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Originally published In Press as doi:10.1074/jbc.M705133200 on September 12, 2007

J. Biol. Chem., Vol. 282, Issue 45, 33181-33191, November 9, 2007
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Glycogen Synthase Kinase-3{alpha} Reduces Cardiac Growth and Pressure Overload-induced Cardiac Hypertrophy by Inhibition of Extracellular Signal-regulated Kinases*Formula

Peiyong Zhai{ddagger}1, Shumin Gao{ddagger}, Eric Holle§, Xianzhong Yu§, Atsuko Yatani{ddagger}, Thomas Wagner§, and Junichi Sadoshima{ddagger}2

From the {ddagger}Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103 and §Oncology Research Institute, Greenville, South Carolina 29605

Received for publication, June 21, 2007 , and in revised form, August 21, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3{alpha} and GSK-3beta. Pressure overload increases expression of GSK-3{alpha} but not GSK-3beta. Despite our wealth of knowledge about GSK-3beta, the function of GSK-3{alpha} in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3{alpha} transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3{alpha} inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3{alpha} inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3{alpha}, but not GSK-3beta, inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3{alpha} increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase C{epsilon} (PKC{epsilon}) inhibitor peptide, suggesting that GSK-3{alpha} inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3{alpha} increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3{alpha}. In conclusion, up-regulation of GSK-3{alpha} inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3{alpha} is mediated through inhibition of ERK.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GSK-33 is a proline-directed serine/threonine kinase that is ubiquitously expressed. It has versatile biological functions, including regulation of metabolism, cell growth/death, development, cytoskeletal organization, transcription, and protein translation (1-3). GSK-3 remains active at resting state and is inactivated by a variety of mitogens, many protein kinases including protein kinase B/Akt, and by the Wnt signaling pathway. Many targets of GSK-3 are negatively regulated by it, and inactivation of GSK-3 stimulates cellular functions by removing the repression (1-3).

GSK-3 has two isoforms, GSK-3{alpha} and GSK-3beta. The molecular mass of GSK-3{alpha} is 51 kDa, and that of GSK-3beta is 47 kDa (4). Structurally, the two isoforms have 97% sequence homology within their kinase domains, but GSK-3{alpha} has an extended N-terminal glycine-rich tail (2), and the two kinases share only 36% in the last 76 C-terminal residues (1). Although both isoforms share substrates, their expression patterns, substrate preferences, and cellular functions are not identical (for review, see Ref. 5). GSK-3{alpha} is not phosphorylated/inhibited by protein kinase C (PKC), whereas the activity of GSK-3beta is decreased by about 50% when phosphorylated by some PKC isotypes, including PKC-{alpha},-beta1, and -{gamma} (6). In neurons, GSK-3{alpha}, but not GSK-3beta, is involved in regulating production of Alzheimer disease amyloid-beta peptides (7). Genetic ablation of GSK-3beta in mice results in an embryonic lethal phenotype (8), suggesting that GSK-3beta and GSK-3{alpha} play different roles in development. Using constitutively active GSK-321A/21A/9A/9A knockin mice, it has recently been shown that inactivation of GSK-3beta, rather than GSK-3{alpha}, is the major route by which insulin activates skeletal muscle glycogen synthase (9). GSK-3beta, but not GSK-3{alpha}, regulates cardiomyocyte mitochondrial permeability transition in culture (10). Inhibition of GSK-3{alpha} more robustly enhances cAMP-response element- and NF{kappa}B-dependent transactivation than that of GSK-3beta (11). Silencing of GSK-3{alpha} down-regulates the binding activity of early growth response-1 (EGR-1) to EGR1-responsive element, whereas silencing of GSK-3beta up-regulates this activity (11). Smad3/4-responsive transcription is enhanced by GSK-3{alpha} small interfering RNA (siRNA) but slightly decreased by GSK-3beta siRNA (11). Thus, GSK-3{alpha} is different from GSK-3beta in many ways, despite many similarities (Table 1).


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  		TABLE 1
The differences between GSK-3{alpha} and GSK-3beta reported in the literature

CRE, cAMP-response element; EGR-1, early growth response-1.

 


Figure 1
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  		FIGURE 1.
A, expression of GSK-3{alpha} and phosphorylation (p-) level of GSK-3{alpha} in mouse hearts in response to 4 weeks of TAC. *, p < 0.05 versus SHAM. B, expression of GSK-3beta and phosphorylation of GSK-3beta in mouse hearts in response to TAC. C, expression of GSK-3{alpha} and its phosphorylation in areas remote from myocardial infarction (MI) 7 days after coronary ligation. *, p < 0.05 versus SHAM. D, expression of GSK-3beta and its phosphorylation in areas remote from myocardial infarction 7 days after coronary ligation.

