.5 U of heat stable-Tli polymerase (Promega, UK). Amplification was as follows: one cycle of 99°C for 4 min, 65°C for 1 min and 75°C for 1 min followed by 30 cycles of 99°C for 1 min, 65°C for 30 s and 75°C for 1 min, using a Techne PHC-3 Thermal Cycler. Duplicate aliquots (10 μl) were then resolved on 9% polyacrylamide gels using pGEM DNA size markers and amplified fragments observed by staining the gel with ethidium bromide. Amplification of mRNA from the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a positive control for these experiments using the following primers: forward primer 5′-CCT TCA TTG ACC TCA AC-3′, reverse primer 5′-AGT TGT CAT GGA TGA CC-3′. The PCR product obtained using the 80K/MARCKS primers was a fragment of 420 bp whilst the GAPDH primers produced a fragment of 396 bp.

2.6 Subcellular fractionation of proteins and Western analysis

Protein samples were prepared both from whole ventricular tissue (2, 5, 6, 7, 8, 9, 10, 11, 14, 17, 21, 28 days old and adult) and from cardiac myocytes (2-day and adult). Samples were lysed in an ice-cold extraction buffer containing 0.05 mol/l Tris HCl (Ph 7.5), 0.005 mol/l ethylenediaminetetraacetic acid (EDTA), 0.01 mol/l ethylene glycol-bis(β-aminoethyl ether) tetraacetic acid (EGTA), 0.001 mol/l dithiothreitol (DTT), 0.01 mol/l benzamidine, 50 μg/ml phenylmethylsulphonylfluoride (PMSF), 10 μg/ml leupeptin and 0.3% Triton X-100, homogenised using a Polytron (whole ventricular samples only) and sonicated. Heat-stable protein samples were prepared by boiling the homogenates for 5 min followed by centrifugation for 5 min at 12 000 rpm (4°C) to pellet heat-labile protein debris and an equal volume of 2× Laemmli buffer added to the supernatant. Subcellular fractionated protein samples (cytosolic and membrane fractions) were prepared by homogenisation of whole ventricular tissue in an ice-cold buffer containing 0.05 mol/l Tris HCl (pH 7.5), 0.001 mol/l DTT, 0.01 mol/l benzamidine, 50 μg/ml PMSF and 10 μg/ml leupeptin followed by ultracentrifugation (200 000×g at 4 C for 1 h). The supernatant fraction was removed and constituted the cytosolic protein fraction. EDTA (0.005 mol/l), EGTA (0.01 mol/l) and Triton X-100 (0.3%) were then added to this fraction to the final concentrations shown in brackets. The pellet was resuspended in ice-cold extraction buffer containing 0.005 mol/l EDTA, 0.01 mol/l EGTA, and 0.3% Triton X-100. One-millilitre samples were concentrated using Amicon microconcentrators (Amicon, UK) and 10 μl samples taken for protein determination prior to the addition of an equal volume of 2× Laemmli buffer to the remaining lysate. Proteins were separated electrophoretically on 8% SDS-PAGE gels and transferred to PVDF membranes (Immobilon-P, 0.45 μm, Millipore, UK) using a semi-dry blotting apparatus (LKB Multiphor II, Pharmacia) and a transfer buffer containing 0.037 mol/l Tris base, 0.039 mol/l glycine, 0.04% SDS and 20% methanol. Membranes were blocked for 2 h at room temperature in phosphate-buffered saline (PBSA)/0.2% Tween 20 containing 5% non-fat milk followed by incubation overnight at 4 C with a rabbit anti-mouse 80K/MARCKS antibody (1:2500 dilution), directed against the carboxy-terminal peptide sequence SPEAPPAPTAE [16], in PBSA/ 0.2% Tween 20 containing 1% non-fat milk. The membrane was then washed 3 times (10 min each) with PBSA/0.2% Tween 20 prior to incubation with either 125I-protein A antibody (1 h at room temperature; 1 μCi/ml) or with donkey anti-rabbit horseradish peroxidase-conjugated antibody (30 min at room temperature, 1:2000 dilution) in PBSA/0.2% Tween 20 containing 1% non-fat milk. Filters were washed 5 times in PBSA/0.2% Tween 20 and either exposed immediately to Kodak X-Omat film with intensifying screens at −70°C for 24–72 h (125I-labelled blots) or incubated for 1 min with ECL Western Blotting Reagents (Amersham International) (peroxidase-conjugated labelled blots), prior to exposure to film. The resultant autoradiographs were quantified using densitometric scanning. All filters were then stained with Coomassie Brilliant Blue solution in order to confirm that equal amounts of protein had been loaded per lane.

