Hemodynamic Overload–Induced Activation of Myocardial Mitogen-Activated Protein Kinases In Vivo
Augmented Responses in Young Spontaneously Hypertensive Rats and Diminished Responses in Aged Fischer 344 Rats
Mitogen-activated protein (MAP) kinases have been shown to be activated by various growth factors in cultured or isolated cardiomyocytes. However, little is known about the regulation of MAP kinases in vivo, especially in clinically important conditions, such as hypertension and senescence. In this study, we assessed mechanical overload–induced activation of myocardial MAP kinases in beating hearts from hypertensive or senescent rats. Fifteen minutes of left ventricular hemodynamic overload activated MAP kinase activity by 2.2-fold (P<0.05) in 4-week-old Wistar-Kyoto rats. The age-matched spontaneously hypertensive rats had greater MAP kinase activity than did Wistar-Kyoto rats both at baseline (1.4 times, P<0.05) and after the hemodynamic overload (1.7 times, P<0.05). Myocardial MAP kinase protein level, assessed by Western blot analysis, was also higher (1.6 times, P<0.01) in spontaneously hypertensive rats. In contrast, aged (18-month-old) Fischer 344 rats, which were known to have a diminished capacity of hypertrophy in response to mechanical stress, had lower MAP kinase activity both at baseline (63%, P<0.01) and after the hemodynamic overload (52%, P<0.05). Their MAP kinase protein level was lower (38%, P<0.01) than that in young (6-month-old) adults. Alterations in MAP kinase may contribute to changes in hypertrophic response in these animals.
Mitogen-activated protein (MAP) kinases,1 2 3 especially the first identified extracellular signal–regulated kinases (ERKs) of molecular sizes 42 and 44 kDa, seem to be involved in cell growth processes, such as proliferation and hypertrophy.4 The ERKs are known to phosphorylate MAP kinase–activated protein kinase-1/90-kDa ribosomal S6 kinase and to regulate its activity.5 6
Most studies involving MAP kinase activation were performed by using growth factor–stimulated cells in culture. Mechanical stretch of cardiomyocytes, which causes cellular hypertrophy, was shown to activate MAP kinases.7 8 In these in vitro conditions, most MAP kinase substrates can be phosphorylated by most MAP kinases, resulting in the confusion in physiological significance of each pathway reported in vitro. In contrast, little is known about the in vivo regulation of MAP kinase activity in response to mechanical stimulation.
In cardiac and skeletal muscle, mechanical load plays a critical role in determining muscle mass and its phenotype. However, myocardial response to mechanical load is significantly different in different animal models. For example, we have previously shown that senescent rat myocardium failed to induce immediate-early genes in response to pressure overload.9 The senescent heart has a marked decrease in hypertrophic capacity in response to either pressure or volume overload.10 11 On the other hand, young spontaneously hypertensive rats (SHR) are known to develop enhanced left ventricular hypertrophy, which is disproportional to the degree of hypertension.12 Because MAP kinases have been shown to play critical roles in the regulation of immediate-early genes and cell growth, we hypothesized that the myocardial MAP kinase activity may be altered in SHR and aged rats. In vivo assessment is necessary to evaluate the physiological roles of the MAP kinase cascade in many clinically important situations, including hypertensive hypertrophy and senescence. Alterations in the protein level of MAP kinase may contribute to changes in hypertrophic response in these animals.
In the present study, we determined the activation of myocardial tissue MAP kinase by left ventricular hemodynamic overload. We assessed MAP kinase activity and its response to hemodynamic overload in 4- and 20-week-old SHR to assess myocardial MAP kinase activation in an early phase and in an established phase of pressure-overload hypertrophy, respectively. We performed the same assessment in Fischer 344 rats to assess the age differences in hemodynamic overload–induced activation of myocardial MAP kinase. Finally, we assessed whether the altered MAP kinase activity in young SHR and aged Fischer 344 rats was associated with potential differences in MAP kinase protein levels.
