Acceleration of growth of cultured cardiomyocytes and translocation of protein kinase C SIMON Sigfried
N. ALLO, and Janet
LOIS L. CARL, AND HOWARD E. MORGAN Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822
Allo, Simon N., Lois L. Carl, and Howard E. Morgan. Acceleration of growth of cultured cardiomyocytes and translocation of protein kinase C. Am. J. Physiol. 263 (CeZZPhysioZ. 32): C319-C325, 1992.-Phorbol 12-myristate 13-acetate (PMA), norepinephrine (NE), and contraction stimulate cardiomyocyte growth (increased protein content). Differences exist in the time course and extent of protein and RNA accumulation. Cells plated at 4 x IOF tells/60-mm dish and arrested with 50 mM KC1 demonstrated no significant growth. Treatment with PMA stimulated growth to a maximum of 17% at 48 h. In contrast, maximal stimulation of growth was 36% at 48 h and 31% at 72 h for contracting and NE-treated cells, respectively. Maximal stimulation of the capacity for protein synthesis (RNA content) was 32% for PMA-treated cells at 24 h compared with 59% and 77% for NE-treated and contracting cells, respectively, at 72 h. In support of a primary role for altered capacity in the regulation of protein synthesis, there was a significant correlation (r = 0.84) between RNA and protein contents that was independent of the stimulus used. Angiotensin II increased RNA content by 28% at 48 h but had no effect on growth up to 72 h. Growth stimulation and increased nuclear protein kinase C (PKC) activity were induced by contraction, NE, and PMA treatment and were inhibited by staurosporine (a PKC inhibitor), suggestive of a central role for PKC. ribosomal nephrine;
ribonucleic angiotensin
acid; hypertrophy; II; staurosporine;
phorbol ester; norepicontraction
OF OUR KNOWLEDGE of cellular changes in cardiac hypertrophy, measured as protein accretion or increased myocyte cross-sectional area, has been obtained in experiments utilizing isolated perfused hearts and primary cardiomyocyte cultures (6, 10, 20, 25, 28). However, studies with isolated myocytes have utilized different culture media, substrata, cell densities, and hypertrophic stimuli such as phorbol esters, norepinephrine (NE), contraction, and angiotensin II (ANG II). These variations become significant in view of the recent demonstration by Bishopric and Kedes (3) that expression of the skeletal cu-actin gene during NE-induced hypertrophic growth was greatly influenced by cell density. When cardiomyocytes were plated at a high density (3-4 x lo6 tells/60-mm dish), NE- and isoproterenol-induced stimulation of skeletal cu-actin gene expression, hyperand contractility were mediated by the trophy, ,&adrenoceptors because these effects were inhibited by ,&adrenergic antagonists but not by cu,-adrenergic antagonists. On the contrary, NE stimulation of cu-myosin heavy chain gene expression in these high-density cultures was mediated by the a,-adrenergic receptor because it was inhibited by al- but not by ,&adrenergic antagonists. The significance of plating density on cellular response was illustrated by the observation that, in contrast to high-density cultures in which NE stimulation of growth was mediated by the fl-adrenergic receptor, the same effect was mediated by the al-adrenoceptor in cardiomyocytes plated at a low density (3).
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In cultures plated at 0.5 X lo6 tells/60-mm dish in minimum essential medium (MEM) containing 10% newborn calf serum for 24 h and maintained in serumsubstituted medium (I), treatment with phorbol l2myristate 13-acetate (PMA) resulted in a 34% increase in both protein and RNA content compared with control values. With the use of the same serum-substituted medium, cells plated at a density of 4 x IO6 tells/60-mm dish and arrested with KC1 were induced to grow by treatment with NE and by contraction (19, 20). Cells plated at 1.5 x lo6 cells/lOO-mm dish in a 1:4 mixture of Dulbecco’s modified Eagle’s medium and medium 199 (GIBCO) demonstrated a 106% and 124% increase in protein content following treatment with PMA and phenylephrine, respectively (12). Therefore, because of differences in the plating densities and culture media used, the comparative hypertrophic effects of the different stimuli remain to be evaluated in the same model. Recently, it was demonstrated that contraction and PMA-induced hypertrophic growth were primarily the result of increased rDNA transcription leading to an increased capacity for protein synthesis, as measured by the RNA content (1, 18). The mechanisms by which these stimuli elicit their hypertrophic effects are not completely understood. Less clear is how the intracellular events initiated by a variety of stimuli may converge to regulate ribosome synthesis at the level of rDNA transcription. A significant role has been suggested for protein kinase C (PKC) in the mechanism of hypertrophic growth stimulation by contraction, PMA, NE, and ANG II (1, 20, 25). The mechanism by which PKC activity and distribution may be altered by these stimuli is not completely understood. PMA acts as an analogue of diacylglycerol (DAG) and directly activates PKC (7). NE and ANG II bind to their respective receptors on the cell surface and result in activation of phospholipase C (PLC). This increase in PLC activity leads to the hydrolysis of phosphoinositol phosphate to yield DAG (the physiological activator of PKC) and inositol trisphosphate. The mechanism by which spontaneous contraction of cultured cardiomyocytes may result in altered PKC activity and distribution remains to be investigated. However, a role for PKC in contractioninduced growth is suggested by the studies of Richter et al. (23), demonstrating contraction-induced PKC translocation to membranes in skeletal muscle. In this study, the hypertrophic effects of several stimuli were compared in the same cultured cardiomyocyte model. Because all of these stimuli are capable of altering cellular PKC activity and distribution, this study begins to address the question of a central role for PKC in the hypertrophic effects of PMA, NE, and con-
0 1992 the American
Physiological
Society
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c319
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traction. Staurosporine, a putative PKC inhibitor, inhibited growth stimulation and decreased nuclear PKC activity. EXPERIMENTAL
PROCEDURES
Cardiomyocyte cultures. Neonatal cardiomyocytes were isolated by enzymatic digestion as previously described (I, 19). Cell number was determined with a Coulter counter, and the cells were resuspended in MEM containing 10% newborn calf serum and 0.1 mM 5bromo-Z’-deoxyuridine. The cells were plated at a density of 4 x IO6 tells/60-mm dish precoated with 0.1% gelatin. After an overnight incubation to permit attachment of viable cardiomyocytes, the cells were maintained in serum-substituted medium containing 50 mM KCl. After incubation for 3 days, hypertrophy was initiated by incubating cells in serumsubstituted media containing 50 mM KC1 and either 10y7 M PMA, 2 x lo-” M NE, or 10y6 M ANG II. Contraction was initiated by incubating cells in media containing 5 mM KC1 (19, 20). Stock solutions of PMA and staurosporine in dimethyl sulfoxide (DMSO) were used, and the final concentration of DMSO in each dish was not more than 0.001%. After 24-48 h of contraction, ATP content was 24 +- 2.5 pmol/g protein, which was unchanged from quiescent cells that were incubated in 50 mM KC1 (23 t 2.1 pmol/g protein). These values are 80% of those measured in isolated perfused rat hearts (14). Maintenance of creatine phosphate content depended upon addition of creatine (5 mM) to the incubation medium. In the absence of creatine, the contents of creatine phosphate were 7 t 1.3 and 7 +- 1.4 pmol/g protein in contracting and quiescent cells, respectively. In the presence of 5 mM creatine, these values increased to 67 t 4.4 and 49 I~I 3.8 pmol/g protein for contracting and quiescent cells, respectively. The creatine phosphate content of isolated perfused rat hearts averaged 45 ,umol/g protein (14). Maintenance of ATP content in cultured cardiomyocytes indicated that growth of these cells was not restrained by energy deficiency. Determination of cellular protein and RNA contents. Cellular protein content was used as a measure of cell growth. Dishes of cells were rinsed three times with phosphate-buffered saline (PBS). The cells were scraped with I ml of sodium citrate buffer containing 0.25% sodium dodecyl sulfate (SDS) [standard saline citrate (SSC)/SDS] and stored at -20°C. The samples were thawed and vortexed, and duplicate aliquots of 25 ~1 were assayed for protein by the method of Lowry et al. (17) using bovine serum albumin (BSA) as a standard. DNA concentration was determined fluorometrically in duplicate loo-p1 aliquots as previously described (8) using calf thymus DNA as a standard. The capacity for protein synthesis was estimated by measuring the RNA content. Briefly, RNA in 0.5-ml aliquot of SSC/ SDS homogenate of cells was precipitated and washed three times with 0.5 ml 0.5 N perchloric acid (PCA). The pellet was hydrolyzed in 750 ~10.3 N NaOH at 37°C for 24 h. The sample was neutralized with 250 ~1 4 N PCA, centrifuged, and the absorbance at 232 and 260 nm of the supernatant was measured. The RNA concentration was calculated as previously described (19,
20).
