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Am J Physiol Heart Circ Physiol 277: H1808-H1816, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 5, H1808-H1816, November 1999

Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo

L. Kim1, T. Lee1, J. Fu1, and M. E. Ritchie1,2,3

1 Division of Cardiology and Cardiovascular Research Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0542; 2 Division of Cardiology, Veterans Affairs Medical Center, Cincinnati, Ohio 45222; and 3 Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation appear important in conferring hypertrophy in vitro. However, the response of PKC and MAP kinase to stimuli known to induce hypertrophy in vivo has not been determined. We recently demonstrated that pressure-overload hypertrophy induced a transiently transfected gene driven by an hypertrophy responsive enhancer (HRE) through a marked increase in binding activity of its interacting nuclear factor (HRF). These data suggested that the HRE/HRF could serve as a target for evaluating the signal transduction events responsible for hypertrophy in vivo. Accordingly, we characterized MAP kinase and PKC isoform activation, injected HRE driven reporter gene expression, and HRF binding activity in rat hearts subjected to ascending aortic clipping or sham operation in the presence of the angiotensin-converting enzyme (ACE) inhibitor fosinopril, hydralazine, or no treatment. Analyses showed that PKC-epsilon and MAP kinase were acutely activated following ascending aortic ligature and that fosinopril significantly inhibited but did not completely abrogate PKC-epsilon and MAP kinase activation. However, fosinopril completely prevented pressure overload-mediated induction of HRE containing constructs and obviated increased HRF binding activity. These results suggest a direct relationship between ACE activity and HRE/HRF-mediated gene activation and imply that PKC-epsilon and MAP kinase may be involved in transducing this signal.

angiotensin-converting enzyme; protein kinase C; mitogen-activated protein kinase; BCK promoter; gene activation; hypertrophy; nuclear factors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STRETCH-INDUCED HYPERTROPHY of cultured neonatal cardiocytes appears to be due to autocrine release of angiotensin II (ANG II) (31). ANG II, through the angiotensin 1 (AT1) receptor, rapidly activates protein kinase C (PKC) (29). Activation of PKC is associated with rapid and abbreviated induction of mitogen-activated protein (MAP) kinase (28). ANG II stimulation also induces the immediate early response genes c-fos and Egr-1 (31). The mechanical stretch responsive region of the c-fos gene in vitro and ex vivo maps to the serum response element and requires the PKC-mediated formation and binding of the SRF/p62TCF ternary complex (1, 28).

ANG II also appears to play a major role in cardiac hypertrophy in rats in vivo. Nonantihypertensive doses of the angiotensin-converting enzyme (ACE) inhibitor fosinopril and the AT1 receptor blocker TCV-116 cause regression of ascending aortic banding-induced and spontaneous hypertension-induced cardiac hypertrophy, respectively (18, 35). In vivo hypertrophy is also associated with changes in PKC: 2 wk of ascending aorta ligature leads to an accumulation of PKC-beta 1,2 and PKC-epsilon isoforms in hypertrophied adult rat hearts (11). However, the initial response of PKC to hypertrophic stimuli in vivo, which in vitro may be directly involved in the signaling events producing hypertrophy, has not been assessed. In addition, the role of MAP kinase in vivo has not been determined. Moreover, a relationship between these signaling events and the genotypic response of hypertrophy in vivo has not been established.

We recently demonstrated that acute pressure overload hypertrophy transcriptionally induces an injected luciferase reporter gene under the control of an enhancer, which we have named the hypertrophy response element (HRE) through a marked increase in binding activity of its interacting nuclear factor (hypertrophy response factor, HRF) (27). Thus we have a model for evaluating some of the events of hypertrophy-mediated gene activation. Accordingly, to begin to determine the signal transduction pathway utilized and identify the DNA elements and nuclear factors responsible for hypertrophy-mediated gene activation in vivo, we characterized MAP kinase and PKC activation, transiently transfected reporter gene expression, and nuclear factor binding activity acutely and subacutely in rat hearts subjected to ascending aortic clipping or sham operation in the presence of fosinopril, hydralazine, or no treatment.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Acute pressure overload hypertrophy was induced by ascending aortic constriction as previously described (11). Sprague-Dawley rats, 10-12 wk old, 250-350 g, were anesthetized (0.37 mg/g ip chloral hydrate), the left thorax was opened at the second intercostal space, and the ascending aorta was isolated. A Weck hemoclip was placed on the ascending aorta proximal to all great vessels and constricted to an area of 1.0 mm2. The wound was closed, a few puffs of positive pressure room air ventilation from a Harvard Rodent Ventilator were given, and the animals recovered. Sham-operated animals underwent the same procedure but the clip was not closed. Injection of DNA-containing solutions into the apex of the left ventricle was performed as described (7, 12-14). Briefly, the left thorax was opened at the sixth intercostal space, the heart was externalized, and a solution containing 50 µg of experimental and 15 µg of pSV2CAT (to control for transfection efficiency) DNA was injected into the apex. The wound was closed, and the animals recovered with positive pressure ventilation for 2-5 min before aortic ligature was performed. All animals were killed 5 days after surgery for analysis. This model allows for the in vivo analysis of gene expression in response to acute pressure overload hypertrophy and has been reported (22, 26, 27). For drug treatment, rats received 50 mg · kg-1 · day-1 fosinopril or 10 mg · kg-1 · day-1 hydralazine in their drinking water for 7 days before the operation and were continued on this regimen until they were killed. Hydralazine was chosen because it is a known afterload-reducing agent that does not function through the renin-angiotensin system. As a result it has been used by numerous other authors as a control for drug treatment with an ACE inhibitor. This dose of fosinopril reduces cardiac ACE activity by 75%, and neither dosing regimen is antihypertensive (18, 35). We found similar results in our experiments: neither fosinopril (systolic blood pressure 99 ± 3.5 mmHg vs. 104 ± 4.3 in water-treated rats) nor hydralazine (systolic blood pressure 102 ± 2.3) significantly affected blood pressure as determined by direct femoral artery measurements.