The first set of study groups consists of SHR13 at 4 and 20 weeks. Age-matched Wistar-Kyoto rats (WKY), the parental strain of SHR, were used as nonhypertensive controls. The second set of study groups consists of Fischer 344 rats at the ages of 6 months (young adult) and 18 months (aged). All the animal experiments were performed in accordance with The Guiding Principles in the Care and Use of Animals.
Left Ventricular Hemodynamic Overload in a Buffer-Perfused Rat Heart
Each rat was anesthetized with intraperitoneally injected pentobarbital (50 mg/kg). The heart was isolated and perfused with the modified Krebs-Henseleit buffer. This isolated perfused rat heart model has been previously described in detail.14 In the present study, the heart was paced at 3.5 Hz, and the temperature was kept at 37°C. Coronary flow rate was first adjusted to obtain a mean coronary perfusion pressure of 90 mm Hg and remained constant throughout the experiment. A small, thin, latex balloon was inserted into the left ventricle through the mitral valve and tied around the atrioventricular groove to impose hemodynamic overload on the left ventricle and to simultaneously measure left ventricular pressure. After 15 minutes of equilibration, hemodynamic overload was imposed for the various predetermined periods by inflating the intraventricular balloon to develop a left ventricular end-diastolic pressure of 25 mm Hg, as we have previously demonstrated by an induction of the immediate-early gene c-fos.15 End-systolic volume, which would be less than half of the end-diastolic volume in an in situ ejecting heart, was fixed to be equal to the end-diastolic volume in this isovolumic contraction mode. Thus, systolic myocardial wall stress was markedly elevated even with moderately elevated end-diastolic pressure (25 mm Hg). Although the pressure overload is a dominant feature of this model, other features include a volume overload by increasing intra–left ventricular balloon volume and an elevated coronary perfusion pressure that is due to constant coronary flow throughout the intra–left ventricular balloon inflation. Strict and independent control of pressure overload, volume overload, and coronary perfusion16 would provide us with more accurate information regarding the effects of hemodynamic overload on the MAP kinase pathway. Although the phenomenon during the balloon inflation was primarily due to the hemodynamic overload, there is a possibility of unexpected myocardial injury. The balloon inflation in this isolated perfused heart model may have caused some ischemia, hypoxia, myocardial edema, and damage to coronary vessels, which we admit as limitations of the model.
In the control hearts, the balloon remained deflated throughout the entire protocol. At the end of the perfusion, the heart was removed from the perfusion apparatus, and the left ventricular myocardium was quickly weighed and frozen in liquid nitrogen. Each tissue sample was homogenized in 1 mL/100 mg tissue of lysis buffer (25 mmol/L Tris-HCl [pH 7.4], 25 mmol/L NaCl, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 0.5 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 10 nmol/L okadaic acid, 1 mmol/L phenylmethylsulfonyl fluoride, 0.8 μg/mL leupeptin, 10 μg/mL aprotinin, and 10 mg/mL p-nitrophenyl phosphate). Aliquots of the supernatant were used for the MAP kinase activity assay and Western blotting.
MAP Kinase Assay
Kinase assays in myelin basic protein (MBP)-containing polyacrylamide gel were performed as previously described.17 Twenty micrograms of myocardial homogenate was electrophoresed on 10% SDS-polyacrylamide gels containing 0.5 mg/mL MBP (rabbit brain, Sigma Chemical Co). SDS was removed from the gel by washing in the solution containing 20% 2-propanol and 50 mmol/L Tris-HCl (pH 8.0) for 1 hour and then in the solution containing 5 mmol/L β-mercaptoethanol and 50 mmol/L Tris-HCl (pH 8.0) for another hour. MAP kinase was denatured by incubating the gel in 6 mol/L guanidine-HCl solution for an hour and then renatured by incubation in 5 mmol/L β-mercaptoethanol, 50 mmol/L Tris-HCl, and 0.04% Tween 40 for 1 hour. The gel was preincubated in 40 mmol/L HEPES (pH 8.0), 2 mmol/L dithiothreitol, and 10 mmol/L MgCl2 at room temperature for 1 hour. The in-gel phosphorylation was performed by incubating the gel with 40 mmol/L HEPES (pH 8.0), 0.5 mmol/L EGTA (pH 8.0), 10 mmol/L MgCl2, 2 μmol/L protein kinase inhibitor (rabbit sequence, Sigma), 40 μmol/L ATP, and 2.5 μCi/mL γ-ATP (6000 Ci/mmol/L) at 25°C for 1 hour. After the phosphorylation reaction, the gel was washed in 5% trichloroacetic acid and 1% sodium pyrophosphate until the radioactivity of the solution become negligible. The gel was dried and subjected to autoradiography. MAP kinase activity was quantified as radioactivity at MAP kinase bands at 44 and 42 kDa by densitometry.