Isolation of nuclei. Nuclei were isolated as previously described (1, 4). Cells were rinsed three times with 3 ml of PBS and once with 1 ml buffer 1 containing 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 8.0, 10 mM NaCl, 2.5 mM MgC1,, and 5 mM dithiothreitol. The cells in each dish were incubated on ice in 0.5 ml of buffer 1 for IO min. After addition of an equal volume of buffer 1 supplemented with 0.6 M sucrose and 0.6% Triton X-100, the cells were scraped and homogenized gently in a Dounce homogenizer with two strokes of an A pestle. The homogenate was layered over an equal volume of buffer 1
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containing 0.6 M sucrose and centrifuged at 1,500 g for 10 min at 4°C. The pellet was resuspended in sucrose buffer [250 mM 100 mM 3-(N-morpholino)propanesulfonic acid sucrose, (MOPS), 2.4 mM ethylene glycol-his@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2.0 mM EDTA, 100 PM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride] containing 0.5% Triton X-100. The nuclear suspension was sonicated five times for IO s each and centrifuged at 100,000 g for 30 min. The supernatant was used to measure PKC activity. Determination of PKC activity. Nuclear PKC activity was measured in a reaction mixture containing 60 pug histone III-S, 15 mM Tris HCl, pH 7.5, 10 mM MgCl,, 1 mM CaCl,, 266 pg/ml phosphatidylserine, 18 pg/ml diolein, and 10 PM [y-:j2P]ATP (6 X IO6 cpm) in a final volume of 300 ~1 (1). The reaction was started by the addition of 4-6 pg sample protein and incubated at 30°C for 5 min. The reaction was terminated by spotting 35 ~1 of the reaction mixture onto a 2.5 x 2 cm Whatman P81 phosphocellulose paper, which was immediately dropped into 75 mM H,,PO,. The filters were washed a total of five times for 15 min each with 75 mM H:,P04, followed by a 100% ethanol wash. The filters were dried and counted for radioactivity. The reaction rate was linear for 10 min, and all experiments were performed for 5 min. Protein concentration was measured with a Bio-Rad protein dye based on the procedure by Bradford (5). PKC activity was determined by subtracting the Ca’+-phospholipid-independent :j2P incorporation measured in the presence of 2 mM EGTA from incorporation measured in the presence of Ca2+, diolein, and phosphatidylserine. Initial experiments were conducted to demonstrate that the nuclear PKC was solubilized following detergent treatment and sonication of the nuclei. No PKC activity was measurable in the 100,000-g pellet from solubilized nuclei of either control or contracting cells. The kinase activity in the absence of Ca2+phospholipid was 64.6 t 8.9 pmol .rngg. minl, while activity in the presence of Ca’+ -phospholipid was 53.9 t 5.0 pm01 . mg+ . min. These activities were not altered in the 100,000-g pellets obtained from contracting cells (63.5 t 2.8 and 55.7 t 5.3 pmol rng-l. min- 1 for activity in the absence and respectively). Therefore, PKC presence of Ca”+ -phospholipid, activity in the 100,000-g supernatant represented the total activity in the nuclei. Determination of adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase activity. CAMP-dependent protein kinase activity was measured in a reaction mixture containing 50 mM MOPS, 10 mM MgC12, 0.25 mg/ml BSA, 75 pg/ml kemptide, and 0.1 mM [r-:j”P]ATP (1 x lo6 cpm) in a total volume of 0.1 ml. After incubation for 5 min at 3O”C, the reaction was started by the addition of 5 pg of sample protein (28). After 5-min incubation, the reaction was terminated by spotting 50 ~1 of mixture onto a 2.5 X 2-cm Whatman P81 phosphocellulose paper. The filters were processed as described above for PKC assays, and the radioactivity was measured. Protein kinase activity was determined in the absence and presence of IO PM CAMP. Statistical analysis. Data are expressed as the means t SE of the indicated number of separate determinations. The statistical analysis of differences between multiple groups was performed by a one-way analysis of variance followed by the Student’s t test. A value of P < 0.05 was taken to indicate a statistically significant difference between the groups compared. RESULTS
Characterization of the effect of stimuli on cardiomyocyte growth and the capacity for protein synthesis. The initial phase of this study characterized hypertrophic
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growth of cardiomyocytes in response to PMA, contracTable 1. Effect of staurosporine on rate of contraction tion, NE, and ANG II using the same cell culture model. of cardiomyocytes In agreement with previous studies, cardiomyocytes plated at a density of 4 x lo6 tells/60-mm dish and arBeats/min Beats/min Staurosporine, nM After 7 h After 48 h rested with 50 mM KC1 did not demonstrate synchronous contraction and exhibited no significant growth (Fig. 1). 63+9 162+16 However, when cardiomyocytes were exposed to 5 mM i.5 49+21 144f7 71+16 112f13* KCI, they began to contract spontaneously. These cells 16+8* .: 69+19* contracted at a rate of 63 of: 9 beats/min after 7 h and Contraction was initiated in cardiomvocvtes bv a reduction of lKCl1 achieved a maximum rate of contraction of 162 + 16 in medium from 50 to 5 mM. Simultaneously, staurosporine was added beats/min after 48 h (Table 1). Incubation of 50 mM to achieve indicated final concentration. Rate of contraction in control KCl-arrested cells with lop7 M PMA or 2 X lop6 M NE (50 mM KCl-arrested cells) was zero. Values are means k SE of 4 dishes. stimulated protein accumulation without induction of * Significant difference from rate in absence of staurosporine. contraction. Reduction of KC1 concentration from 50 to 5 and, in this case, by ANG II (Fig. 2). A significant inmM KC1 induced spontaneous contraction and resulted crease of RNA content by PMA was observed after 24 h. in increased protein accumulation. Significant differences in the extent and time course of the stimulation of This increase in RNA content, 31% over control, was the stimulation observed for PMA-treated cells. protein accumulation by PMA, NE, and contraction were maximum RNA content in NE-treated and contracting myocytes observed (Fig. 1). Increased protein content by PMA, contraction, and NE were initially observed after 48 h. In was significantly elevated after 48 h. In contrast to PMAtreated cells, maximal increases of 59% and 77% were contrast to PMA and contraction, where longer incubations did not result in further increases in protein con- observed after 72 h for NE-treated and contracting cells, respectively (Fig. 2). RNA content in ANG II-treated tent, a significant further increase was obtained with NE. 28% over control Maximal stimulation of growth by PMA treatment or cells was increased by approximately contraction were 17 and 36%, respectively, after 48 h. In after 48 h. In support of the hypothesis that increased contrast, maximal stimulation of growth by NE was 31% capacity for protein synthesis plays a central role in the growth of cardiomyocytes, a significant correlation (r = observed after 72 h. ANG II was not an effective growth 0.84) between RNA and protein content was observed stimulus in that protein content was not significantly increased after either 48 or 72 h of treatment with low6 M (Fig. 3). Effect of staurosporine on growth and the capacity for ANG II. protein synthesis. The regulation of several cellular proIn previous studies, it was demonstrated that contraccesses by PKC has been examined with the use of several tion and PMA-induced hypertrophic growth were primarily the result of increased capacity for protein syn- potent inhibitors (11, 21, 27). A role for PKC in PMA-, growth of cardiomyocytes thesis, as measured by RNA content (1,19,20). Similar to NE-, and contraction-induced was examined with the use of the PKC inhibitor, staurothe effects on protein content, significant differences were observed in the extent and time course of accelera- sporine. To correct for possible reductions in protein and RNA contents that may be due to the cytotoxic effects of tion of RNA accumulation by PMA contraction, NE, high concentrations of staurosporine, the data were nor-5OmM K 50mM K 05mM K 050mM K D50mM K malized to the DNA content. The concentrations of stau+ PMA +NE +ANG II rosporine used in this study (0.5-15 nM) did not reduce cell numbers. Similarly, treatment of KCl-arrested cells -5OmM
g 1100 5 25 2 1000 .G 2 2 900 a 3 ij 800 I-
K 050mM
+ PMA
K 05mM
K (50mM
+ NE
K (50mM
-ANG
K II
40
i, T n 0 hr
24 hr
48 hr
i
72 I
Time (h) Fig. 1. Effect of stimuli on cardiomyocyte protein accumulation. Cardiomyocyte hypertrophic growth was initiated on day 4 in culture (time 0) by exposure to 50 mM KC1 (control), 50 mM KC1 + 10m7 M phorbol 12-myristate Is-acetate (PMA), 5 mM KC1 (contracting), 50 mM KC1 + 2 x 10mfiM norepinephrine (NE), and 50 mM KC1 + 10-a M ANG II. Protein content was measured over 3 days of treatment. DNA content did not change as result of these treatments (19, 20). Values are means i SE of 4-7 separate preparations. * Significant difference from control cells (P < 0.05)
0 hr
24
r
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r
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Time (h) Fig. 2. Effect of stimuli on capacity for protein synthesis. Capacity for protein synthesis (RNA content) was determined in cells treated as in Fig. 1. Data points are means + SE of 4-8 separate preparations. * Significant difference from control (P < 0.05).