Plasmid DNA preparation. BCK57LUC consists of that portion of the BCK promoter (from -92 to +57) previously shown to contain the element (+25 to +57) necessary for developmental expression of BCK in the heart in vitro and in vivo (26). Subsequent analyses have shown that the +25 to +57 element is capable of conferring hypertrophy responsiveness to its cognate as well as an heterologous promoter (27). The element was therefore termed a hypertrophy responsive element (HRE) and the factor interacting with it, hypertrophy responsive factor (HRF). pSV2CAT was generously donated by Dr. Muthu Periasamy.

Cytoplasmic protein and nuclear extract preparation. All protein work was performed at 4°C. Procedures are derivations of standard protocols (2, 11, 23). Hearts were washed immediately with ice-cold PBS, and the left ventricle was isolated and weighed. The left ventricular apex was divided into three equal portions. Two portions were used for PKC analyses (see below) and the remainder was used for MAP kinase analyses. Hearts isolated 5 days after injection were similarly treated and were also used for transient transfection and electromobility shift assays (EMSA) analyses. For MAP kinase analyses, heart was minced in whole heart homogenization buffer [WHHB (in mM): 25 glycylglycine, 15 MgSO4, 4 EGTA, pH 8.0, 1 dithiothreitol (DTT), 0.2 phenylmethylsulfonyl fluoride (PMSF) (2, 22, 23)]. This minced solution was homogenized with 10-12 strokes of a motor-driven Teflon homogenizer. The homogenate was removed and pelleted at 6,000 g for 10 min in a microcentrifuge. The supernatant was transferred to a fresh tube, and the volume was quantified. This supernatant was retained for luciferase and chloramphenicol acetyltransferase (CAT) assays. The pelleted cells were then resuspended in hypotonic buffer (in mM, 10 HEPES, pH 7.9, 1.5 magnesium chloride, 10 potassium chloride, 0.2 PMSF, and 0.5 DTT) five times the packed cellular volume and quickly centrifuged at 1,850 g for 5 min. The supernatant was discarded, and the pellet was resuspended in hypotonic buffer three times the original packed cellular volume. After 15 min of swelling on ice, cells were transferred to a glass Dounce homogenizer, homogenized with 25-30 strokes of a type B pestle, and centrifuged for 15 min at 3,300 g. The cytoplasmic protein containing supernatant was kept for MAP kinase analyses. The pelleted nuclei were resuspended in one-half packed nuclear volume of low-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM magnesium chloride, 0.02 M potassium chloride, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT). In a drop-wise fashion, a high-salt buffer (20 mM HEPES, pH 7.9, 5% glycerol, 1.5 mM magnesium chloride, 1.2 M potassium chloride, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT) of an equal volume was then added. Nuclear proteins were extracted for 30-45 min on a shaker at 0-4°C. The extracted nuclei were pelleted by centrifuging for 30 min at 25,000 g. The nuclear protein containing supernatant was then dialyzed, precipitated proteins were pelleted by centrifugation for 5 min in a microcentrifuge, and the nuclear proteins in the supernatant were kept. All proteins were quantified by standard Bradford assay.

Isolation of proteins for PKC analysis. A portion of the isolated tissue was minced and homogenized in PKC WHHB (WHHB plus 2 mM EDTA, 25 µg/ml leupeptin, and 10 µg/ml aprotinin). The homogenate was removed and pelleted at 6,000 g for 10 min in a microcentrifuge. The supernatant was transferred to a fresh tube and frozen at -80°C. The pelleted cells were then resuspended in PKC hypotonic buffer (hypotonic buffer plus 2 mM EDTA, 5 mM EGTA, 25 µg/ml leupeptin, and 10 µg/ml aprotinin) five times and then three times the packed cellular volume as above and allowed to swell on ice. Cells were dounced and centrifuged as above, and the cytoplasmic protein containing supernatant was kept for analysis. The pelleted nuclei and membrane fragments were resuspended in PKC hypotonic buffer plus 1% vol/vol Triton X-100 and extracted for 30 min. This solution was then centrifuged at 25,000 g for 15 min. The supernatant contains membrane-associated PKC isoforms and was kept for analysis. For determination of total PKC, 1% vol/vol Triton X-100 was added to PKC hypotonic buffer. The protocol was followed as above except supernatants were combined and protein content was quantified.

Immunoblots. Ten micrograms of isolated protein were separated by 7.5% SDS-PAGE using standard methods. For determination of MAP kinase activation, separated proteins were transferred to Immobilon-P (polyvinylidene difluoride) transfer membranes (Millipore) and evidence for MAP kinase activation was determined using a commercially available MAP kinase kit (New England Biolab). For PKC analyses separated proteins were transferred to Immunolite blotting membrane (Bio-Rad). Isoform specific PKC antibodies were obtained from Promega and detected using an Immunolite Assay Kit (Bio-Rad). PKC isoform translocation to the membrane fraction was taken as evidence of PKC activation. MAP kinase and PKC activation were quantified using Adobe Photoshop software.