2.7 PKC activation assay

The following method was used to prepare active PKC enzyme from whole ventricular tissue from rats aged 5, 6, 7, 8, 9, 10, 11 days of age and adult (42-day). Briefly, hearts were excised rapidly, rinsed in ice-cold Krebs-Henseleit bicarbonate buffer (pH 7.4) and the ventricular tissue homogenised immediately in 1 ml ice-cold buffer containing 0.05 mol/l Tris-HCl (pH 7.5), 0.001 mol/l DTT, 0.01 mol/l benzamidine, 50 μg/ml PMSF and 10 μg/ml leupeptin, and sonicated. The lysate was fractionated into cytosolic and membrane samples by ultracentrifugation (200 000×g at 4 C for 1 h) in the presence (membrane fraction) or absence (cytosolic fraction) of 0.005 mol/l EDTA (pH 8.0), 0.01 mol/l EGTA (pH 8.0) and 0.3% (v/v) Triton X-100 exactly as described previously [19]. These components were added to the cytosolic fraction prior to further biochemical analyses. The subcellular fractions were incubated for 1 h at 4 C with DEAE-cellulose (DE-52), pre-equilibrated with Buffer A (0.02 mol/l Tris-HCl, pH 7.5, 0.5 mmol/l EDTA, 0.5 mmol/l EGTA, and 0.001 mol/l DTT). DEAE-cellulose columns were prepared using this material and PKC eluted from the column with 0.3 mol/l NaCl. Fractions (4 ml) were collected, concentrated using Microcon microconcentrators (Amicon, UK) and the concentrate stored in 50% glycerol overnight at −70°C. The following day, samples were assayed in triplicate for total PKC activity as described previously [13]. Briefly, PKC activity in 25 μl of each concentrated sample was measured by calculating the amount of 32P-labelled phosphate transferred to a specific peptide substrate (based upon the pseudosubstrate domain of PKCε which is phosphorylated by all diacylglycerol/phorbol ester-activated isoforms), in 15 min at 25°C. The assay was performed in a total volume of 50 μl containing: 0.05 mol/l Tris; 0.001 mol/l calcium acetate; 2 μg/ml phorbol 12-myristate 13-acetate (PMA); 0.0025 mol/l DTT; 0.075 mmol/l substrate peptide; 0.66 mole % Lα phosphatidyl-l-serine; 0.015 mol/l magnesium acetate; 0.05 mmol/l adenosine-5′-triphosphate (ATP) and 0.2 μCi γ32P-ATP, terminated by the addition of 100 μl ‘stop reagent’ after incubation for 15 min at 25°C and 125 μl spotted onto P81 protein binding paper. After washing twice in 500 ml 5% v/v acetic acid for 10 min to remove any unincorporated radioactivity, the papers were allowed to air-dry, placed in poly Q scintillation vials with 10 ml ‘Ready Safe’ liquid scintillation fluid and counted in a scintillation counter (Beckman, UK).

2.8 Protein determination

Protein concentrations in homogenised tissue and/or cell preparations were determined according to the method of Bradford [20]using BSA type V as a standard.