In our previous work, we performed immunoprecipitation with anti-ERK1/2 before the MAP kinase assay, resulting in similar positioning of the MBP phosphorylating activity at 42 and 44 kDa. These results support the feasibility of the in-gel kinase assay to detect the ERK1/2 activity apart from the activities of other MAP kinase families, such as c-Jun N-terminal kinase and p38 kinase,18 19 which also phosphorylate MBP. In addition to ERK1 and ERK2, other kinases, such as c-Jun N-terminal kinase and p38 kinase,18 19 could mediate hypertrophic responses of myocardium, which should be studied in the near future.
MAP kinase activity was also determined by incubating myocardial homogenates in 20 μL of the MAP kinase reaction buffer (25 mmol/L Tris-HCl [pH 7.4], 10 mmol/L MgCl2, 1 mmol/L dithiothreitol, 50 μmol/L ATP, 2 μmol/L protein kinase inhibitor, 0.5 mmol/L EGTA, 1 mg/mL MBP, and 5 μCi/mL [γ-32P]ATP [6000 Ci/mmol/L]) at 25°C for 10 minutes. Under this condition, activities of protein kinase A and protein kinase C are known to be inhibited.7 The reaction was stopped by adding Laemmli loading buffer, and the samples were separated by SDS-PAGE. The gel was dried, and the radioactivity at MBP bands was quantified by autoradiography to assess MAP kinase activity.
Anti–MAP Kinase Immunoblotting
Twenty micrograms of myocardial homogenate described above was electrophoresed on 10% SDS-polyacrylamide gels and transferred to a membrane (Clear Blot Membrane-P, ATTO Tokyo) by use of a semidry blotting system (Horizon Blot, ATTO Tokyo). Blots were probed by sequential incubation with rabbit anti-rat MAP kinase (UBI) and peroxidase-linked donkey anti-rabbit IgG. The enhanced chemiluminescence (ECL, Amersham) light-emitting nonradioactive Western blotting kit was used, and light intensity was quantified by a short exposure to an x-ray film.
Multiple comparisons within groups were analyzed by a repeated-measures ANOVA. Comparisons between groups, such as effects of age, strain, and pressure overload intervention, were analyzed by a factorial ANOVA. Results were described as mean±SE, and a value of P<0.05 was taken as significant.
Myocardial MAP Kinase Activation by Left Ventricular Hemodynamic Overload
Hearts from 4-week-old WKY underwent left ventricular hemodynamic overload by inflating the intra–left ventricular balloon to develop 25 mm Hg of left ventricular end-diastolic pressure for 5, 10, 15, and 30 minutes (Figure 1⇓). Left ventricular peak systolic pressure went up from a baseline value of 108±19 mm Hg to 152±17 mm Hg after 5 minutes of balloon inflation and remained elevated (162±15, 158±15, and 162±16 mm Hg at 10, 15, and 30 minutes, respectively).