Statistical analyses. Statistical significance of PKC and MAP kinase experiments were determined by ANOVA.

EMSAs. EMSAs were performed using a 1/2× Tris-borate-EDTA buffer 5% polyacrylamide gel electrophoresed for 2.5 h at 160 mV at +4°C. Typically 10 µg of nuclear protein were incubated with 2 µg of poly(dI/dC), 10,000 counts/min of radiolabeled probe in an aqueous solution with a final concentration of 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 1 mM EDTA, 60 mM KCl, 1 mM DTT, and 5% glycerol. For competition experiments, an indicated molar excess amount of unlabeled oligonucleotide was added to the solution and incubated for 10 min before the addition of the labeled probe. The probe is listed below and the proposed HRF binding site is double underlined
+25+41+57

HRE:agctgccgacggacgg<UNL>agcgcccccgcccccgc</UNL>

Transient transfection assays. After measurement of the supernatant volume, samples were isolated for CAT and luciferase assays. The supernatant (100 µl) was heated to 60°C for 7 min, cooled immediately on ice, and centrifuged for 5 min in a microcentrifuge. For assessment of transfection efficiency 30 µl were used in a standard CAT assay. Only samples demonstrating a degree of transfection efficiency reflected by CAT conversion greater than 0.5% were included for analysis. CAT conversion averaged 4.3% with a range of 0.5-14%. Of note, background CAT conversion of sham or pSV0CAT injected samples was always much less than 0.1%. Hence, all data reported in this paper are derived from CAT conversions within the linear range. For luciferase assays, Triton was added to 200 µl of the supernatant for a final Triton concentration of 0.27%. Absolute luciferase activity was determined on 10 µl of this solution by standard assay (Promega). Relative luciferase activity of individual constructs was determined by normalizing for transfection efficiency and volume of extract (7, 12-14, 26, 27).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Analyses of PKC isoforms. The effect of surgery and acute pressure overload on isoform abundance was determined initially. Two weeks of hypertrophy results in PKC-beta 1,2 and PKC-epsilon accumulation (11). However, isoform abundance has not been determined in the acute setting in vivo. In addition to PKC-beta 1,2 and PKC-epsilon , PKC-alpha has been described in isolated adult rat ventricular myocytes (3, 25). Hence, abundance of PKC-alpha , -beta 1,2, and -epsilon was assessed. PKC-gamma was also analyzed and served as a negative control. Cell lysates from adult rat hearts subjected to 5, 15, and 30 min of ascending aortic ligature or sham operation were analyzed for total isoform specific abundance. Interestingly, there were distinct differences in isoform abundance with surgery alone (Fig. 1, note "minus" lanes). PKC-alpha was easily detected at the 5-min time point and was more abundant at both the 15- and 30-min sham time points with no difference between the latter two. PKC-alpha also tended toward an increase in isoform abundance in response to 30 min of aortic ligature (note "plus" vs. "minus" lane at 30 min of ligature). Later time points were not assessed to determine if this subtle accumulation persisted or increased with duration of pressure overload. PKC-beta 1,2 was barely detectable at each time point and is not shown. PKC-epsilon was easily detected at each time point but showed no change in abundance with time or in response to aortic ligature.


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  		Fig. 1.   Differences in protein kinase C (PKC) isoform accumulation following surgery. Representative (n = 4) immunoblot analyses of total PKC-epsilon (A) and PKC-alpha (B) 5, 15, and 30 min after sham operation (-) or ascending aortic ligature (+).

The influence of acute pressure overload on isoform specific activation was then determined. PKC-epsilon is the isoform activated by the hypertrophy inducing stimulus phenylephrine in isolated adult rat ventricular myocytes (25). PKC-epsilon is also activated in hanging whole adult rat heart preparations through increased coronary artery perfusion pressure (17). The PKC isoform specific activation response to acute pressure overload in vivo has not been assessed. Accordingly, to determine if PKC isoform specific activation occurred acutely in response to acute pressure overload in vivo, cell lysates from adult rat hearts subjected to 5, 15, and 30 min of ascending aortic ligature or sham operation were analyzed for evidence of PKC-alpha , -beta 1,2, and -epsilon membrane translocation. Of these isoforms, only PKC-epsilon demonstrated a significant increase in translocation with acute pressure overload. PKC-epsilon activation was significantly greater with aortic ligature than with sham operation at the 5-min time point (Fig. 2). PKC-epsilon membrane translocation returned to sham operation levels following 30 min of aortic ligature. These data show that ascending aortic ligature acutely activates PKC-epsilon .


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  		Fig. 2.   PKC-epsilon isoform specific activation in acute pressure overload in vivo. Bar graph showing time course of PKC-epsilon isoform specific activation. Fold activation of PKC-epsilon represents amount of PKC-epsilon isoform specific activation as determined by membrane translocation in hearts subjected to ascending aortic ligature relative to that amount in sham-operated animals. Values are means ± SE; n = no. of rats. * Statistically significant (P < 0.05 vs. sham) increase in PKC-epsilon activation with ligature. Representative immunoblot analysis of PKC-epsilon in cytosolic (C) and membrane (M) fractions isolated from adult rat hearts 5 and 15 min after ascending aortic clipping (+) or sham (-) operation is shown below bar graph.