 
Both GSK-3{alpha} and GSK-3beta exist in the heart (4). The function of GSK-3beta has been studied in cultured cardiac myocytes in vitro and in transgenic animal models in vivo. GSK-3beta negatively regulates beta-adrenergic- and endothelin-induced cardiac hypertrophy in neonatal rat cardiac myocytes (NRCMs) (12-14). Cardiac-specific overexpression of constitutively active GSK-3beta (GSK-3beta(S9A)) in transgenic mice inhibits cardiac hypertrophy in response to pressure overload and isoproterenol infusion (15, 16). Cardiac-specific overexpression of wild type GSK-3beta in mice caused reduced cardiac growth with diastolic dysfunction (17). These studies provided invaluable insights regarding the function of GSK-3beta in cardiac hypertrophy. However, the function of GSK-3{alpha} in the heart remains to be elucidated. The present study was designed to study the function of GSK-3{alpha} in the heart and the underlying signaling mechanisms.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Primary Culture of Neonatal Rat Ventricular Myocytes—Primary cultures of ventricular cardiac myocytes were prepared from 1-day-old Crl: (WI) BR-Wistar rats (Charles River Laboratories, Wilmington, MA) as previously described (18). A cardiac myocyte-rich fraction was obtained by centrifugation through a discontinuous Percoll gradient.

Construction of Adenoviral Vectors—Recombinant adenovirus was constructed using an Adeno-X adenovirus construction kit (Clontech Laboratories Inc., Palo Alto, CA). We made replication-defective human adenovirus type 5 (devoid of E1 and E3) harboring GSK-3{alpha} (Ad-GSK-3{alpha}), GSK-3beta (Ad-GSK-3beta), and shRNA against GSK-3{alpha} (Ad-shRNA GSK-3{alpha}).

Transgenic Mice—A cDNA clone of GSK-3{alpha} was kindly provided by Dr. J. R. Woodgett. Using this clone and the mouse {alpha}MHC promoter (provided by Dr. J. Robbins), we made transgenic mice with cardiac-specific expression of GSK-3{alpha}.

GSK-3 Kinase Activity Assay—GSK-3{alpha} or GSK-3beta was immunoprecipitated from tissue lysates with equal protein content. The immunoprecipitate was collected, washed, and resuspended in a reaction buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerol phosphate, 12 mM MgCl2, 2 mM dithiothreitol, 0.1 M Na3VO4, and 200 µM ATP. One hundred ng of Tau was added as a substrate and mixed well. The reaction was carried out for 30 min at 30 °C. Phosphorylation of Tau by GSK-3 was detected by standard immunoblotting using a phospho-Tau antibody (Sigma).

Quantitative Reverse Transcription-PCR—Total RNA was prepared using the RNeasy fibrous tissue kit (Qiagen Inc., Valencia, CA), and first-strand cDNA was synthesized using the ThermoScript reverse transcription-PCR system (Invitrogen). Real-time PCR was then carried out on a DNA Engine Opticon 2 system (Bio-Rad) using the DyNAmo HS SYBR Green qPCR kit (Bio-Rad). The specific oligonucleotide primers for atrial natriuretic factor and {alpha}-skeletal actin have been previously reported (18).

Echocardiography—Mice were anesthetized using 12 µl/g of body weight of 2.5% avertin (Sigma), and echocardiography was performed using ultrasonography (Acuson Sequoia C256, Siemens Medical Solutions USA Inc., Malvern, PA) as previously described (18). A 13-MHz linear ultrasound transducer was used. Two-dimension guided M-mode measurements of LV internal diameter were taken from three or more beats and averaged. Left ventricular end-diastolic dimension was measured at the time of the apparent maximal LV diastolic dimension, whereas LV end-systolic dimension was measured at the time of the most anterior systolic excursion of the posterior wall.


Figure 2
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  		FIGURE 2.
A, expression of GSK-3{alpha} in line 28. *, p < 0. 01 versus NTg. B, GSK-3{alpha} kinase activity in line 28 using Tau as a substrate. *, p < 0.05 versus NTg. C, expression of GSK-3beta in line 28. *, p < 0.01 versus NTg. D, GSK-3beta kinase activity in line 28. *, p < 0.05 versus NTg. p-phosphorylated.