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800 .z s 600 \E z 400 0 200 15
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CONTENT
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Fig. 3. Relationship between protein and RNA content. Protein content was plotted against corresponding RNA content for each dish of cells. A significant correlation (r = 0.84) was observed between protein and RNA contents.
with staurosporine did not significantly alter growth, suggesting that staurosporine concentrations up to 15 nM were not toxic for arrested cells (Fig. 4). Treatment with PMA increased protein content, measured as protein/DNA, by 18% (Fig. 4A). This PMAinduced increase in protein content was completely inhibited by 10 nM staurosporine (Fig. 4A). As expected, the capacity for protein synthesis was significantly increased by PMA treatment. Treatment with low concentrations of staurosporine (0.5-5 nM) had no significant effect on PMA-induced RNA accumulation (Fig. 4B). However, this increase was partially inhibited by 10 nM staurosporine. A 55
c +--Control +Contracting ..O..pMA ..f-J...NE
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B
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I 4 Staurosporine,
I 6
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+Control -W-Contracting ..O..pMA ..a..NE
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Fig. 4. Effect of staurosporine on growth induced by PMA, NE, and contraction. Quiescent cardiomyocytes (50 mM KCl) were treated with 10y7 M PMA or 2 x 10B6 M NE in presence of increasing concentrations of staurosporine for 72 h. Contraction was initiated in cells by incubation in serum-substituted medium containing 5 mM KC1 for 72 h. Protein/DNA (A) and RNA/DNA (B) were determined as described in EXPERIMENTAL PROCEDURES. Data points are means t SE of 4-9 separate preparations except at 10 nM staurosporine where value is mean of 2 separate experiments for contracting cells. * Significant difference from control (P < 0.05).
CONTROL PMA ICY-PHORBOL Fig. 5. Effect of PMA and 4cu-phorbol l&13-didecanoate nuclear protein kinase C (PKC) activity after 20 min. control and treated cells were solubilized with 0.5% Triton 100,000-g supernatant was used for PKC assays. PKC measured and expressed as Ca2+ -phospholipid-dependent ity. Values are means t SE of 3-4 separate preparations. difference from control (P < 0.05).
treatment on Nuclei from X-100, and activity was kinase activ* Significant
Treatment with NE for 72 h increased protein/DNA (Fig. 4A) and RNA/DNA (Fig. 4B) by 17% and 25%, respectively. These growth effects were completely inhibited by 0.5-10 nM staurosporine. When myocytes were incubated in media containing 5 mM KC1 to induce contraction, protein and RNA contents were increased by 32% and 56%, respectively, after 72 h (Fig. 4, A and B). Treatment of contracting myocytes with 0.5 and I nM staurosporine did not significantly alter growth stimulation. However, higher concentrations of staurosporine (5 and 10 nM) inhibited protein and RNA accumulation. Because PKC activation by PMA has been shown to inhibit cardiac contraction (29), the effect of staurosporine on spontaneous contraction was determined (Table 1). Treatment with 5 nM staurosporine significantly reduced the rate of contraction after 7 h. After 48 h, the rate of contraction was significantly reduced by 1 and 5 nM staurosporine. Because staurosporine inhibition of growth and contraction occurred at the same concentrations, it was not possible to determine if staurosporine had a direct effect on growth. Effect of contraction, NE, and PMA on nuclear PKC activity. After 20 min exposure of cells to PMA, nuclear
PKC activity increased 5.3-fold over activity in nuclei from control cells (Fig. 5). In earlier experiments (l), - 17% of the PKC activity in control cells was membrane associated and remained unaltered over 48 h of incubation. The percent of PKC activity in the membrane fraction rapidly increased to 49 t 14% after 1 min of PMA treatment (1). A corresponding decrease in cytosolic PKC activity from 83 t 12 to 51 t 14% of total PKC activity was observed after 1 min of treatment. The biologically inactive phorbol ester, 4a-phorbol 12,13-didecanoate, which has previously been shown to lack a growth-stimulating effect (l), did not alter nuclear PKC activity (Fig. 5). After 48 h of contraction, nuclear PKC activity was 218.1 t 27.9 pmol*mg-l*min-l, representing a 107% increase over PKC activity in nuclei from control KClarrested cells, which was 105.6 t 13.3 pmol*mg-l.min-l (Fig. 6A). Nuclear PKC activity was also increased following 48 h of NE treatment. Contrary to the increase in nuclear PKC activity observed after 20 min of PMA
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Fig. 6. Effect of contraction, NE, and PMA on nuclear PKC activity after 48 h. Nuclei from control cells and cells treated for 48 h in absence (A) and presence (B) of indicated concentrations of staurosporine were isolated and PKC activity was assayed as in Fig. 5. Values are means t SE of 4-9 separate preparations. * Significant difference from control
(P c 0.05).
treatment, PKC activity after 48 h of PMA treatment was not elevated (Fig. 6A). This result also was observed in earlier experiments (1). Significantly, increases in nuclear PKC activity after 48 h of contraction and NE treatment were completely inhibited by the addition of staurosporine to the culture medium at concentrations that inhibited growth stimulation (Fig. 6B). Effect of staurosporine on CAMP-dependent protein nase activity. A major problem with the use of PKC
ki-
inhibitors is their ability to inhibit other kinases, albeit at higher concentrations (11). Therefore, total cellular and nuclear CAMP-dependent protein kinase activities were measured to determine their role in the growth-inhibiting effects of staurosporine. Nuclear CAMP-dependent protein kinase activity was unaltered by contraction (3.4 t 0.4 and 2.9 t 0.4 nmolmg-l emin-l for control and contracting cells, respectively). Most importantly ‘, when control and contracting cells were incubated with 5 nM staurosporine, nuclear CAMP-dependent protein kinase activity was unaltered (3.1 t 0.03 and 3.1 t 0.6 nmol mg-l min- l for control and contracting cells, respectively). The ratio of 32P incorporation measured in the absence of CAMP to that measured in the presence of 10 ,uM CAMP averaged 0.037 t 0.005 and was not altered by treatment with 5 nM staurosporine. To further investigate the specificity of staurosporine for PKC, total cellular PKC and CAMP-dependent protein kinase activities were determined in Triton X-100 extracts of cells treated with O-15 nM staurosporine for 48 h. Total kinase activity was solubilized from the cells with 0.5% Triton X-100, followed by sonication and the 100,000-g supernatant used for kinase assays. Cellular PKC activity was 208.9 pmol mg-’ min-l and was reduced in cells incubated with increasing concentrations of staurosporine (Fig. 7). Maximal inhibition of 76% of cellular PKC activity was achieved with 15 nM staurosporine. Incorporation of 32P measured in the absence of l
l
l
9 3 6 Staurosporins
12 15 (nM1
Fig. 7. Effect of staurosporine on total cellular PKC and CAMP-dependent protein kinase activities. Contracting myocytes were treated with indicated concentrations of staurosporine for 48 h. After treatment with 0.5% Triton X-100 and sonication, 100,000-g supernatant was used for kinase assays. PKC activity was measured as Ca2+-phospholipid-dependent 32P incorporation into lysine-rich histone (o), while CAMP-dependent protein kinase activity was measured as 32P incorporation into kemptide in absence (A) and presence (A) of 10 PM CAMP.