MAP kinase activation. Mechanical stretch of neonatal cardiocytes causes the sequential activation of MAPKKK, MAPKK, and MAP kinase (36). The importance of this pathway in hypertrophy of neonatal cardiocytes is underscored by the demonstration that depletion of p42 and p44 MAP kinase isoform mRNAs with antisense oligodeoxynucleotides inhibits development of the morphological features of hypertrophy (10). The activation of MAP kinase in vivo has not been determined. Accordingly, to assess if MAP kinase activation occurred in vivo in response to acute pressure overload, isolates from the same hearts analyzed for PKC isoform specific activation were characterized. As shown in Fig. 3, acute pressure overload resulted in a significant increase in MAP kinase activation of both the 44- and 42-kDa target proteins. MAP kinase activation occurred within 5 min of clipping. The level of activation subsequently fell, returning to baseline by 30 min following ligature. One, two, and five-day postaortic constriction time points were also assayed but were devoid of evidence for MAP kinase activation. These data show that MAP kinase activation occurs rapidly and briefly following acute pressure overload in vivo.


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  		Fig. 3.   Mitogen-activated protein (MAP) kinase activation occurs with acute pressure overload in vivo. Bar graph showing time course of MAP kinase activation. Fold change in MAP kinase activity refers to that amount of MAP kinase activation in hearts subjected to ascending aortic ligature relative to that of sham-operated animals. Values are means ± SE. * Statistically significant (P < 0.05 vs. sham) increase in MAP kinase activation with ligature. Below graph is representative immunoblot analysis of MAP kinase in protein extracts isolated from adult rat hearts 5 and 15 min after ascending aortic clipping (+) or sham (-) operation. Number to left of blots refers to molecular mass of control Erk2.

Influence of ACE inhibition on MAP kinase and PKC activation in vivo. ACE inhibition is known to prevent and/or regress hypertrophy in several in vivo models of cardiac hypertrophy and prevents MAP kinase and PKC activation in vitro (19, 30, 38). AT1 receptor blockade inhibits increased coronary artery perfusion pressure-induced PKC-epsilon membrane translocation (17). These data suggest that the endogenous renin-angiotensin system plays a role in the hypertrophic program and may do so through its influence on PKC-epsilon and MAP kinase activation. Accordingly, the effect of ACE inhibition on MAP kinase and PKC-epsilon isoform specific activation was assessed.

As shown in Fig. 4, ACE inhibition with fosinopril significantly decreased aortic ligature-induced PKC-epsilon activation. However, fosinopril did not fully prevent PKC-epsilon activation as determined by membrane translocation. This decrease was a specific response of treatment on aortic ligature-induced PKC-epsilon activation as there was no difference between treatment groups in sham-operated animals. These data suggest that the increase in membrane translocation of PKC-epsilon with aortic ligature is associated with but not dependent on an ACE-related pathway.


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  		Fig. 4.   Effect of fosinopril or hydralazine on aortic ligature-mediated PKC-epsilon activation. Bar graph showing influence of fosinopril or hydralazine on ascending aortic ligature-mediated PKC-epsilon activation. Units of PKC activation represent amount of PKC isoform specific activation as determined by membrane translocation in hearts subjected to ascending aortic ligature relative to that amount in sham-operated animals. Values are means ± SE. *Statistically significant (P < 0.05 vs. control treated). Shown below graph is representative immunoblot analysis of PKC-epsilon in cytosolic (C) and membrane (M) fractions isolated from adult rat hearts 5 min after ascending aortic clipping after 7 days of fosinopril (F), hydralazine (H), or water alone (W) treatment.

Similarly, fosinopril significantly decreased aortic ligature-mediated MAP kinase activation (Fig. 5). Also like its effect on PKC-epsilon , fosinopril did not completely prevent MAP kinase activation. These data suggest that activation of MAP kinase in the adult rat heart by acute pressure overload is in part but not solely due to an ACE specific event.


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  		Fig. 5.   MAP kinase activation is inhibited by fosinopril. Bar graph showing influence of fosinopril or hydralazine on ascending aortic ligature-mediated MAP kinase activation as determined by phosphorylation of 44-kDa target protein. Fold change in MAP kinase activity refers to that amount of MAP kinase activation in hearts subjected to ascending aortic ligature relative to that of sham-operated animals. Values are means ± SE. * Statistically significant (P < 0.05 vs. control treated) inhibition of aortic ligature-mediated MAP kinase activation. Below graph are representative immunoblots of MAP kinase activation in protein extracts isolated from adult rat hearts 5 or 15 min after ascending aortic clipping (+) or sham (-) operation following 7 days of fosinopril (F), hydralazine (H), or water alone (W) treatment. Number to left of blots refers to molecular mass of control Erk2.

Effect of treatment conditions on transient transfection assays. Sustained pressure overload from ascending aortic ligature ultimately results in cardiac hypertrophy (11, 21, 27, 35). Inhibition of the renin-angiotensin system with ACE inhibitors or AT1 receptor blockers prevents the genotypic and phenotypic changes of cardiac hypertrophy induced in this model (18, 35). We have demonstrated that 5 days of ascending aortic ligature (sufficient to induce cardiac hypertrophy) results in induction of transfected constructs containing a 33-bp enhancer (HRE) and that this induction is associated with a marked increase in binding activity of a 60-kDa nuclear factor (HRF) with the HRE (27). It was hypothesized that the HRE/HRF could serve as a model for studying the mechanism of pressure overload hypertrophy-mediated induction of gene expression. Accordingly, the influence of ACE inhibition on hypertrophy-mediated induction of constructs containing the HRE was determined.