 


Figure 3
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  		FIGURE 3.
A, hearts from age-matched Tg (line 28) and NTg. Tg have a smaller heart at both 3 and 7 months old. B, LVW/BW in Tg (line 28) and NTg. p < 0.01 (*) and p < 0.05 (#) are versus NTg. C, LVW/TL in Tg (line 28) and NTg p < 0.01 (*) and p < 0.05 (#) are versus NTg. D, cross-sectional views of cardiac myocytes in wheat germ agglutinin-Texas Red-stained tissue sections. Cardiac myocytes in Tg (line 28) are smaller at both 3 and 7 months old. E, cardiac myocyte cross-sectional area (line 28). *, p < 0.01 versus NTg. F, isolated cardiac myocyte capacitance at 3 months old. Data were obtained from 50 to 100 cells from 5 animals in each group. *, p < 0.05 versus NTg.

 
Histological Analysis—Histological analyses of the heart sections were conducted as described previously (18). Heart specimens were fixed with 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 6-µm thickness. Interstitial fibrosis was evaluated by picric acid Sirius red staining. The positively stained (red) fibrotic area was expressed as a percentage of total area. Total cardiac myocyte numbers were estimated using a published method (19). Briefly, the number of nuclei per unit area of myocardium (N(n)A) was determined in 20 fields (40x). The average nuclear length (Dn) was determined in longitudinally oriented myocytes. The number of myocyte nuclei per unit volume of myocardium (N(n)v) was calculated using the equation N(n)v = N(n)A/Dn. The estimated total number of myocyte nuclei in the left ventricle was calculated as the product of the number of myocyte nuclei per unit volume, N(n)v, and the total ventricular volume, VT, which was derived from the left ventricular weight using the specific gravity of muscle tissue, 1.06 g/ml.

Evaluation of Apoptosis in Tissue Sections—DNA fragmentation was detected in situ using TUNEL as described (18). Briefly, deparaffinized sections were incubated with proteinase K, and DNA fragments were labeled with fluoresce-in-conjugated dUTP using TdT (Roche Applied Science). Nuclear density was determined by manual counting of 4',6-diamidino-2-phenylindole-stained nuclei in 20 fields from each animal using the 40x objective and of TUNEL-positive nuclei in the same fields using the same power objective. Limiting counting of total nuclei and the TUNEL-positive nuclei to areas with a true cross-section of myocytes made it possible to selectively count only those nuclei that clearly were within myocytes.


Figure 4
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  		FIGURE 4.
Left ventricular fibrosis and apoptosis. A, picric acid Sirius red staining of heart sections obtained from Tg (line 28) and age-matched NTg mice. B, morphometry of myocardial fibrosis at 3 and 7 months old. *, p < 0.05 versus NTg. C and D, representative images of TUNEL staining of myocardial sections. Arrows indicate positive nuclei. DAPI, 4',6-diamidino-2-phenylindole. E, morphometry of TUNEL staining of myocardial sections from Tg (line 28) and age-matched NTg. p < 0.05 (*) and p < 0.01 (#) are versus NTg.

 
Transverse Aortic Constriction (TAC)—TAC was performed according to the method published previously (20). Permanent ligation of the left anterior descending coronary artery was conducted as described (21).

Immunoblotting Analysis—Cardiac tissue homogenates and cell lysates were made in CHAPS buffer (Sigma-Aldrich). We used anti-phospho-specific and corresponding non-phospho-specific antibodies against GSK-3{alpha} (S21), GSK-3beta (S9), Akt (S473), p70 S6 kinase (T389), and extracellular signal-regulated kinases (ERKs) (T202/Y204) (Cell Signaling Technology) and anti-phospho-Tau antibody (Sigma-Aldrich).

Statistical Analysis—Data are reported as the mean ± S.E. Statistical analyses between groups were done by one-way analysis of variance, and when p values were significant, differences among group means were evaluated using the Bonferroni t test. A p value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of GSK-3{alpha} Was Increased in Hypertrophied Myocardium Due to Chronic Pressure Overload or Myocardial Infarction—To examine the expression of GSK-3{alpha} under pathological conditions, the protein level of GSK-3{alpha} was measured in hearts subjected to pressure overload and in the remote area from myocardial infarction. The level of GSK-3{alpha} was increased by 2-fold, whereas phospho-GSK-3{alpha} was not significantly changed in the heart 4 weeks after TAC (Fig. 1A), indicating that GSK-3{alpha} was up-regulated during cardiac hypertrophy. In contrast, the expression of GSK-3beta and the phosphorylation of GSK-3beta were not changed in the heart 4 weeks after TAC (Fig. 1B). Similarly, GSK-3{alpha} expression was increased by 1.6-fold, and phosphorylation of GSK-3{alpha} was significantly decreased in myocardium remote from myocardial infarction 7 days after coronary ligation (Fig. 1C). In contrast, neither total expression nor phosphorylation of GSK-3beta was significantly changed in the remote area of the infarcted heart (Fig. 1D).