CAMP was less than 3.5% of activity in the presence of 10 PM CAMP. In contrast t.o PKC activity, CAMP-dependent protein kinase activity was inhibited only by 16% in cells treated with 15 nM staurosporine (Fig. 7). DISCUSSION
This study demonstrates significant differences in the hypertrophic effects of PMA, NE, contraction, and ANG II when evaluated in the same cell culture model. Significant differences were observed in the time course and degree of growth stimulation in response to these stimuli. The stimulation of growth by these stimuli is in agreement with earlier studies (1, 2, 13, 19, 20). However, the significance of this study is that these growth effects were compared in the same cell culture model. The use of the same culture model and culture media eliminates differences that may be dependent on ceil density or culture media (3) . Contraction was the most potent growth stimulus, resulting in the greatest extent of growth stimulation after 48 h. NE was less potent than contraction, requiring 72 h for maximal growth stimulation. The growth-stimulating effect of PMA was rapid, with maximal effect occurring after 48 h. However, PMA stimulation of growth was less than stimulation by contraction and NE. The mechanism by which these differences in extent and time course of growth stimulation may be elicited is not known but may be related to their relative abilities to activate PKC activity. A common denominator between NE, PMA, ANG II, and contraction is their reported ability to alter PKC activity and distribution (13,22). The existence of type II (p) PKC isozyme in rat liver nuclei (24) and ras-oncogene-induced translocation of PKC to nuclei (9) suggests role for nuclear PKC in the regulation of an important cell growth. Several methods can be used to determine the role of PKC in the growth of cardiomyocytes induced by these stimuli. These include the measurement of PKC activity and d .istribution follow ing treatment with the various agents, demonstra tion th .at addition of exogenous PKC will directly activate transcription, and the use of PKC inhibitors to block growth stimulation. As shown in Figs. 5 and 6, a role for PKC in the growth-stimulating
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effects of contraction, NE, and PMA was supported by an increase in nuclear PKC activity in treated cells compared with KCl-arrested control cells. Addition of exogenous PKC did not stimulate rDNA transcription rate in nuclei isolated from control cells (data not shown). The lack of effect was probably due to the fact that nuclear run-on experiments measure the completion of previously initiated pre-rRNA chains and not the initiation of chains in vitro. The third method by which a role for PKC in growth stimulation can be demonstrated is by the use of putative PKC inhibitors (11, 14, 21, 27). Despite the existence of several putative inhibitors of PKC, their use in studies of the role of PKC in cellular processes is limited, primarily because of lack of specificity of the inhibitors (11). Studies with staurosporine, the most potent PKC inhibitor, have largely been limited to short-term use at high concentrations, 50400 nM (16,27). However, the use of such high concentrations to study slowly developing phenomena such as hypertrophic growth over 48 h is limited by the cytotoxicity of the inhibitors. Initial experiments that involved 50400 nM staurosporine revealed that the drug significantly reduced the number of cells. Staurosporine has been reported to inhibit rat brain PKC and bovine heart PKA with 50% inhibitory concentration values of 10 and 120 nM, respectively (11, 26). Therefore, low concentrations of staurosporine (0.545 nM) were used that did not reduce cell number. These low concentrations also had greater specificity because total cellular and nuclear PKC activity were greatly inhibited without significantly altering CAMP-dependent protein kinase activity. Despite the suggestion of a central role for PKC in growth stimulation, it is possible that under certain circumstances, CAMP-dependent protein kinase may mediate growth stimulation. A role for a CAMP-dependent pathway for acceleration of ribosome formation following increased aortic pressure in perfused hearts was suggested by the observation that increasing aortic pressure resulted in increased CAMP content, CAMP-dependent protein kinase activity, and ribosome formation (28). This hypothesis was further supported by the fact that these effects could be blocked by inhibiting pressure-induced increase in CAMP content with methacholine. The results in this study indicate that, unlike nuclear PKC, whose activity was increased by contraction, nuclear CAMP-dependent protein kinase activity was unchanged. Total cellular and nuclear PKC activities were greatly inhibited by staurosporine with little or no inhibition of CAMP-dependent protein kinase activity. These results suggest that inhibition of CAMP-dependent protein kinase does not play a significant role in the growth-inhibiting effect of staurosporine. The hypothesis that PKC plays a significant role in growth stimulation by PMA, NE, and contraction was supported by the observation that growth could be inhibited by treatment with staurosporine. This hypothesis was further supported by the demonstration that these growth stimuli significantly increased nuclear PKC activity and that this increase was blocked by staurosporine at concentrations that inhibited growth. The differences
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in sensitivity to staurosporine suggest that the differences in the extent and time course of growth stimulation represent differences in the activation of PKC by these growth stimuli. The authors thank Theresa Vrona and Donna Morgan manuscript and Brian Shoop for assistance in preparing This work was supported by the Geisinger Clinic and Heart Association (Pennsylvania Affiliate) fellowship to Address reprint requests to H. E. Morgan. Received
2 October
1991; accepted
in final
form
10 March
for typing this the figures. an American S. N. Allo. 1992.
REFERENCES 1. Allo, S. N., P. J. McDermott, L. L. Carl, and H. E. Morgan. Phorbol ester stimulation of PKC activity and ribosomal DNA transcription: role in hypertrophic growth of cardiomyocytes. J. BioZ. Chem. 266: 22003-22009, 1991. 2. Baker, K. M., and J. F. Aceto. Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H610-H618, 1990. 3. Bishopric, N. H., and L. Kedes. Adrenergic regulation of the skeletal cu-actin gene promoter during myocardial cell hypertrophy. Proc. Natl. Acad. Sci. USA 88: 2132-2136, 1991. 4. Bowman, L. H. rDNA transcription and pre-rRNA processing during the differentiation of a mouse myoblast cell line. Dev. BioZ. 119: 152-163, 1987. 5. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. 6. Camacho, J. A., C. J. Peterson, G. J. White, and H. E. Morgan. Accelerated ribosome formation and growth in neonatal pig hearts. Am. J. Physiol. 258 (Cell Physiol. 27): C86-C91, 1990. 7. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa, and Y. Nishizuka. Direct activation of calciumactivated phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. BioZ. Chem. 257: 7847-7851, 1982. 8. Cesarone, C. F., C. Bolognesi, and L. Santi. Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal. Biochem. 100: 188-197, 1979. 9. Chiarugi, V., L. Magnelli, F. Pasquali, S. Vannucchi, P. Buni, A. Quattrone, G. Basi, S. Capaccioli, and M. Ruggiero. Transformation by ras oncogene induces nuclear shift of protein kinase C. Biochem. Biophys. Res. Commun. 173: 528-533, 1990. 10. Cooper, G., IV, R. L. Kent, C. E. Uboh, E. W. Thompson, and T. A. Marino. Hemodynamic versus adrenergic control of cat right ventricular hypertrophy. J. CZin. Invest. 75: 1403-1414, 1985. 11. Davis, P. D., C. H. Hill, E. Keech, G. Lawton, J. S. Nixon, A. D. Sedgwick, J. Wadsworth, D. Westmacott, and S. E. Wilkinson. Potent selective inhibitors of protein kinase C. FEBS Lett. 259: 61-63, 1989. 12. Drunnmon, P. M., K. Iwaki, S. A. Henderson, A. Sen, and K. R. Chien. Phorbol esters induce immediate-early genes and activate cardiac gene transcription in neonatal rat myocardial cells. J. MoZ. Cell. CardioZ. 22: 901-910, 1990. 13. IIenrich, C. J., and P. C. Simpson. Differential acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes of alpha l-adrenergic and phorbol ester stimulation. J. Mol. Cell. CardioZ. 20: 1081-1085, 1988. 14. Kira, Y., P. J. Kochel, E. E. Gordon, and H. E. Morgan. Aortic perfusion pressure as a determinant of cardiac protein synthesis. Am. J. Physiol. 246 (Cell Physiol. 15): C247-C258, 1984. 15. Kobayashi, E., H. Nakano, M. Morimoto, and T. Tamaoki. Calphostin C (UCN-l028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 159: 548-553, 1989. I., Y. Katoh, T. Kaida, Y. Shibazaki, M. Kuraba16 Komuro, yashi, E. Hoh, F. Takaku, and Y. Yazaki. Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. Possible role of protein kinase C activation. J. BioZ. Chem. 266: 1265-1268, 1991.