As detailed in MATERIALS AND METHODS, for these experiments rats were pretreated for 1 wk with fosinopril or hydralazine. The well-characterized BCK57LUC plasmid, which consists of the BCK promoter containing the HRE enhancer driving the luciferase reporter gene, was then coinjected with pSV2CAT (to control for transfection efficiency) into hearts subsequently subjected to ascending aortic ligature. Fosinopril and hydralazine were continued. Five days later, hearts were isolated, weighed, and analyzed for luciferase and CAT activity. As expected, fosinopril prevented ascending aorta ligature-mediated left ventricular hypertrophy (Table 1). Fosinopril, but not hydralazine, also prevented hypertrophy-mediated induction of BCK57LUC (Table 2). This suggests that induction of HRE-containing constructs by pressure overload is an ACE-dependent event.

                              
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  		Table 1.   Effects of fosinopril and hydralazine on ability of ascending aorta ligature to induce left ventricular hypertrophy


                              
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  		Table 2.   Fosinopril inhibits ascending aorta ligature-mediated induction of hypertrophy responsive element-containing construct transiently transfected in rat left ventricle

Effect of treatment conditions on HRF binding activity. The influence of ACE inhibition on HRF binding activity was then investigated by EMSA using radiolabeled HRE incubated with nuclear proteins isolated from rat hearts used for gene expression analyses. As seen in Fig. 6, fosinopril completely and specifically prevented HRF binding. The decrease in HRF binding with fosinopril was not due to protein degradation or a faulty nuclear extract, as the same tissue was used successfully in determining MAP kinase and PKC activation and an EMSA using a different probe (Sp1) produced a band. These changes were not affected by DNA injection as the same EMSA result occurred using nuclear proteins isolated from animals subjected to 5 days of aortic ligature alone. Thus ACE inhibition specifically prevents the increase in HRF binding activity induced by pressure overload, suggesting that increased HRF binding activity by aortic ligature is mediated by an ACE-mediated pathway. Furthermore, because fosinopril specifically obviated binding of HRF and prevented induction of HRE containing constructs, these data confirm our conjecture that induction of HRE containing constructs with aortic ligature is due to a hypertrophy-mediated increase in HRF binding activity (27).


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  		Fig. 6.   Electromobility shift assays analysis showing obviation of hypertrophy response factor binding with fosinopril treatment. Radiolabeled hypertrophy responsive enhancer (HRE) was incubated with nuclear extracts isolated from hearts subjected to 5 days of ascending aortic ligature in rats in which fosinopril (F), hydralazine (H), or nothing was added to their water (W). Arrow identifies the expected band (lane 1) that is easily competed by 50-fold molar excess of unlabeled HRE (lane 2). Treatment with fosinopril (lane 3) but not hydralazine (lane 4) obviates shifted band.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report we demonstrate for the first time in vivo that MAP kinase activation occurs in acute pressure overload. This report is also the first to characterize PKC isoform specific accumulation and to demonstrate PKC-epsilon activation in acute pressure overload in vivo. We show evidence that activation by ascending aortic ligature of both PKC-epsilon and MAP kinase is in part regulated by an ACE-related pathway and display data exhibiting that ACE inhibition prevents gene activation by obviating induction of a nuclear factor responsible for gene induction. Though the signaling response of the heart to acute pressure overload- and sustained pressure overload-mediated gene activation may not be related, because in vitro data suggest such a connection exists it is interesting to speculate that similar events occur in vivo. If so, our data imply that ACE inhibitors prevent gene activation through obviation of nuclear factor binding via blockade of cell surface signaling that in part utilizes PKC-epsilon and MAP kinase.

PKC analyses. In this study, only PKC-alpha demonstrated an increase in accumulation following surgery and in response to subacute aortic ligature. Conversely, only PKC-epsilon showed evidence of activation with aortic ligature. Interestingly, at later time points there was a trend toward activation of PKC-beta 1,2 (not shown), a previously observed result (11). When combined with prior results, these data suggest that pressure overload of the heart causes temporally defined PKC isoform specific activation and accumulation (23). Such regulation could contribute to stimulus-specific responses (4).

Our investigation on the influence of ACE inhibition on PKC isoform specific activation focused solely on PKC-epsilon and showed that fosinopril significantly decreased acute pressure overload activation of PKC-epsilon . The response of PKC-epsilon to fosinopril supports in vivo work by other investigators, implicating the renin-angiotensin system in PKC-epsilon activation (17). However, our data differ in that ACE inhibition did not prevent PKC-epsilon activation, implicating the presence of other signals involved in acute pressure overload activation of PKC-epsilon . Numerous explanations, including the use of different agents and different model systems, could be forwarded to account for this discrepancy.