Generation of GSK-3{alpha} Transgenic Mice—To study the effect of GSK-3{alpha} up-regulation on cardiac phenotype in vivo, two lines of transgenic mice with cardiac-specific overexpression of GSK-3{alpha} (Tg), line 13 and line 28, were established. Cardiac tissue transgene expression was assessed at the protein level using 2-3-month-old mice. The expression of GSK-3{alpha} in Tg mice was 4.3 and 3.7 times that of NTg mice in line 28 and line 13, respectively (Fig. 2A and supplemental Fig. IA). Interestingly, the expression of GSK-3beta was down-regulated in Tg mice in both lines (Fig. 2B and supplemental Fig. IB). The GSK-3{alpha} kinase activity in Tg was increased by 1.7-fold in line 28, and the GSK-3beta activity was decreased by 40% (Fig. 2, C and D). We primarily characterized line 28 in this study.


Figure 5
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  		FIGURE 5.
The effect of 4 weeks of aortic banding on hypertrophy and fetal gene expression. A, LVW/BW. p < 0.001 (*) and p < 0.01 (#) are versus SHAM. p < 0.05 ($) and p < 0.01 (&) are versus the corresponding NTg. B, LVW/TL). p < 0.001 (*) and p < 0.01 (#) are versus SHAM. p < 0.05 ($) and p < 0.01 (&) are versus the corresponding NTg. C, wheat germ agglutinin-Texas Red staining of cardiac sections. D, myocyte cross-sectional area. p < 0.001 (*) and p < 0.01 (#) are versus SHAM. p < 0.05 ($) and p < 0.01 (&) are versus the corresponding NTg. E, atrial natriuretic factor (ANF) expression. p < 0.001 (*) and p < 0.01 (#) are versus SHAM. $, p < 0.05 versus NTg. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. F, {alpha}-skeletal actin (ASA) expression. p < 0.001 (*) and p < 0.01 (#) are versus SHAM. $, p < 0.05 versus NTg.

 
GSK-3{alpha} Down-regulates Cardiac Physiological Growth—To examine the effect of GSK-3{alpha} on physiological growth of the heart, heart size, LV weight, and cardiac myocyte size were compared between NTg and Tg mice at both 3 and 7 months old. Tg mice had smaller hearts than NTg mice at both ages (Fig. 3A). The LV weight (LVW)/body weight (BW) and the LVW/tibia length (TL) in Tg mice were significantly lower than those in NTg mice at both ages (Fig. 3, B and C). The LV myocyte cross-sectional area was significantly smaller in Tg mice than in NTg mice at 3 and 7 months old (Fig. 3, D and E), indicating smaller myocyte size in Tg mice. To further examine the effect of GSK-3{alpha} on cardiac myocyte size, myocytes were isolated from 3-month-old mice. Cardiac myocyte capacitance, proportional to the cell surface area, was significantly decreased in Tg (Fig. 3F), indicating that the overall size of cardiac myocytes was smaller in Tg than in NTg. The estimated total number of cardiac myocytes was not significantly different between NTg and Tg at either 3 or 7 months old (supplemental Fig. II, A and B), suggesting that the smaller cardiac myocyte size rather than changes in the total number of myocytes is an important basis for the smaller heart in Tg. In the other line of Tg mice (line 13), LVW/BW and LVW/TL in Tg were significantly lower than those in littermate NTg (supplemental Fig. III, A and B). These data indicate that GSK-3{alpha} negatively regulates physiological growth of the heart.