Aliquots of the left ventricular myocardial homogenate were loaded onto an MBP-containing gel, and their MAP kinase activity was evaluated. As shown in Figure 1⇑, autoradiography revealed 2 major bands, a dominant band at 44 kDa and a weaker band at 42 kDa, which correspond to ERK1 and ERK2, respectively.17 Because 44-kDa species represent the major MAP kinase activity in this model, quantification of kinase activity was performed by densitometric analysis of the 44-kDa bands. The time course analysis showed that MAP kinase activity increased within 5 minutes of hemodynamic overload and began to decrease at 30 minutes in each experimental group (Figure 1⇑). Therefore, the comparison of MAP kinase activity between the groups was performed at the 15-minute time point in the subsequent studies.
Augmented Response of MAP Kinase Activity to Hemodynamic Overload in Young SHR
(Figure 2⇓) shows MAP kinase activity by hemodynamic overload in SHR and WKY. The SHR (Charles River, Ibaragi, Japan) is known to develop hypertension: systolic blood pressure starts to increase at 4 weeks and reaches >180 mm Hg at 12 weeks. As shown in the Table⇓, ratios of left ventricular weight (in milligrams) to body weight (in grams) were significantly (P<0.01) greater in 4- and 20-week-old SHR groups than in the age-matched WKY and SHR groups, respectively. These degrees of hypertrophy were compatible with those reported in a previous study.12
The basal MAP kinase activity in 4-week-old SHR hearts was 1.4±0.2 times greater (P<0.05) than that in control WKY hearts. Hemodynamic overload for 15 minutes on 4-week-old WKY hearts increased MAP kinase activity by 2.2±0.2-fold compared with control activity (P<0.05). In 4-week-old SHR, hemodynamic overload increased MAP kinase activity by 2.7±0.5 (P<0.01), which was 3.8±0.7 times greater (P<0.01) than that in the control WKY without hemodynamic overload (Figure 2A⇑). Two-factor ANOVA with strain and pressure overload as independent factors showed that 4-week-old SHR had significantly greater MAP kinase activity in response to hemodynamic overload than did WKY (P<0.05).
As shown in Figure 2B⇑, hemodynamic overload for 15 minutes on 20-week-old SHR and WKY hearts increased MAP kinase activity by 2.0-fold (P<0.01) and 2.1-fold (P<0.01), respectively. In contrast to the 4-week-old rats, there was no significant strain difference in MAP kinase activity at 20 weeks. Therefore, in the early phase of hypertension, but not in the established phase, SHR hearts had a higher baseline MAP kinase activity and a greater response to mechanical overload than did WKY hearts.
Elevated Myocardial MAP Kinase Protein Level in Young SHR
To investigate the mechanism of the higher MAP kinase activity at baseline and its greater response to the left ventricular hemodynamic overload in young SHR, we performed immunoblotting analysis by using an anti–MAP kinase antibody on the myocardial homogenate (Figure 3⇓). The anti–rat MAP kinase antibody recognized 2 bands (a major band at 44 kDa and a minor band at 42 kDa) in the SHR and WKY heart homogenate (Figure 3⇓, top). Figure 3⇓, bottom, shows the quantification of 44-kDa MAP kinase protein. The level of MAP kinase protein was greater by 1.6-fold (P<0.01) in SHR hearts compared with WKY hearts at 4 weeks. In 20-week-old rats, the average value was 1.3-fold greater in SHR, but the difference was not statistically significant.
Depressed MAP Kinase Responses to Hemodynamic Overload and Protein Level in Aged Fischer 344 Rats
We assessed MAP kinase activity in aged Fischer 344 rats, in which depressed hypertrophic responses10 and depressed immediate-early gene induction9 to pressure overload have been demonstrated. We performed the same hemodynamic-overload intervention on the isolated buffer-perfused hearts of young adult (6-month-old) and aged (18-month-old) Fischer 344 rats (Figure 4⇓). The in-gel kinase assay revealed lower myocardial MAP kinase activity both at baseline (63% of the young adult rats, P<0.01) and after the pressure overload (52% of the young adult rats, P<0.05) in aged Fischer 344 rats (Figure 4A⇓). Western blotting revealed markedly decreased levels (38% of the young adult rats, P<0.01) of myocardial MAP kinase protein of both 44- and 42-kDa species in the 18-month-old Fischer 344 rats (Figure 4B⇓).