MAP kinase analyses. MAP kinase activation, particularly phosphorylation of p44- and p42-kDa target proteins, is a consistent occurrence that appears functionally important in in vitro models of cardiac hypertrophy (10, 28, 34). Although we do not address the practical consequence of MAP kinase activation, we do show that MAP kinase activation occurs in adult rat hearts in vivo in response to acute pressure overload. We also demonstrate that fosinopril, an agent capable of preventing or regressing hypertrophy in vivo, significantly inhibits MAP kinase activation and does so by >75% (18, 35). Interestingly, these data are similar to in vitro analyses showing AT1 receptor blockade inhibition of MAP kinase though the magnitude differs (19, 30). The larger reduction in MAP kinase activation in our model may be due to the lack of selectivity of fosinopril.

Although these results suggest that MAP kinase activation is a pivotal event in cardiac hypertrophy, a direct relationship between immediate events that occur after acute stimulation and the more chronic adaptive pheno- and genotypic changes of hypertrophy that result from prolonged, continuous stimulation have yet to be established. Moreover, MAP kinase activation is not solely responsible for the development of hypertrophy in vitro and can occur in response to agents incapable of producing a hypertrophic phenotype (8, 10, 24, 38). Furthermore, our data show that fosinopril does not completely prevent MAP kinase activation. When combined with our PKC analyses and those of other investigators, these data suggest that multiple bifurcating signals originating from both acute and subacute-chronic stressors confer the hypertrophic phenotype (6, 33). Other pathways that could be involved in signaling hypertrophy include the c-Jun NH2-terminal stress-activated protein kinase (JNK/SAPK) pathway and the Janus kinase (JAK)/signal transduction and activation of transcription (STAT) cascade. The former is activated by stretch in cultured cardiocytes and the latter is activated in response to hypertrophic stimuli in vitro and in vivo with acute pressure overload showing molecule-specific temporal responsiveness (2, 9, 12, 19, 21). Analyses also indicate the existence of ANG II-dependent and independent JAK-STAT activation.

PKC-MAP kinase relationship. The previously observed concurrence of PKC activation and MAP kinase activation with a hypertrophic stimulus is also supported by our data (28-30). However, our results differ from in vitro analyses in that we do not demonstrate a direct relationship between PKC and MAP kinase. Rather we show that both are rapidly activated following aortic ligature with each being significantly affected but not completely abolished by ACE inhibition. Our results are not due to differences in experimental conditions because analyses of PKC and MAP kinase activation were performed on isolates from the same hearts. Possible explanations for the difference between our data and that of other investigators are that we focused on specific PKC isoform changes, whereas others have looked at overall PKC activation, and that we used an in vivo whole heart model, while others analyze pure cultures of specific cell types (17, 25, 28-30). Also, the PKC isoform we tested (PKC-epsilon ) may not be solely responsible for transferring the signal to modulate gene expression.

Regulation of gene expression in hypertrophy. Only recently have the DNA-protein interactions responsible for mediating hypertrophy responsiveness in vivo been identified. Separate groups have demonstrated that GATA and AP1 sites of the beta -myosin heavy chain and atrial natriuretic factor genes, respectively, function as hypertrophy responsive elements in vivo (12-14). Using the same experimental approach, we have also identified a hypertrophy responsive element/factor (HRE/HRF) and in this study demonstrate an association between this element/factor and signaling events (27). Although the signaling cascade responsible has not been established, our results indicate a link between PKC-epsilon -MAP kinase activation, nuclear factor binding, and gene activation.

Problems with study. Because we do not directly address the cellular location of PKC or MAP kinase, we cannot with certainty attribute our data as being derived solely from cardiocytes. This is important because ANG II activates MAP kinase and PKC in both cardiac fibroblasts and cardiac myocytes in vitro (5, 28, 32). However, based on the isoforms studied it is likely that the PKC data reflect changes specific for the cardiomyocyte (2, 11, 25). The correlation between our results and in vitro analyses, particularly regarding MAP kinase, further supports that MAP kinase and PKC data are derived from cardiocytes (10, 19, 28-30, 34-36). Our analyses of gene induction and inhibition also imply that changes occur within the cardiocyte. For example, the HRE is not important for transfected BCK expression in cultured fibroblasts or skeletal muscle cells but is critical for expression in cultured neonatal cardiocytes and in the adult rat heart in vivo (26, 27, 37). Thus injected HRE-containing construct expression, basal, induced by hypertrophy, or limited by ACE inhibition, likely stems from the cardiocyte. Hence, it could be surmised that concurrent changes in MAP kinase and PKC activation that are similarly influenced by hypertrophy and ACE inhibition also arise from alterations of the cardiocyte.

Another problem is that a relationship between the acute events that occur following aortic ligature and the adaptive changes resulting from sustained pressure overload has not been established. Hence, it is difficult to correlate PKC and MAP kinase analyses with gene expression even if they are similarly influenced by ACE inhibition. Moreover, activation of signaling events may not necessarily lead to changes in gene expression; the phenotypic changes of hypertrophy include cytoskeletal reorganization as well (16). Regardless, our results are still relevant and can serve as bases for further investigation.


    ACKNOWLEDGEMENTS

We thank Margaret Collins for outstanding technical assistance in the early portions of this work, Sandy Nagel for secretarial support, and Nancy Baird and Muthu Periasamy for critically evaluating the manuscript.


    FOOTNOTES

This work was supported in part by a Veterans Affairs Research Advisory Group award and a Grant-in-Aid from the American Heart Association-Ohio affiliate (M. E. Ritchie). L. Kim was supported by an American Heart Association Summer Student Research Award.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. E. Ritchie, Krannert Institute of Cardiology, Indiana Univ. School of Medicine, 1111 W. 10th St., Indianapolis, IN 46202 (E-mail: miritchi{at}iupui.edu).