GSK-3{alpha} Modestly Increases Apoptosis and Fibrosis but Does Not Cause Cardiac Dysfunction at Base Line—The percent fibrosis in Tg mouse hearts was slightly but significantly increased as compared with that in NTg at 3 and 7 months old (Fig. 4, A and B). Similarly, the TUNEL-positive nuclei were slightly but significantly increased in Tg mouse hearts at both ages (Fig. 4C). Despite the modest increase in apoptosis and fibrosis, Tg mice did not develop any signs of heart failure. The lung weight/BW and lung weight/TL were not significantly different between NTg and Tg mice (data not shown). The ejection fraction and fractional shortening (FS), determined by echocardiographic measurement, in Tg mice were not significantly different from those in NTg mice (Table 2). The +dP/dt, -dP/dt, and LV end-diastolic pressure (LVEDP), determined by hemodynamic analysis, were also not significantly different between NTg and Tg mice (Table 3). After up to 7 months of follow-up, no significant increase in premature mortality was found in either line 28 or line 13 Tg mice. These data indicate that Tg mice did not develop cardiac dysfunction at base line up to 7 months of age despite a modest increase in apoptosis and fibrosis.


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  		TABLE 2
Echocardiographic analysis of GSK-3{alpha} Tg mice

DSEP WT, diastolic septal wall thickness; SSEP WT, systolic septal wall thickness; DPW WT, diastolic posterior wall thickness; SPW WT, systolic posterior wall thickness; EF, ejection fraction; FS, fractional shortening; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; N, number; HR, heart rate.

 


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  		TABLE 3
Hemodynamic measurements of GSK-3{alpha} Tg mice

MAP, mean aortic pressure; LVSP, LV systolic pressure; N, number; HR, heart rate.

 


Figure 6
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  		FIGURE 6.
The effect of 4 weeks of aortic banding on LV function. A, lung weight/BW. *, p < 0.01 versus SHAM; #, p < 0.05 versus NTg. B, lung weight/TL. p < 0.05 ($) and p < 0.01 (*) are versus SHAM. #, p < 0.05 versus NTg. C, ejection fraction (EF). *, p < 0.05 versus SHAM; #, p < 0.05 versus NTg. D, fractional shortening (FS). *, p < 0.05 versus SHAM; #, p < 0.05 versus NTg. E, radius/wall thickness (r/h) ratio. *, p < 0.05 versus NTg. F, LVEDP/EDD. p < 0.05 (*) and p < 0.01 (#) are versus SHAM. $, p < 0.05 versus NTg. G, LV end-diastolic stress (LVED stress, LVEDP*EDD/(two times end-diastolic posterior wall thickness)). p < 0.05 (*) and p < 0.01 (#) are versus SHAM. $, p < 0.05 versus NTg. H, LV end-systolic stress (LVES stress, LVESP*ESD/(two times end-systolic posterior wall thickness)).

 
GSK-3{alpha} Inhibits Pressure Overload-induced Cardiac Hypertrophy but Exacerbates Cardiac Dysfunction Caused by Pressure Overload—To examine the effect of GSK-3{alpha} on pressure overload-induced cardiac hypertrophy, 3-month-old Tg mice and NTg mice underwent TAC operation. Two to 4 weeks after TAC, Tg mice developed less severe cardiac hypertrophy than NTg mice. The increases in LVW/BW (2 weeks of banding, 37.5 versus 48.1%, p < 0.01; 4 weeks of banding, 48.7 versus 59.1%, p < 0.01) and LVW/TL (2 weeks of banding, 36.2 versus 55.3%, p < 0.01; 4 weeks of banding, 50 versus 65.9%, p < 0.01) were significantly smaller in Tg mice than in NTg mice (Fig. 5, A and B, supplemental Fig. IV), and the increases in cardiac myocyte cross-sectional area 4 weeks after TAC (21% versus 40%, p < 0.01) were also significantly smaller in Tg mice than in NTg mice (Fig. 5, C and D). Expression of fetal-type genes, such as atrial natriuretic factor and {alpha}-skeletal actin, was significantly less in Tg than in NTg 4 weeks after TAC (Fig. 5, E and F), although it was not significantly different between SHAM-operated NTg and Tg. These data suggest that GSK-3{alpha} overexpressed in the heart attenuates pressure overload-induced cardiac hypertrophy. Interestingly, despite the inhibition of cardiac hypertrophy, Tg mice developed more severe cardiac dysfunction than NTg mice after TAC. Although no (of 11) NTg mice died after the operation, 2 (of 11) Tg mice died 3 days after the operation, and postmortem pathological measurements revealed severe lung congestion (data not shown), indicating that the mice died due to heart failure. More severe lung congestion (higher lung weight/BW and lung weight/TL) was observed in Tg mice 2 and 4 weeks after TAC (Fig. 6, A and B, supplemental Fig. V, A and B). Echocardiographic measurements indicated that ejection fraction (EF) and fractional shortening (FS) in Tg mice were significantly lower than those in NTg mice 2 and 4 weeks after TAC (Fig. 6, C and D, supplemental Fig. V, C and D). The radius/wall thickness (r/h) ratio, an indicator of LV wall stress, was significantly higher in Tg than in NTg 4 weeks after TAC (Fig. 6E). LVEDP/EDD, an indicator of LV wall stiffness, was significantly greater in Tg than in NTg 4 weeks after TAC (Fig. 6F). Hemodynamic measurements indicated that, although LV end-systolic stress was not different between NTg and Tg either at base line or after TAC, LV end-diastolic stress was significantly higher in Tg than in NTg 4 weeks after TAC (Fig. 6, G and H). LV +dP/dt and -dP/dt in Tg mice were significantly decreased compared with those in NTg mice, and the decreases were significantly greater in Tg than in NTg after 2 and 4 weeks of TAC (Table 4, supplemental Fig. VE).