3 Results

3.1 80K/MARCKS mRNA and protein expression in developing rat ventricular tissue

To determine whether 80K/MARCKS mRNA was expressed in cardiac tissue, poly A+ RNA was prepared from both whole ventricular tissue and cardiac myocytes obtained from neonatal (2-day) and adult rats and analysed by RT-PCR using primers against the mouse 80K/MARCKS sequence. Fig. 1 shows a representative polyacrylamide gel of an RT-PCR experiment which demonstrates the expression of 80K/MARCKS mRNA in neonatal and adult myocytes and in whole ventricular tissue. The results show that 80K/MARCKS mRNA is expressed in Swiss 3T3 fibroblasts (positive control) and in all cardiac samples examined. Comparison of the levels of the 80K/MARCKS 420 bp amplified fragment relative to the levels of the GAPDH 396 bp amplified fragment (duplicate samples) suggested that expression of 80K/MARCKS mRNA in neonatal myocytes may be higher than in adult myocytes, whilst in whole ventricular samples there appears to be very little difference in the mRNA expression of this molecule during development. Analysis of the levels of 80K/MARCKS protein expression during myocardial development is shown in Fig. 2. The upper panel of Fig. 2 shows a representative Western blot of 80K/MARCKS protein expression during development of the rat myocardium. The specificity of antibody binding was confirmed by competitive inhibition with the immunizing peptide which abolished specifically the immunoreactive band at 80 kDa (data not shown). The lower panel of Fig. 2 represents data obtained from the densitometric scan of the immunoblot shown in the upper panel. All values are expressed relative to those observed at 2-day of age. The period of myocardial development from 2 to 5 days of age showed an increase in protein expression to 175% of control levels. This is followed by a large transient, but consistent, decrease in 80K/MARCKS protein expression between 6 and 8 days of age (61% of the protein expressed in 2-day-old samples). Subsequently, levels returned by 11 days of age to those observed at 5 days and then gradually decreased throughout the 14–21-day period from 168% (at 11 days) to 52% (at 21 days) of the levels expressed at 2 days of age and remained low from 21 days of age through to adulthood. In subsequent experiments we have shown that the decrease in expression of the 80K/MARCKS protein during the 6–8-day developmental period is independent of the sex of the animals, with males and females producing similar patterns of expression (data not shown). Additionally, we have observed that the loss in 80K/MARCKS protein is temporally shifted to later periods of development (7–9 days) in some samples. The reason(s) for this variation is unclear at the present time, but we propose that differences in litter size and/or accuracy of mating/birth times may account for these slight variations. However, the decrease in 80K/MARCKS protein expression was a reproducible phenomenon in more than 6 separate experiments using hearts from different animals. Thus, in summary we have identified a ‘window’ of 80K/MARCKS protein down-regulation in myocardial tissue that occurs during the 6–9-day developmental period.

Fig. 2

Changes in 80K/MARCKS protein expression in the developing rat myocardium. Twenty-five micrograms of heat-stable protein from whole ventricular tissue was loaded onto each lane of an 8% SDS/PAGE gel and electrophoresed at 45 V overnight. Separated proteins were transferred to a PVDF membrane, incubated overnight at 4°C with a murine anti-80K/MARCKS antibody (1:2500 dilution) and detected by incubation with 125I-labelled protein A (0.1 μCi/ml) for 1 h at room temperature. Results were analysed by autoradiography. Lower graph shows the % change in levels of 80K/MARCKS protein expression during myocardial development. The autoradiographs were quantified by laser densitometry and results presented as % change relative to those values expressed in 2-day-old myocardial tissue.

Fig. 1

Expression of 80K/MARCKS mRNA in neonatal and adult myocytes and whole ventricles, by RT-PCR. One microlitre of diluted cDNA, produced by reverse transcription of mRNA obtained from neonatal (2-day) and adult myocyte and whole ventricle samples, was amplified and the resultant 80K/MARCKS or GAPDH PCR products visualised on 9% acrylamide gels stained with ethidium bromide. Although the 80K/MARCKS PCR product (420 bp) is expressed in all cardiac samples, levels are higher in neonatal (2-day) than in adult cardiac myocytes.