Received 17 December 1998; accepted in final form 10 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aoyagi, T., and S. Izumo. Mapping of the pressure response element of the c-fos gene by direct DNA injection into beating hearts. J. Biol. Chem. 268: 27176-27179, 1993[Abstract/Free Full Text].

2.   Bhat, G., J. Thekkumkara, W. Thomas, K. Conrad, and K. Baker. Angiotensin II stimulates cis-inducing factor-like DNA binding activity. J. Biol. Chem. 169: 31443-31449, 1994.

3.   Bogoyevitch, M., P. Parker, and P. Sugden. Characterization of protein kinase C isotype expression in adult rat heart. Circ. Res. 72: 757-767, 1993[Abstract/Free Full Text].

4.   Bogoyevitch, M., and P. Sugden. The role of protein kinases in adaptational growth of the heart. Int. J. Biochem. Cell Biol. 28: 1-12, 1996[Medline].

5.   Booz, G. W., D. E. Dostal, H. A. Singer, and K. Baker. Involvement of protein kinase C and Ca2+ in angiotensin II-induced mitogenesis of cardiac fibroblasts. Am. J. Physiol. 267 (Cell Physiol. 36): C1308-C1318, 1994[Abstract/Free Full Text].

6.   Bowman, J. C., S. F. Steinberg, T. Jiang, D. L. Geenen, G. I. Fishman, and P. M. Buttrick. Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates. J. Clin. Invest. 100: 2189-2195, 1997[Medline].

7.   Buttrick, P. M., M. L. Kaplan, R. N. Kitsis, and L. A. Leinwand. Distinct behavior of cardiac myosin heavy chain gene constructs in vivo. Discordance with in vitro results. Circ. Res. 72: 1211-1217, 1993[Abstract/Free Full Text].

8.   Clerk, A., M. Bogoyevitch, S. Fuller, A. Lazou, P. Parker, and P. Sugden. Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response. Biochem. J. 317: 109-118, 1996.

9.   Force, T., C. M. Pombo, J. A. Avruch, J. V. Bonventre, and J. M. Kyriakis. Stress activated protein kinases in cardiovascular disease. Circ. Res. 78: 947-953, 1996[Free Full Text].

10.   Glennon, P. E., S. Kaddoura, E. M. Sale, G. J. Sale, S. J. Fuller, and P. H. Sugden. Depletion of mitogen-activated protein kinase using an antisense oligodeoxynucleotide approach downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes. Circ. Res. 78: 954-961, 1996[Abstract/Free Full Text].

11.   Gu, X., and S. P. Bishop. Increased protein kinase C and isozyme redistribution in pressure-overload cardiac hypertrophy in the rat. Circ. Res. 75: 926-931, 1994[Abstract/Free Full Text].

12.   Harsdorf, R., J. G. Edwards, Y. T. Shen, R. K. Kudej, R. Dietz, L. A. Leinward, B. Nadal-Ginard, and S. F. Vatner. Identification of a cis-acting regulatory element conferring inducibility of a atrial natriuretic factor gene in acute pressure overload. J. Clin. Invest. 100: 1294-1304, 1997[Medline].

13.   Hasegawa, K., S. J. Lee, S. M. Jobe, B. E. Markham, and R. N. Kitsis. cis-Acting sequences that mediate induction of beta -myosin heavy chain gene expression during left ventricular hypertrophy due to aortic construction. Circulation 96: 3943-3953, 1997[Abstract/Free Full Text].

14.   Herzig, T. C., S. M. Jobe, H. Aoki, J. D. Molkentin, A. W. Cowley, S. Izumo, and B. E. Markham. Angiotensin II type 1a receptor expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload. Proc. Natl. Acad. Sci. USA 94: 7543-7548, 1997[Abstract/Free Full Text].

15.   Hirota, H., K. Yoshida, T. Kishimoto, and T. Taga. Continuous activation of gpl30, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice. Proc. Natl. Acad. Sci. USA 92: 4862-4866, 1995[Abstract/Free Full Text].

16.   Iwaki, K., V. P. Sukhatme, H. E. Shubeita, and K. R. Chien. alpha - and beta -Adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells: fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an alpha 1-mediated response. J. Biol. Chem. 265: 13809-13817, 1990[Abstract/Free Full Text].

17.   Kang, P. M., A. Nakouzi, T. Simpson, J. Scheuer, and P. M. Buttrick. Role of endogenous renin-angiotensin system in c-fos activation and PKC-epsilon translocation in adult rat hearts. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H2177-H2183, 1996[Abstract/Free Full Text].

18.   Kojima, M., I. Shiojima, T. Tamazaki, I. Komuro, Z. Yunzen, W. Ying, T. Mizuno, K. Jueki, K. Tobe, T. Kadowaki, R. Nagai, and Y. Tazaki. Angiotensin II receptor antagonist TCV-116 induces regression of hypertensive left ventricular hypertrophy in vivo and inhibits the intracellular signaling pathway of stretch-mediated cardiomyocyte hypertrophy in vitro. Circulation 89: 2204-2211, 1994[Abstract/Free Full Text].