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  		TABLE 4
Hemodynamic analysis of GSK-3{alpha} mice 4 weeks after TAC

PG, pressure gradient. Changes = TAC-SHAM. Please note that the lower PG in Tg TAC is due to heart failure rather than less severe constriction.

 


Figure 7
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  		FIGURE 7.
Cardiac fibrosis and apoptosis after 4 weeks of aortic banding. A, picric acid Sirius red staining of cardiac sections. The presence of significantly more fibrosis in Tg after TAC is shown. B, percentage of PASR positive areas. p < 0.05 (*) and p < 0.01 are (&) versus the corresponding NTg. p < 0.05 ($) and p < 0.01 (#) are versus SHAM. C, representative images of TUNEL staining of myocardial sections after TAC. Arrows indicate positive nuclei. DAPI, 4',6-diamidino-2-phenylindole. D, percent TUNEL-positive myocytes. p < 0.05 (*) and p < 0.01 (#) are versus SHAM. p < 0.05 ($) and p < 0.01 (&) are versus corresponding NTg.

 
GSK-3{alpha} Further Increases Cardiac Fibrosis and Apoptosis in Response to Pressure Overload—After TAC, cardiac fibrosis was increased in both NTg and Tg mice, with significantly more fibrosis in Tg mice (Fig. 7, A and B). The increase in percent fibrosis after 4 weeks of TAC was significantly greater in Tg than in NTg (1.52-versus 1.17-fold, p < 0.01). Similarly, TUNEL-positive nuclei were more numerous in both NTg and Tg mice 4 weeks after TAC, but Tg mouse hearts had significantly more positive nuclei than NTg (Fig. 7, C and D). The increase in percent TUNEL-positive nuclei was significantly greater in Tg than in NTg in response to 4 weeks of TAC (2.34-versus 1.72-fold, p < 0.01). The more severe fibrosis and apoptosis in Tg mouse hearts may, at least in part, explain more severe cardiac dysfunction in Tg mice after TAC.

GSK-3{alpha} Inhibits Growth and Promotes Apoptosis in Cardiac Myocytes in Vitro—To study whether GSK-3{alpha} directly acts on cardiac myocytes to inhibit cardiac growth and increase apoptosis, we performed in vitro experiments using cultured NRCMs. We made an adenovirus harboring GSK-3{alpha} (Ad-GSK-3{alpha}) and an adenovirus harboring a shRNA against GSK-3{alpha} (Ad-shRNA-GSK-3{alpha}). Ad-GSK-3{alpha} and Ad-shRNA-GSK-3{alpha} specifically increased and knocked down the expression of GSK-3{alpha}, respectively, without influencing the expression level of GSK-3beta in NRCMs (Fig. 8A). Overexpression of GSK-3{alpha} decreased cardiac myocyte size, whereas knockdown of GSK-3{alpha} increased cardiac myocyte size (Fig. 8B). Protein content in cardiac myocytes, another indicator of cardiac myocyte growth, was significantly decreased in a dose-dependent manner in cells transduced with Ad-GSK-3{alpha} but was significantly increased dose-dependently in cells transduced with Ad-shRNA-GSK-3{alpha} (Fig. 8C). These results suggest that GSK-3{alpha} negatively regulates cardiac myocyte growth. To study whether GSK-3{alpha} inhibits agonist-induced hypertrophy, we treated GSK-3{alpha}-overexpressing cardiac myocytes with phenylephrine. Increased cell size and protein content caused by phenylephrine were abolished in cardiac myocytes overexpressing GSK-3{alpha} (Fig. 8, D and E). Cytoplasmic accumulation of mono- and oligonucleosomes, detected by Cell Death ELISA, was dose-dependently increased by overexpression of GSK-3{alpha} but was dose-dependently inhibited by knockdown of GSK-3{alpha} (Fig. 8F). These results suggest that GSK-3{alpha} promotes cardiac myocyte apoptosis.