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17. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 18. McDermott, P. J., L. L. Carl, K. J. Conner, and S. N. Allo. Transcriptional regulation of ribosomal RNA synthesis during growth of cardiac myocytes in culture. J. Biol. Chem. 266: 44094416, 1991. 19. McDermott, P. J., and H. E. Morgan. Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture. Circ. Res. 64: 542-553, 1989. 20. McDermott, P. J., L. I. Rothblum, S. D. Smith, and H. E. Morgan. Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture. J. Biol. Chem. 264: 18220-18227, 1989. 21. Merrill, A. H., Jr., and V. L. Stevens. Modulation of protein kinase C and diverse cell functions by sphingosine-a pharmacologically interesting compound linking sphingolipids and signal transduction. Biochim. Biophys. Acta 1010: 131-139, 1989. 22. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature Lond. 308: 693-698, 1984. 23. Richter, E. A., P. J. Cleland, S. Rattigan, and M. G. Clark. Contraction-associated translocation of protein kinase C in rat skeletal muscle. FEBS Lett. 217: 232-236, 1987.
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24. Rogue, P., G. Labourdette, A. Masmoudi, Y. Yoshida, F. L. Huang, K. P. Huang, J. Zwiller, G. Vincendon, and A. N. Malviya. Rat liver nuclei protein kinase C is the isozyme type II. J. Biol. Chem. 265: 4161-4165, 1990. 25. Simpson, P., A. McGrath, and S. Savion. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ. Res. 51: 787-801, 1982. 26 Tamaoki, T., H. Nomoto, I. Takahashi, Y. Kato, M. Morim’ oto, and F. Tomita. Staurosporine, a potent inhibitor of phospholipid/Ca”+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135: 397-402, 1986. Twomey, B. M., K. Clay, and M. M. Dale. The protein kinase 27m C inhibitor, K252a, inhibits superoxide production in human neutrophils activated by both PIP,-dependent and -independent mechanisms. Biochem. Pharmacol. 41: 1449-1454, 1991. P. A., T. Haneda, and H. E. Morgan. Effect of 2% Watson, higher aortic pressure on ribosome formation and CAMP content in rat heart. Am. J. Physiol. 256 (Cell Physiol. 25): (X257-C1261, 1989. S., F. A. Sunahara, and A. K. Sen. Tumor-promoting 29. Yuan, phorbol esters inhibit cardiac functions and induce redistribution of protein kinase C in perfused beating rat heart. Circ. Res. 61: 372-378, 1987.
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N. ALLO, and Janet
LOIS L. CARL, AND HOWARD E. MORGAN Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822
Allo, Simon N., Lois L. Carl, and Howard E. Morgan. Acceleration of growth of cultured cardiomyocytes and translocation of protein kinase C. Am. J. Physiol. 263 (CeZZPhysioZ. 32): C319-C325, 1992.-Phorbol 12-myristate 13-acetate (PMA), norepinephrine (NE), and contraction stimulate cardiomyocyte growth (increased protein content). Differences exist in the time course and extent of protein and RNA accumulation. Cells plated at 4 x IOF tells/60-mm dish and arrested with 50 mM KC1 demonstrated no significant growth. Treatment with PMA stimulated growth to a maximum of 17% at 48 h. In contrast, maximal stimulation of growth was 36% at 48 h and 31% at 72 h for contracting and NE-treated cells, respectively. Maximal stimulation of the capacity for protein synthesis (RNA content) was 32% for PMA-treated cells at 24 h compared with 59% and 77% for NE-treated and contracting cells, respectively, at 72 h. In support of a primary role for altered capacity in the regulation of protein synthesis, there was a significant correlation (r = 0.84) between RNA and protein contents that was independent of the stimulus used. Angiotensin II increased RNA content by 28% at 48 h but had no effect on growth up to 72 h. Growth stimulation and increased nuclear protein kinase C (PKC) activity were induced by contraction, NE, and PMA treatment and were inhibited by staurosporine (a PKC inhibitor), suggestive of a central role for PKC. ribosomal nephrine;
ribonucleic angiotensin
acid; hypertrophy; II; staurosporine;
phorbol ester; norepicontraction
OF OUR KNOWLEDGE of cellular changes in cardiac hypertrophy, measured as protein accretion or increased myocyte cross-sectional area, has been obtained in experiments utilizing isolated perfused hearts and primary cardiomyocyte cultures (6, 10, 20, 25, 28). However, studies with isolated myocytes have utilized different culture media, substrata, cell densities, and hypertrophic stimuli such as phorbol esters, norepinephrine (NE), contraction, and angiotensin II (ANG II). These variations become significant in view of the recent demonstration by Bishopric and Kedes (3) that expression of the skeletal cu-actin gene during NE-induced hypertrophic growth was greatly influenced by cell density. When cardiomyocytes were plated at a high density (3-4 x lo6 tells/60-mm dish), NE- and isoproterenol-induced stimulation of skeletal cu-actin gene expression, hyperand contractility were mediated by the trophy, ,&adrenoceptors because these effects were inhibited by ,&adrenergic antagonists but not by cu,-adrenergic antagonists. On the contrary, NE stimulation of cu-myosin heavy chain gene expression in these high-density cultures was mediated by the a,-adrenergic receptor because it was inhibited by al- but not by ,&adrenergic antagonists. The significance of plating density on cellular response was illustrated by the observation that, in contrast to high-density cultures in which NE stimulation of growth was mediated by the fl-adrenergic receptor, the same effect was mediated by the al-adrenoceptor in cardiomyocytes plated at a low density (3).