3.2 Subcellular distribution of 80K/MARCKS protein during myocardial development

When in its unphosphorylated form, the 80K/MARCKS protein is found in association with the cell membrane [21, 22]whereas phosphorylation of the protein by activated PKC results in the translocation of 80K/MARCKS from the membrane to the cytosolic fraction [23, 24]. However, this response is transient such that levels of 80K/MARCKS protein return to the membrane if the stimulus is removed whereas in the presence of chronic stimulation (more than 6 h), 80K/MARCKS levels are down-regulated [25]. Thus, subcellular compartmentalisation of the 80K/MARCKS protein may be used as an indirect measure of PKC activation [26]. Total membrane and cytosolic protein extracts were prepared from whole ventricular tissue from animals ranging from 2 to 11 days of age and the levels of 80K/MARCKS protein examined by immunoblot analysis using the anti-80K/MARCKS antibody. Fig. 3 shows representative Western blots of the membrane and cytosolic fractions prepared from the same hearts. It should be noted that 50 μg membrane protein and 100 μg cytosolic protein were loaded per lane. The data show a down-regulation of membrane-bound 80K/MARCKS protein during the 6–7-day period of development, concomitant with a slight increase in the levels of cytosolic 80K/MARCKS protein at this time indicative of phosphorylation and subsequent down-regulation of 80K/MARCKS during this time period. Interestingly, the 80K/MARCKS antibody binds to both non-phosphorylated (membrane-bound) and phosphorylated (cytosolic) 80K/MARCKS with equal immunoreactivity [Brooks, S.F. personal communication] and so the low level of detection of cytosolic 80K/MARCKS in these samples is unlikely to be due to a lower affinity of the antibody for the phosphorylated cytosolic protein.

Fig. 3

Subcellular distribution of the 80K/MARCKS protein during myocardial development. One hundred micrograms of subfractionated heat stable protein prepared from ventricular tissue (2- and 5–11-day-old animals) were loaded onto an 8% SDS/PAGE gel, electrophoresed and transferred to PVDF membrane as described in the legend of Fig. 2. The membranes were then probed with an anti-80K/MARCKS antibody (1:2500 dilution) overnight at 4°C. Reacting antigens were detected by incubation of the blot with a donkey anti-rabbit horseradish peroxidase antibody (1:2000 dilution) for 30 min followed by incubation with Amersham ECL reagents. Similar results were obtained in 2 separate experiments.

3.3 80K/MARCKS protein expression in cardiac myocytes

Since myocardial tissue consists of a variety of cell types, including cardiac myocytes, fibroblasts, endothelial cells and smooth muscle cells, we next investigated whether 80K/MARCKS protein is expressed in cardiac myocytes. Fig. 4 shows an immunoblot of 80K/MARCKS protein expression in control Swiss 3T3 mouse fibroblasts, neonatal and adult whole ventricular tissue and cardiac myocytes. The results show that 80K/MARCKS expression is highest in whole ventricular tissue extracts obtained from both neonatal and adult hearts. In addition, neonatal cardiac myocytes express significant levels of 80K/MARCKS protein whereas expression was not detected in adult cardiac myocytes, even after overexposure of the autoradiogram. Thus, there is a distinct shift in the cellular distribution of 80K/MARCKS protein during development such that 80K/MARCKS protein expression is down-regulated in adult cardiac myocytes compared with levels expressed in neonatal myocytes.

Fig. 4

An autoradiograph showing 80K/MARCKS protein expression in cardiac myocytes. Twenty micrograms of heat-stable protein obtained from neonatal (2-day) and adult cardiac myocytes and whole ventricular tissue extracts were analysed by Western analysis as described in Fig. 3. Similar results were obtained in 2 separate experiments. 3T3 = Swiss 3T3 fibroblasts; NM = neonatal myocytes; AM = adult myocytes; NWV = neonatal whole ventricle; AWV = adult whole ventricle.