GSK-3{alpha} Inhibits Phosphorylation of ERK through PKC{delta}/{epsilon}-dependent Mechanisms—To study the signaling mechanisms involved in down-regulation of cardiac growth by GSK-3{alpha}, activities of various signaling molecules were screened. Among them, phosphorylation of ERK was decreased by 40% in Tg hearts (Fig. 9A). After TAC, phosphorylation of ERK was increased in both NTg and Tg, but the increase in Tg (2-fold) was significantly smaller than that in NTg (2.7-fold) (Fig. 9B). In NRCMs in vitro, adenovirus-mediated overexpression of GSK-3{alpha}, but not GSK-3beta, markedly reduced ERK phosphorylation (Fig. 9C), indicating that the effect on ERK phosphorylation may be specific to GSK-3{alpha}. On the other hand, knockdown of GSK-3{alpha} increased the phosphorylation of ERK in NRCMs (Fig. 9D). To study the signaling mechanism through which GSK-3{alpha} inhibits ERK phosphorylation, a MEK1 inhibitor and inhibitors for PKC{delta} and PKC{epsilon} were applied to NRCMs transduced with the Ad-shRNA-GSK-3{alpha}. The selective and cell-permeable MEK inhibitor, PD98059, dose-dependently inhibited ERK phosphorylation caused by GSK-3{alpha} knockdown (Fig. 9E), indicating that GSK-3{alpha} negatively regulates ERK phosphorylation through MEK. Both the PKC{delta} inhibitor, rottlerin, and the PKC{epsilon} inhibitor peptide also dose-dependently decreased ERK phosphorylation induced by GSK-3{alpha} silencing (Fig. 9, F and G), indicating that GSK-3{alpha} inhibits ERK phosphorylation through PKC{delta}/PKC{epsilon}.

GSK-3{alpha} Inhibits Cardiac Myocyte Growth and Promotes Apoptosis through Inhibition of ERK—We examined the mechanism through which down-regulation of GSK-3{alpha} induces cardiac hypertrophy. The MEK inhibitor, PD98059, significantly attenuated the effect of GSK-3{alpha} silencing on NRCM protein content (Fig. 10A), indicating that MEK-ERK-dependent mechanisms are the major pathway through which GSK-3{alpha} inhibits cardiac growth. Both the PKC{delta} inhibitor, rottlerin, and PKC{epsilon} inhibitor peptide also significantly inhibited the increase in protein content due to GSK-3{alpha} silencing in NRCMs (Fig. 10B), indicating that GSK-3{alpha} may inhibit cardiac growth through PKC{delta}/PKC{epsilon}-MEK-ERK-dependent mechanisms. Knockdown of GSK-3{alpha} significantly reduced apoptosis caused by serum starvation as expected (Fig. 10C). PD98059 significantly enhanced apoptosis caused by serum starvation and abolished the protective effects of GSK-3{alpha} silencing against serum starvation (Fig. 10C). These results suggest that endogenous GSK-3{alpha} stimulates apoptosis through inhibition of MEK-ERK-dependent mechanisms.


Figure 8
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  		FIGURE 8.
A, expression of GSK-3{alpha} in cardiac myocytes transduced with a control adenovirus (LacZ, 30 m.o.i.), adenovirus harboring GSK-3{alpha} (GSK-3{alpha}, 30 m.o.i.) or adenovirus harboring a shRNA against GSK-3{alpha} (shRNA, 30 m.o.i.). B, cardiac myocyte size when myocytes were transduced with LacZ (30 m.o.i.), GSK-3{alpha} (30 m.o.i.), or shRNA (30 m.o.i.). *, p < 0.05 versus LacZ. n = 3. C, protein content in cardiac myocytes transduced with LacZ, GSK-3{alpha}, or shRNA at the indicated doses. The protein content of LacZ at 10 m.o.i. is designated as 1. p < 0.05 (*) and p < 0.01 ($) are versus LacZ; n = 3. D and E, cell size and protein content of cardiac myocytes transduced with LacZ (30 m.o.i.) or GSK-3{alpha} (30 m.o.i.) after phenylephrine (PE, 10 µM) stimulation. p < 0.05 (*) and p < 0.01 (#) are versus LacZ. $, p < 0.05 versus control. n = 3. F, cytoplasmic accumulation of mono- and oligonucleosomes, a sensitive indicator of DNA fragmentation due to apoptosis, was quantitated by Cell Death ELISA Plus. Cardiac myocytes were transduced with LacZ, GSK-3{alpha}, or shRNA at the indicated doses. The apoptosis of LacZ at 10 m.o.i. is designated as 1. p < 0.05 (*) and p < 0.01 (#) are versus LacZ; n = 3.