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0363-6143/92
$2.00 Copyright
In cultures plated at 0.5 X lo6 tells/60-mm dish in minimum essential medium (MEM) containing 10% newborn calf serum for 24 h and maintained in serumsubstituted medium (I), treatment with phorbol l2myristate 13-acetate (PMA) resulted in a 34% increase in both protein and RNA content compared with control values. With the use of the same serum-substituted medium, cells plated at a density of 4 x IO6 tells/60-mm dish and arrested with KC1 were induced to grow by treatment with NE and by contraction (19, 20). Cells plated at 1.5 x lo6 cells/lOO-mm dish in a 1:4 mixture of Dulbecco’s modified Eagle’s medium and medium 199 (GIBCO) demonstrated a 106% and 124% increase in protein content following treatment with PMA and phenylephrine, respectively (12). Therefore, because of differences in the plating densities and culture media used, the comparative hypertrophic effects of the different stimuli remain to be evaluated in the same model. Recently, it was demonstrated that contraction and PMA-induced hypertrophic growth were primarily the result of increased rDNA transcription leading to an increased capacity for protein synthesis, as measured by the RNA content (1, 18). The mechanisms by which these stimuli elicit their hypertrophic effects are not completely understood. Less clear is how the intracellular events initiated by a variety of stimuli may converge to regulate ribosome synthesis at the level of rDNA transcription. A significant role has been suggested for protein kinase C (PKC) in the mechanism of hypertrophic growth stimulation by contraction, PMA, NE, and ANG II (1, 20, 25). The mechanism by which PKC activity and distribution may be altered by these stimuli is not completely understood. PMA acts as an analogue of diacylglycerol (DAG) and directly activates PKC (7). NE and ANG II bind to their respective receptors on the cell surface and result in activation of phospholipase C (PLC). This increase in PLC activity leads to the hydrolysis of phosphoinositol phosphate to yield DAG (the physiological activator of PKC) and inositol trisphosphate. The mechanism by which spontaneous contraction of cultured cardiomyocytes may result in altered PKC activity and distribution remains to be investigated. However, a role for PKC in contractioninduced growth is suggested by the studies of Richter et al. (23), demonstrating contraction-induced PKC translocation to membranes in skeletal muscle. In this study, the hypertrophic effects of several stimuli were compared in the same cultured cardiomyocyte model. Because all of these stimuli are capable of altering cellular PKC activity and distribution, this study begins to address the question of a central role for PKC in the hypertrophic effects of PMA, NE, and con-
0 1992 the American
Physiological
Society
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traction. Staurosporine, a putative PKC inhibitor, inhibited growth stimulation and decreased nuclear PKC activity. EXPERIMENTAL
PROCEDURES
Cardiomyocyte cultures. Neonatal cardiomyocytes were isolated by enzymatic digestion as previously described (I, 19). Cell number was determined with a Coulter counter, and the cells were resuspended in MEM containing 10% newborn calf serum and 0.1 mM 5bromo-Z’-deoxyuridine. The cells were plated at a density of 4 x IO6 tells/60-mm dish precoated with 0.1% gelatin. After an overnight incubation to permit attachment of viable cardiomyocytes, the cells were maintained in serum-substituted medium containing 50 mM KCl. After incubation for 3 days, hypertrophy was initiated by incubating cells in serumsubstituted media containing 50 mM KC1 and either 10y7 M PMA, 2 x lo-” M NE, or 10y6 M ANG II. Contraction was initiated by incubating cells in media containing 5 mM KC1 (19, 20). Stock solutions of PMA and staurosporine in dimethyl sulfoxide (DMSO) were used, and the final concentration of DMSO in each dish was not more than 0.001%. After 24-48 h of contraction, ATP content was 24 +- 2.5 pmol/g protein, which was unchanged from quiescent cells that were incubated in 50 mM KC1 (23 t 2.1 pmol/g protein). These values are 80% of those measured in isolated perfused rat hearts (14). Maintenance of creatine phosphate content depended upon addition of creatine (5 mM) to the incubation medium. In the absence of creatine, the contents of creatine phosphate were 7 t 1.3 and 7 +- 1.4 pmol/g protein in contracting and quiescent cells, respectively. In the presence of 5 mM creatine, these values increased to 67 t 4.4 and 49 I~I 3.8 pmol/g protein for contracting and quiescent cells, respectively. The creatine phosphate content of isolated perfused rat hearts averaged 45 ,umol/g protein (14). Maintenance of ATP content in cultured cardiomyocytes indicated that growth of these cells was not restrained by energy deficiency. Determination of cellular protein and RNA contents. Cellular protein content was used as a measure of cell growth. Dishes of cells were rinsed three times with phosphate-buffered saline (PBS). The cells were scraped with I ml of sodium citrate buffer containing 0.25% sodium dodecyl sulfate (SDS) [standard saline citrate (SSC)/SDS] and stored at -20°C. The samples were thawed and vortexed, and duplicate aliquots of 25 ~1 were assayed for protein by the method of Lowry et al. (17) using bovine serum albumin (BSA) as a standard. DNA concentration was determined fluorometrically in duplicate loo-p1 aliquots as previously described (8) using calf thymus DNA as a standard. The capacity for protein synthesis was estimated by measuring the RNA content. Briefly, RNA in 0.5-ml aliquot of SSC/ SDS homogenate of cells was precipitated and washed three times with 0.5 ml 0.5 N perchloric acid (PCA). The pellet was hydrolyzed in 750 ~10.3 N NaOH at 37°C for 24 h. The sample was neutralized with 250 ~1 4 N PCA, centrifuged, and the absorbance at 232 and 260 nm of the supernatant was measured. The RNA concentration was calculated as previously described (19,
20).
Isolation of nuclei. Nuclei were isolated as previously described (1, 4). Cells were rinsed three times with 3 ml of PBS and once with 1 ml buffer 1 containing 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 8.0, 10 mM NaCl, 2.5 mM MgC1,, and 5 mM dithiothreitol. The cells in each dish were incubated on ice in 0.5 ml of buffer 1 for IO min. After addition of an equal volume of buffer 1 supplemented with 0.6 M sucrose and 0.6% Triton X-100, the cells were scraped and homogenized gently in a Dounce homogenizer with two strokes of an A pestle. The homogenate was layered over an equal volume of buffer 1
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containing 0.6 M sucrose and centrifuged at 1,500 g for 10 min at 4°C. The pellet was resuspended in sucrose buffer [250 mM 100 mM 3-(N-morpholino)propanesulfonic acid sucrose, (MOPS), 2.4 mM ethylene glycol-his@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2.0 mM EDTA, 100 PM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride] containing 0.5% Triton X-100. The nuclear suspension was sonicated five times for IO s each and centrifuged at 100,000 g for 30 min. The supernatant was used to measure PKC activity. Determination of PKC activity. Nuclear PKC activity was measured in a reaction mixture containing 60 pug histone III-S, 15 mM Tris HCl, pH 7.5, 10 mM MgCl,, 1 mM CaCl,, 266 pg/ml phosphatidylserine, 18 pg/ml diolein, and 10 PM [y-:j2P]ATP (6 X IO6 cpm) in a final volume of 300 ~1 (1). The reaction was started by the addition of 4-6 pg sample protein and incubated at 30°C for 5 min. The reaction was terminated by spotting 35 ~1 of the reaction mixture onto a 2.5 x 2 cm Whatman P81 phosphocellulose paper, which was immediately dropped into 75 mM H,,PO,. The filters were washed a total of five times for 15 min each with 75 mM H:,P04, followed by a 100% ethanol wash. The filters were dried and counted for radioactivity. The reaction rate was linear for 10 min, and all experiments were performed for 5 min. Protein concentration was measured with a Bio-Rad protein dye based on the procedure by Bradford (5). PKC activity was determined by subtracting the Ca’+-phospholipid-independent :j2P incorporation measured in the presence of 2 mM EGTA from incorporation measured in the presence of Ca2+, diolein, and phosphatidylserine. Initial experiments were conducted to demonstrate that the nuclear PKC was solubilized following detergent treatment and sonication of the nuclei. No PKC activity was measurable in the 100,000-g pellet from solubilized nuclei of either control or contracting cells. The kinase activity in the absence of Ca2+phospholipid was 64.6 t 8.9 pmol .rngg. minl, while activity in the presence of Ca’+ -phospholipid was 53.9 t 5.0 pm01 . mg+ . min. These activities were not altered in the 100,000-g pellets obtained from contracting cells (63.5 t 2.8 and 55.7 t 5.3 pmol rng-l. min- 1 for activity in the absence and respectively). Therefore, PKC presence of Ca”+ -phospholipid, activity in the 100,000-g supernatant represented the total activity in the nuclei. Determination of adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase activity. CAMP-dependent protein kinase activity was measured in a reaction mixture containing 50 mM MOPS, 10 mM MgC12, 0.25 mg/ml BSA, 75 pg/ml kemptide, and 0.1 mM [r-:j”P]ATP (1 x lo6 cpm) in a total volume of 0.1 ml. After incubation for 5 min at 3O”C, the reaction was started by the addition of 5 pg of sample protein (28). After 5-min incubation, the reaction was terminated by spotting 50 ~1 of mixture onto a 2.5 X 2-cm Whatman P81 phosphocellulose paper. The filters were processed as described above for PKC assays, and the radioactivity was measured. Protein kinase activity was determined in the absence and presence of IO PM CAMP. Statistical analysis. Data are expressed as the means t SE of the indicated number of separate determinations. The statistical analysis of differences between multiple groups was performed by a one-way analysis of variance followed by the Student’s t test. A value of P < 0.05 was taken to indicate a statistically significant difference between the groups compared. RESULTS
Characterization of the effect of stimuli on cardiomyocyte growth and the capacity for protein synthesis. The initial phase of this study characterized hypertrophic