Fifteen minutes of left ventricular hemodynamic overload activated MAP kinase in 4-week-old WKY. SHR at the ages of 4 and 20 weeks had augmented MAP kinase activation by hemodynamic overload compared with the aged-matched WKY. Protein levels of MAP kinase were also higher in SHR compared with age-matched WKY. The senescent Fischer 344 rats showed lower baseline MAP kinase activity and protein levels and diminished activation on response to the hemodynamic overload.
In the present study, MAP kinase was rapidly activated by a physiological stimulus (hemodynamic overload) in an intact organ. It is now important to address whether each signal transduction pathway observed in cultured cells is significantly involved in the in vivo setting. To our knowledge, this is the first study to demonstrate significant alterations in MAP kinase activity in a pathological (SHR) and a physiological (senescent) animal model with important clinical implications.
Augmented Response of Myocardial MAP Kinase Activity to Hemodynamic Overload and Elevated Myocardial MAP Kinase Protein Levels in Young SHR
We found higher baseline MAP kinase activity as well as augmented responses to acute hemodynamic overload in young SHR compared with age-matched WKY. In SHR, a stable compensated hypertrophic phase is maintained for a long period (≈12 months).20 21 We demonstrated an increase of MAP kinase in SHR before the establishment of hypertension (at 4 weeks of age). Even with the increased MAP activity at this prehypertensive stage, the SHR is known to have significant left ventricular hypertrophy that is not in parallel with its blood pressure12 as well as increased heart rate and cardiac output.22 The results indicate that augmented MAP kinase activity may play one of the pathogenic roles in the initiation and development of cardiac hypertrophy in SHR rather than the secondary phenomenon associated with established hypertrophy. The increased MAP kinase activity in SHR can be accounted for largely by increased levels of MAP kinase proteins, because there was a significant direct correlation (r=0.87, P<0.05) between kinase activity and the protein levels in each sample (data not shown). However, we note that this increase in protein levels of MAP kinase in 4-week-old and 20-week-old SHR is not necessarily secondary to hypertensive hypertrophy but would be a primary genetic characteristic of the SHR model.
Depressed MAP Kinase Responses to Hemodynamic Overload and Protein Levels in Aged Fischer 344 Rats
The diminished myocardial MAP kinase activation by acute left ventricular hemodynamic overload in aged rats seems to be primarily due to the decreased MAP kinase protein level. It is well known that when subjected to similar hemodynamic stress, old patients develop heart failure more frequently than do younger patients,23 which may, in part, be due to a diminished ability in the aged to develop hypertrophy. Previous experimental studies have shown that the aged Fischer 344 rats have a diminished capacity for left ventricular hypertrophy in response to hemodynamic or volume overload10 11 associated with the decreased expression of immediate-early genes in response to hemodynamic overload9 . The results from the present study indicate that diminished levels of MAP kinase may be one of the mechanisms of the diminished hypertrophic response and the impaired immediate-early gene induction in the aged Fischer rats. We must note that the hypertrophic responses of the senescent heats are model specific (reviewed in Swynghedauw24 ). Capasso et al25 showed less hypertrophic response in the senescent Fischer rats with renovascular hypertension, whereas Besse et al26 reported slow but eventually full hypertrophic response in the senescent rats with deoxycorticosterone acetate-salt hypertension. The present observations in the senescent Fischer rats with acute and severe hemodynamic overload that were made with the use of the isolated perfused heat preparation may not be applicable to other models of hemodynamic overload, especially in aged patients with hemodynamic overload.
Future inclusion of aged SHR would provide us with further information regarding interaction between the effects of hypertrophy and those of aging.
We thank Dr J. Sadoshima for helpful advice on MAP kinase assay and Dr J.W. Wei for providing us with the aged Fischer 344 rats.
- Received March 27, 2000.
- Revision received April 19, 2000.
- Accepted August 4, 2000.
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