3.4 PKC activation during development of the rat myocardium

The shift in the subcellular distribution of 80K/MARCKS from the membrane to the cytosolic fraction observed during the 6–7-day developmental period (Fig. 3) and the observed transient decrease in membrane 80K/MARCKS protein expression at this time (Figs. 2 and 3) suggested to us that PKC activation may be involved in regulating expression of the 80K/MARCKS protein. Accordingly, we measured the changes in subcellular distribution of PKC enzyme activity in myocardial tissue obtained from animals during the 5–11-day developmental period and in adult rats in order to determine a possible mechanism for down-regulation of 80K/MARCKS during this period. Fig. 5a shows that dramatic changes in total PKC activity occur during development of the rat myocardium such that there is a 160% increase in membrane-associated PKC activity compared to cytosolic PKC activity between day 6 and day 9 of post-natal development (n=3 for each experiment). This is consistent with the activation of protein kinase C isoenzymes and subsequent translocation from the cytosol to the membrane [29]. Interestingly, there was no significant change in total PKC activity during this period (Fig. 5a). By 9 days of age the levels of PKC activity had returned to those present at 5 days of age. It is interesting to note that whereas the ratio of membrane/cytosolic PKC activity in the adult ventricle is similar to the levels seen in the early (5–6 days old) neonatal heart samples (Fig. 5a), the total protein kinase C activity in the adult myocardium was less than 30% of the total kinase activity obtained from all neonatal heart values (Fig. 5b). Although the assay system used in this study does not provide data on individual PKC isoforms, it does provide overall PKC activity levels for both Ca2+-dependent (α, β and γ) and Ca2+-independent (δ, ϵ, η and θ) isoforms [Amersham International, personal communication], both families of which are represented in cardiac myocytes [5–9].

Fig. 5

Changes in PKC activity in the developing rat myocardium. (a) Changes in total PKC activity during myocardial development. PKC activity levels were measured as described in Section 2. Values are expressed as pmol/min/mg total protein. (b) Changes in cytosolic and membrane PKC activity levels during myocardial development. PKC was extracted from ventricular tissue and separated into membrane and cytosolic fractions as described in Section 2. Activatable PKC was purified by binding to a DEAE-cellulose column and eluted with 0.3 mol/l NaCl. Eluted PKC was concentrated using Microcon filters (Amicon, UK) and enzyme activity was assayed using the Amersham PKC assay system kit in accordance with the manufacturer's instructions. Results are expressed as a ratio of cytosolic/membrane PKC activity levels and show the mean±standard deviation values obtained from 3 separate experiments. *P<0.05, ANOVA and Tukey's t-test.

4 Discussion

In the present study we have determined the expression and regulation of 80K/MARCKS, a major substrate protein of PKC, during development of the rat myocardium. 80K/MARCKS is a specific substrate of the co-factor-dependent enzyme, PKC, and activation of this enzyme results in a rapid phosphorylation of the 80K/MARCKS protein [12, 13]. In this study we have determined the potential regulation of 80K/MARCKS protein expression during the 5–11-day post-natal period by PKC. Previous studies by Rozengurt and colleagues have shown that stimulation of PKC by biologically active phorbol esters leads to the rapid phosphorylation and subsequent down-regulation of 80K/MARCKS mRNA within 5–7 h and protein within 14–18 h of activation [16]. Furthermore, in cells containing down-regulated levels of PKC, 80K/MARCKS protein levels recovered to normal within 72 h [16]. Phosphorylation of 80K/MARCKS leads to the dissociation of the molecule from the membrane to the cytosolic compartment [23, 24]and this has been suggested to serve as a suitable marker of PKC activation [27]. When we investigated the levels of PKC activity during the 5–11-day developmental period in the rat myocardium, we observed a significant increase in the amount of membrane bound PKC which reached a maximum at day 8 of development (Fig. 5a), indicative of the translocation of, and an increase in, PKC enzyme activity [3, 26]. Interestingly, there was no change in the total levels of PKC activity during the 5–11-day post-natal period although in adult myocardium levels of total PKC activity fell to less than 30% of the levels measured in neonatal hearts (Fig. 5b). This is in accordance with a recent report by Rybin and Steinberg [8]who showed that there was an age-dependent decline in immunoreactivity for PKC α, δ, ϵ and ζ isoforms during myocardial development in the rat, from the late embryonic period through to adulthood. Consistent with the increase in PKC activity during the 5–11-day post-natal period, we observed a down-regulation in the membrane 80K/MARCKS protein levels in addition to a small shift in the subcellular distribution of 80K/MARCKS from membrane to cytosol during the 6–7-day post-natal period as measured by immunoblotting (Fig. 3) suggestive of an increase in phosphorylated 80K/MARCKS protein followed by subsequent down-regulation of the protein during this period of development. The reason why only small amounts of cytosolic 80K/MARCKS protein were detected is probably due to the fact that translocated cytosolic 80K/MARCKS is rapidly degraded following chronic stimulation [25]and in this study we monitored expression at 24-h time intervals and so were unlikely to observe optimal cytosolic levels. Thus, we propose that the transient drop in 80K/MARCKS protein expression at 6–8 days of age is due to the activation of PKC followed by phosphorylation and subsequent down-regulation of 80K/MARCKS protein levels as has been reported previously in Swiss 3T3 and Rat 1 fibroblasts [16, 25]. The fact that levels of 80K/MARCKS protein in the heart return by day 11 of development to those observed at day 5 could be due to the fact that PKC activity levels also return to basal and the enzyme then does not stimulate 80K/MARCKS phosphorylation and subsequent down-regulation. Although recent reports have determined the distribution of PKC isoforms in the rat heart [5, 6, 8, 9, 28]our results extend these studies since we show, for the first time, that the PKC activity profile alters significantly during the early post-natal period and declines to low levels in the adult rat myocardium.