19.   Komuro, I., Y. Yamazaki, Y. Katoh, I. Shiojima, K. Tobe, T. Kadowaki, R. Nagai, and Y. Yazaki. Protein kinase cascade activated by mechanical stress in cardiocytes: possible involvement of angiotensin II. Eur. Heart J. 16: 8-11, 1995.

20.   Kunisada, K., H. Hirota, Y. Fujio, H. Matsui, Y. Tani, K. Yamauchi-Takihara, and T. Kishimoto. Activation of JAK-STAT and MAP kinase by leukemia inhibitory factor through gp130 in cardiac myocytes. Circulation 94: 2626-2632, 1996[Abstract/Free Full Text].

21.   Molkentin, J. D., and B. E. Markham. An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha -cardiac myosin heavy chain gene in vivo. Mol. Cell. Biol. 14: 5056-5065, 1994[Abstract/Free Full Text].

22.   Pan, J., K. Fukuda, H. Kodama, S. Makino, T. Takahashi, M. Sano, S. Hori, and S. Ogawa. Role of angiotensin II in activation of the JAK/STAT pathway induced by acute pressure overload in the rat heart. Circ. Res. 81: 611-617, 1997[Abstract/Free Full Text].

23.   Paul, K., N. A. Ball, G. W. Dorn, and R. A. Walsh. Left ventricular stretch stimulates angiotensin II-mediated phosphatidylinositol hydrolysis and protein kinase C epsilon isoform translocation in adult guinea pig hearts. Circ. Res. 81: 643-650, 1997[Abstract/Free Full Text].

24.   Post, G. R., D. Goldstein, D. J. Thuerauf, C. C. Glembotski, and J. H. Brown. Dissociation of p44 and p42 mitogen-activated protein kinase activation from receptor-induced hypertrophy in neonatal rat ventricular myocytes. J. Biol. Chem. 271: 8452-8457, 1996[Abstract/Free Full Text].

25.   Puceat, M., R. Hilal-Dandan, B. Strulovici, L. L. Brunton, and J. Heller Brown. Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes. J. Biol. Chem. 269: 16938-16944, 1994[Abstract/Free Full Text].

26.   Ritchie, M. E. Characterization of human B creatine kinase gene regulation in the heart in vitro and in vivo. J. Biol. Chem. 217: 25485-25491, 1996.

27.   Ritchie, M. E. A 60 kD nuclear phosphoprotein mediates induction of brain creatine kinase (BCK) in in vivo hypercritophy (Abstract). Circulation 94: 1-530, 1996[Free Full Text].

28.   Sadoshima, J., and S. Izumo. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J. 12: 1681-1692, 1993[Medline].

29.   Sadoshima, J., and S. Izumo. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro. Circ. Res. 73: 424-438, 1993[Abstract/Free Full Text].

30.   Sadoshima, J., Z. Qiu, J. Morgan, and S. Izumo. Angiotensin II and other hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes. The critical role of Ca2+-dependent signaling. Circ. Res. 76: 1-15, 1995[Abstract/Free Full Text].

31.   Sadoshima, J., Y. Xu, H. S. Slayter, and S. Izumo. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell 75: 977-984, 1993[Medline].

32.   Schorb, W., T. C. Peeler, N. N. Madigan, K. M. Conrad, and K. M. Baker. Angiotensin II-induced protein tyrosine phosphorylation in neonatal rat cardiac fibroblasts. J. Biol. Chem. 269: 19626-19632, 1994[Abstract/Free Full Text].

33.   Takeishi, Y., G. Chu, D. M. Kirpatrick, Z. Li, H. Wakasaki, E. G. Kranias, G. L. King, and R. A. Walsh. In vivo phosphorylation of cardiac troponin I by protein kinase C beta2 decreases cardiomyocyte calcium responsiveness and contactility in transgenic mouse hearts. J. Clin. Invest. 102: 72-78, 1998[Medline].

34.   Thornburn, J., J. Frost, and A. Thornburn. Mitogen-activated protein kinase mediate changes in gene expression but not cytoskeletal organization associated with cardiac muscle cell hypertrophy. J. Cell Biol. 126: 1565-1572, 1994[Abstract/Free Full Text].

35.   Weinberg, E. O., F. J. Scheon, D. George, Y. Kagaya, P. S. Douglas, S. E. Litwin, H. Schunkert, C. R. Benedict, and B. H. Lorell. Angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis. Circulation 90: 1410-1422, 1994[Abstract/Free Full Text].

36.   Yamazuki, T., I. Komuro, S. Kudoh, Y. Zou, I. Shiojima, T. Mizuno, H. Takano, Y. Hiroi, K. Ueki, K. Tobe, T. Kadowaki, R. Nagai, and Y. Yazaki. Mechanial stress activates protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes. J. Clin. Invest. 96: 438-446, 1995.

37.   Zhang, J., J. E. Wilks, and J. J. Billadello. Characterization of a nuclear protein that interacts with regulatory elements in the human B creatine kinase gene. J. Biol. Chem. 270: 16134-16139, 1995[Abstract/Free Full Text].

38.   Zheng, J.-S., M. O. Boluyt, X. Long, L. O'Neill, E. G. Lakatta, and M. T. Crow. Extracellular ATP inhibits adrenergic agonist-induced hypertrophy of neonatal cardiac myocytes. Circ. Res. 78: 525-535, 1996[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 277(5):H1808-H1816
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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