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growth of cardiomyocytes in response to PMA, contracTable 1. Effect of staurosporine on rate of contraction tion, NE, and ANG II using the same cell culture model. of cardiomyocytes In agreement with previous studies, cardiomyocytes plated at a density of 4 x lo6 tells/60-mm dish and arBeats/min Beats/min Staurosporine, nM After 7 h After 48 h rested with 50 mM KC1 did not demonstrate synchronous contraction and exhibited no significant growth (Fig. 1). 63+9 162+16 However, when cardiomyocytes were exposed to 5 mM i.5 49+21 144f7 71+16 112f13* KCI, they began to contract spontaneously. These cells 16+8* .: 69+19* contracted at a rate of 63 of: 9 beats/min after 7 h and Contraction was initiated in cardiomvocvtes bv a reduction of lKCl1 achieved a maximum rate of contraction of 162 + 16 in medium from 50 to 5 mM. Simultaneously, staurosporine was added beats/min after 48 h (Table 1). Incubation of 50 mM to achieve indicated final concentration. Rate of contraction in control KCl-arrested cells with lop7 M PMA or 2 X lop6 M NE (50 mM KCl-arrested cells) was zero. Values are means k SE of 4 dishes. stimulated protein accumulation without induction of * Significant difference from rate in absence of staurosporine. contraction. Reduction of KC1 concentration from 50 to 5 and, in this case, by ANG II (Fig. 2). A significant inmM KC1 induced spontaneous contraction and resulted crease of RNA content by PMA was observed after 24 h. in increased protein accumulation. Significant differences in the extent and time course of the stimulation of This increase in RNA content, 31% over control, was the stimulation observed for PMA-treated cells. protein accumulation by PMA, NE, and contraction were maximum RNA content in NE-treated and contracting myocytes observed (Fig. 1). Increased protein content by PMA, contraction, and NE were initially observed after 48 h. In was significantly elevated after 48 h. In contrast to PMAtreated cells, maximal increases of 59% and 77% were contrast to PMA and contraction, where longer incubations did not result in further increases in protein con- observed after 72 h for NE-treated and contracting cells, respectively (Fig. 2). RNA content in ANG II-treated tent, a significant further increase was obtained with NE. 28% over control Maximal stimulation of growth by PMA treatment or cells was increased by approximately contraction were 17 and 36%, respectively, after 48 h. In after 48 h. In support of the hypothesis that increased contrast, maximal stimulation of growth by NE was 31% capacity for protein synthesis plays a central role in the growth of cardiomyocytes, a significant correlation (r = observed after 72 h. ANG II was not an effective growth 0.84) between RNA and protein content was observed stimulus in that protein content was not significantly increased after either 48 or 72 h of treatment with low6 M (Fig. 3). Effect of staurosporine on growth and the capacity for ANG II. protein synthesis. The regulation of several cellular proIn previous studies, it was demonstrated that contraccesses by PKC has been examined with the use of several tion and PMA-induced hypertrophic growth were primarily the result of increased capacity for protein syn- potent inhibitors (11, 21, 27). A role for PKC in PMA-, growth of cardiomyocytes thesis, as measured by RNA content (1,19,20). Similar to NE-, and contraction-induced was examined with the use of the PKC inhibitor, staurothe effects on protein content, significant differences were observed in the extent and time course of accelera- sporine. To correct for possible reductions in protein and RNA contents that may be due to the cytotoxic effects of tion of RNA accumulation by PMA contraction, NE, high concentrations of staurosporine, the data were nor-5OmM K 50mM K 05mM K 050mM K D50mM K malized to the DNA content. The concentrations of stau+ PMA +NE +ANG II rosporine used in this study (0.5-15 nM) did not reduce cell numbers. Similarly, treatment of KCl-arrested cells -5OmM
g 1100 5 25 2 1000 .G 2 2 900 a 3 ij 800 I-
K 050mM
+ PMA
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K (50mM
+ NE
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-ANG
K II
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i, T n 0 hr
24 hr
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Time (h) Fig. 1. Effect of stimuli on cardiomyocyte protein accumulation. Cardiomyocyte hypertrophic growth was initiated on day 4 in culture (time 0) by exposure to 50 mM KC1 (control), 50 mM KC1 + 10m7 M phorbol 12-myristate Is-acetate (PMA), 5 mM KC1 (contracting), 50 mM KC1 + 2 x 10mfiM norepinephrine (NE), and 50 mM KC1 + 10-a M ANG II. Protein content was measured over 3 days of treatment. DNA content did not change as result of these treatments (19, 20). Values are means i SE of 4-7 separate preparations. * Significant difference from control cells (P < 0.05)
0 hr
24
r
48
r
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*1
Time (h) Fig. 2. Effect of stimuli on capacity for protein synthesis. Capacity for protein synthesis (RNA content) was determined in cells treated as in Fig. 1. Data points are means + SE of 4-8 separate preparations. * Significant difference from control (P < 0.05).
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800 .z s 600 \E z 400 0 200 15
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Fig. 3. Relationship between protein and RNA content. Protein content was plotted against corresponding RNA content for each dish of cells. A significant correlation (r = 0.84) was observed between protein and RNA contents.
with staurosporine did not significantly alter growth, suggesting that staurosporine concentrations up to 15 nM were not toxic for arrested cells (Fig. 4). Treatment with PMA increased protein content, measured as protein/DNA, by 18% (Fig. 4A). This PMAinduced increase in protein content was completely inhibited by 10 nM staurosporine (Fig. 4A). As expected, the capacity for protein synthesis was significantly increased by PMA treatment. Treatment with low concentrations of staurosporine (0.5-5 nM) had no significant effect on PMA-induced RNA accumulation (Fig. 4B). However, this increase was partially inhibited by 10 nM staurosporine. A 55
c +--Control +Contracting ..O..pMA ..f-J...NE
30
B
I 2
’ 0
I 4 Staurosporine,
I 6
I 8
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nM
2.0 1.8
T
+Control -W-Contracting ..O..pMA ..a..NE
< 1.6 6 Il.4 5 K 1.2 1.0 0.8
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4 Staurosporine,
I
6
I
I
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Fig. 4. Effect of staurosporine on growth induced by PMA, NE, and contraction. Quiescent cardiomyocytes (50 mM KCl) were treated with 10y7 M PMA or 2 x 10B6 M NE in presence of increasing concentrations of staurosporine for 72 h. Contraction was initiated in cells by incubation in serum-substituted medium containing 5 mM KC1 for 72 h. Protein/DNA (A) and RNA/DNA (B) were determined as described in EXPERIMENTAL PROCEDURES. Data points are means t SE of 4-9 separate preparations except at 10 nM staurosporine where value is mean of 2 separate experiments for contracting cells. * Significant difference from control (P < 0.05).
CONTROL PMA ICY-PHORBOL Fig. 5. Effect of PMA and 4cu-phorbol l&13-didecanoate nuclear protein kinase C (PKC) activity after 20 min. control and treated cells were solubilized with 0.5% Triton 100,000-g supernatant was used for PKC assays. PKC measured and expressed as Ca2+ -phospholipid-dependent ity. Values are means t SE of 3-4 separate preparations. difference from control (P < 0.05).
treatment on Nuclei from X-100, and activity was kinase activ* Significant
Treatment with NE for 72 h increased protein/DNA (Fig. 4A) and RNA/DNA (Fig. 4B) by 17% and 25%, respectively. These growth effects were completely inhibited by 0.5-10 nM staurosporine. When myocytes were incubated in media containing 5 mM KC1 to induce contraction, protein and RNA contents were increased by 32% and 56%, respectively, after 72 h (Fig. 4, A and B). Treatment of contracting myocytes with 0.5 and I nM staurosporine did not significantly alter growth stimulation. However, higher concentrations of staurosporine (5 and 10 nM) inhibited protein and RNA accumulation. Because PKC activation by PMA has been shown to inhibit cardiac contraction (29), the effect of staurosporine on spontaneous contraction was determined (Table 1). Treatment with 5 nM staurosporine significantly reduced the rate of contraction after 7 h. After 48 h, the rate of contraction was significantly reduced by 1 and 5 nM staurosporine. Because staurosporine inhibition of growth and contraction occurred at the same concentrations, it was not possible to determine if staurosporine had a direct effect on growth. Effect of contraction, NE, and PMA on nuclear PKC activity. After 20 min exposure of cells to PMA, nuclear
PKC activity increased 5.3-fold over activity in nuclei from control cells (Fig. 5). In earlier experiments (l), - 17% of the PKC activity in control cells was membrane associated and remained unaltered over 48 h of incubation. The percent of PKC activity in the membrane fraction rapidly increased to 49 t 14% after 1 min of PMA treatment (1). A corresponding decrease in cytosolic PKC activity from 83 t 12 to 51 t 14% of total PKC activity was observed after 1 min of treatment. The biologically inactive phorbol ester, 4a-phorbol 12,13-didecanoate, which has previously been shown to lack a growth-stimulating effect (l), did not alter nuclear PKC activity (Fig. 5). After 48 h of contraction, nuclear PKC activity was 218.1 t 27.9 pmol*mg-l*min-l, representing a 107% increase over PKC activity in nuclei from control KClarrested cells, which was 105.6 t 13.3 pmol*mg-l.min-l (Fig. 6A). Nuclear PKC activity was also increased following 48 h of NE treatment. Contrary to the increase in nuclear PKC activity observed after 20 min of PMA
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HYPERTROPHIC m
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50mM
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. I s 1E %
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Fig. 6. Effect of contraction, NE, and PMA on nuclear PKC activity after 48 h. Nuclei from control cells and cells treated for 48 h in absence (A) and presence (B) of indicated concentrations of staurosporine were isolated and PKC activity was assayed as in Fig. 5. Values are means t SE of 4-9 separate preparations. * Significant difference from control
(P c 0.05).