Since its discovery more than a decade ago [29], investigators have been seeking a role for 80K/MARCKS in cell growth since it is a major and specific in vivo substrate of PKC, which itself has been shown to modulate the growth and differentiation of a variety of cell types [1, 13]. Recently, we have shown that levels of 80K/MARCKS appear to play a pivotal role in modulating melanocyte proliferation and tumorigenesis [15]and changes in the expression of the 80K/MARCKS protein have been implicated in the regulation of cell entry and exit from the quiescent G0-phase of the cell cycle in response to serum-derived growth factors [30]. However, nothing is known about the role of this protein in the heart or in the cardiac myocyte. However, it is possible that 80K/MARCKS could be associated with the known changes in growth potential that occurs in the cardiac myocyte through development (for review, see [31]) since data from gene targeting experiments in the mouse indicate that 80K/MARCKS is necessary for normal development of the central nervous system and for extrauterine survival [32]. However, a similar role for this protein in cardiac myocytes remains to be demonstrated since abnormalities in organs other than the brain and the eye in these mice were not reported by these investigators. In addition, it is possible that 80K/MARCKS regulates calmodulin-dependent responses in the myocyte since it has been proposed that 80K/MARCKS complexes calmodulin [33, 34], thereby reducing calcium signalling in the cell. Possibly, the decrease in the expression of 80K/MARCKS in the adult heart could result in less sequestration of calmodulin, leading to perturbation of regulation of calmodulin-dependent enzymes since calmodulin has been shown to regulate a number of cellular events in cardiac muscle including calcium ion homeostasis, cyclic nucleotide metabolism, energy metabolism and pH regulation [28, 35, 36]. In addition, an indirect role for 80K/MARCKS in cardiac myocyte growth could result from the fact that calmodulin has been shown to modulate this response since constitutive overexpression of calmodulin in cardiac myocytes produces cardiac myocyte hypertrophy in adult transgenic mice and also may enhance myocyte proliferation at early developmental stages [36, 37]. Therefore, it is possible that alterations in 80K/MARCKS expression in the rat heart may produce effects on cardiac growth via a calmodulin-dependent mechanism such that reduced expression of 80K/MARCKS in the adult myocyte could lead to increased levels of ‘free’ calmodulin that could lead to hypertrophy. Additionally, 80K/MARCKS binds actin [38]and has been suggested to co-localise with vinculin and talin in focal adhesion plaques [39]. Thus, a decrease in the abundance of 80K/MARCKS could affect both the calcium signalling and cytoskeletal organisation of developing cardiac cells.

In summary, we have shown that protein (and possibly mRNA) expression levels of the specific PKC substrate, 80K/MARCKS, decline during development in the rat and that the levels of 80K/MARCKS protein may be regulated by PKC. Whether 80K/MARCKS is important in controlling growth and differentiation of the cardiac myocyte remains to be demonstrated.

Acknowledgements

We wish to thank The British Heart Foundation, The Leopold Muller Foundation and STRUTH for financial support, and Professor David J. Hearse for his continual support and encouragement.

References

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