treatment, PKC activity after 48 h of PMA treatment was not elevated (Fig. 6A). This result also was observed in earlier experiments (1). Significantly, increases in nuclear PKC activity after 48 h of contraction and NE treatment were completely inhibited by the addition of staurosporine to the culture medium at concentrations that inhibited growth stimulation (Fig. 6B). Effect of staurosporine on CAMP-dependent protein nase activity. A major problem with the use of PKC
ki-
inhibitors is their ability to inhibit other kinases, albeit at higher concentrations (11). Therefore, total cellular and nuclear CAMP-dependent protein kinase activities were measured to determine their role in the growth-inhibiting effects of staurosporine. Nuclear CAMP-dependent protein kinase activity was unaltered by contraction (3.4 t 0.4 and 2.9 t 0.4 nmolmg-l emin-l for control and contracting cells, respectively). Most importantly ‘, when control and contracting cells were incubated with 5 nM staurosporine, nuclear CAMP-dependent protein kinase activity was unaltered (3.1 t 0.03 and 3.1 t 0.6 nmol mg-l min- l for control and contracting cells, respectively). The ratio of 32P incorporation measured in the absence of CAMP to that measured in the presence of 10 ,uM CAMP averaged 0.037 t 0.005 and was not altered by treatment with 5 nM staurosporine. To further investigate the specificity of staurosporine for PKC, total cellular PKC and CAMP-dependent protein kinase activities were determined in Triton X-100 extracts of cells treated with O-15 nM staurosporine for 48 h. Total kinase activity was solubilized from the cells with 0.5% Triton X-100, followed by sonication and the 100,000-g supernatant used for kinase assays. Cellular PKC activity was 208.9 pmol mg-’ min-l and was reduced in cells incubated with increasing concentrations of staurosporine (Fig. 7). Maximal inhibition of 76% of cellular PKC activity was achieved with 15 nM staurosporine. Incorporation of 32P measured in the absence of l
l
l
9 3 6 Staurosporins
12 15 (nM1
Fig. 7. Effect of staurosporine on total cellular PKC and CAMP-dependent protein kinase activities. Contracting myocytes were treated with indicated concentrations of staurosporine for 48 h. After treatment with 0.5% Triton X-100 and sonication, 100,000-g supernatant was used for kinase assays. PKC activity was measured as Ca2+-phospholipid-dependent 32P incorporation into lysine-rich histone (o), while CAMP-dependent protein kinase activity was measured as 32P incorporation into kemptide in absence (A) and presence (A) of 10 PM CAMP.
CAMP was less than 3.5% of activity in the presence of 10 PM CAMP. In contrast t.o PKC activity, CAMP-dependent protein kinase activity was inhibited only by 16% in cells treated with 15 nM staurosporine (Fig. 7). DISCUSSION
This study demonstrates significant differences in the hypertrophic effects of PMA, NE, contraction, and ANG II when evaluated in the same cell culture model. Significant differences were observed in the time course and degree of growth stimulation in response to these stimuli. The stimulation of growth by these stimuli is in agreement with earlier studies (1, 2, 13, 19, 20). However, the significance of this study is that these growth effects were compared in the same cell culture model. The use of the same culture model and culture media eliminates differences that may be dependent on ceil density or culture media (3) . Contraction was the most potent growth stimulus, resulting in the greatest extent of growth stimulation after 48 h. NE was less potent than contraction, requiring 72 h for maximal growth stimulation. The growth-stimulating effect of PMA was rapid, with maximal effect occurring after 48 h. However, PMA stimulation of growth was less than stimulation by contraction and NE. The mechanism by which these differences in extent and time course of growth stimulation may be elicited is not known but may be related to their relative abilities to activate PKC activity. A common denominator between NE, PMA, ANG II, and contraction is their reported ability to alter PKC activity and distribution (13,22). The existence of type II (p) PKC isozyme in rat liver nuclei (24) and ras-oncogene-induced translocation of PKC to nuclei (9) suggests role for nuclear PKC in the regulation of an important cell growth. Several methods can be used to determine the role of PKC in the growth of cardiomyocytes induced by these stimuli. These include the measurement of PKC activity and d .istribution follow ing treatment with the various agents, demonstra tion th .at addition of exogenous PKC will directly activate transcription, and the use of PKC inhibitors to block growth stimulation. As shown in Figs. 5 and 6, a role for PKC in the growth-stimulating
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effects of contraction, NE, and PMA was supported by an increase in nuclear PKC activity in treated cells compared with KCl-arrested control cells. Addition of exogenous PKC did not stimulate rDNA transcription rate in nuclei isolated from control cells (data not shown). The lack of effect was probably due to the fact that nuclear run-on experiments measure the completion of previously initiated pre-rRNA chains and not the initiation of chains in vitro. The third method by which a role for PKC in growth stimulation can be demonstrated is by the use of putative PKC inhibitors (11, 14, 21, 27). Despite the existence of several putative inhibitors of PKC, their use in studies of the role of PKC in cellular processes is limited, primarily because of lack of specificity of the inhibitors (11). Studies with staurosporine, the most potent PKC inhibitor, have largely been limited to short-term use at high concentrations, 50400 nM (16,27). However, the use of such high concentrations to study slowly developing phenomena such as hypertrophic growth over 48 h is limited by the cytotoxicity of the inhibitors. Initial experiments that involved 50400 nM staurosporine revealed that the drug significantly reduced the number of cells. Staurosporine has been reported to inhibit rat brain PKC and bovine heart PKA with 50% inhibitory concentration values of 10 and 120 nM, respectively (11, 26). Therefore, low concentrations of staurosporine (0.545 nM) were used that did not reduce cell number. These low concentrations also had greater specificity because total cellular and nuclear PKC activity were greatly inhibited without significantly altering CAMP-dependent protein kinase activity. Despite the suggestion of a central role for PKC in growth stimulation, it is possible that under certain circumstances, CAMP-dependent protein kinase may mediate growth stimulation. A role for a CAMP-dependent pathway for acceleration of ribosome formation following increased aortic pressure in perfused hearts was suggested by the observation that increasing aortic pressure resulted in increased CAMP content, CAMP-dependent protein kinase activity, and ribosome formation (28). This hypothesis was further supported by the fact that these effects could be blocked by inhibiting pressure-induced increase in CAMP content with methacholine. The results in this study indicate that, unlike nuclear PKC, whose activity was increased by contraction, nuclear CAMP-dependent protein kinase activity was unchanged. Total cellular and nuclear PKC activities were greatly inhibited by staurosporine with little or no inhibition of CAMP-dependent protein kinase activity. These results suggest that inhibition of CAMP-dependent protein kinase does not play a significant role in the growth-inhibiting effect of staurosporine. The hypothesis that PKC plays a significant role in growth stimulation by PMA, NE, and contraction was supported by the observation that growth could be inhibited by treatment with staurosporine. This hypothesis was further supported by the demonstration that these growth stimuli significantly increased nuclear PKC activity and that this increase was blocked by staurosporine at concentrations that inhibited growth. The differences
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in sensitivity to staurosporine suggest that the differences in the extent and time course of growth stimulation represent differences in the activation of PKC by these growth stimuli. The authors thank Theresa Vrona and Donna Morgan manuscript and Brian Shoop for assistance in preparing This work was supported by the Geisinger Clinic and Heart Association (Pennsylvania Affiliate) fellowship to Address reprint requests to H. E. Morgan. Received
2 October
1991; accepted
in final
form
10 March
for typing this the figures. an American S. N. Allo. 1992.
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