 
GSK-3{alpha} Inhibits p70 S6 Kinase through ERK-dependent Mechanisms—Because protein synthesis is regulated through Akt/mTOR signaling, we examined the impact of GSK-3{alpha} overexpression upon this signaling pathway both in vitro and in vivo. Although phospho-Akt and Akt were not different between NTg and Tg, phosphorylation of p70 S6 kinase, a downstream target of mTOR, was decreased in Tg (Fig. 11A). In cultured cardiac myocytes, overexpression of GSK-3{alpha} inhibited, whereas knockdown of GSK-3{alpha} increased, the phosphorylation of p70 S6 kinase (Fig. 11B). Furthermore, increased phosphorylation of p70 S6 kinase induced by GSK-3{alpha} knockdown was abolished by the MEK1 inhibitor PD98059 (Fig. 11C), suggesting that GSK-3{alpha} inhibits p70 S6 kinase through a MEK/ERK-dependent mechanism.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results suggest that GSK-3{alpha} negatively regulates cardiac growth and promotes apoptosis in cardiac myocytes and that GSK-3{alpha} is up-regulated by clinically important pathologic insults such as pressure overload and myocardial infarction. Cardiac-specific overexpression of GSK-3{alpha} in mice resulted in small hearts and cardiac myocyte size, increases in apoptosis and fibrosis, and normal cardiac function at base line. In response to pressure overload, GSK-3{alpha} transgenic mice exhibited less severe cardiac hypertrophy, enhanced apoptosis and fibrosis, and markedly reduced cardiac function with increased wall stress. Unlike GSK-3beta transgenic mice, which also show inhibited cardiac growth (17), GSK-3{alpha} mice have normal base-line cardiac function up to 7 months of age and inhibition of ERK. In response to pressure overload, GSK-3{alpha} mice developed cardiac dysfunction, in contrast to GSK-3beta (S9A) transgenic mice, which demonstrated well maintained cardiac function despite inhibition of hypertrophy (15).

In GSK-3{alpha} transgenic mice, expression of GSK-3beta was significantly reduced. The molecular mechanism mediating the interplay between GSK-3{alpha} and GSK-3beta is unknown at present. We believe that the cardiac phenotype observed in Tg-GSK-3{alpha} is caused primarily by increases in GSK-3{alpha} rather than decreases in GSK-3beta, because in transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta, decreases in GSK-3beta activity induce cardiac hypertrophy and improve cardiac function.4 We speculate that the GSK-3{alpha} dominant environment overrides any cardiac phenotype induced by the 40% reduction in GSK-3beta activity in our transgenic mouse model.


Figure 9
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  		FIGURE 9.
A, phosphorylation of ERK (pERK) in mouse heart (line 28) at base line. *, p < 0.01 versus NTg. B, phosphorylation of ERK after TAC. *, p < 0.05 versus SHAM. #, p < 0.05 versus NTg. S, SHAM; T, TAC. C and D, phosphorylation of ERK in cardiac myocytes transduced with the indicated adenovirus (30 m.o.i.). *, p < 0.01 versus LacZ. E, phosphorylation of ERK in cardiac myocytes transduced with the indicated adenovirus (30 m.o.i.) with or without PD98059 at the indicated doses. PD98059 dose-dependently attenuated shRNA-GSK-3{alpha}-induced phosphorylation of ERK; n = 3. F, phosphorylation of ERK in cardiac myocytes transduced with indicated adenovirus (30 m.o.i.) with or without PKC{delta} inhibitor, rottlerin, at the indicated doses. Rottlerin dose-dependently inhibited shRNA-GSK-3{alpha}-induced ERK phosphorylation; n = 3. G, phosphorylation of ERK in cardiac myocytes transduced with indicated adenovirus (30 m.o.i.) with or without PKC{epsilon} inhibitor peptide at the indicated doses. The PKC{epsilon} inhibitor peptide dose-dependently decreased shRNA-GSK-3{alpha}-induced ERK phosphorylation; n = 3.

 
Mitogen-activated protein kinases (MAPKs) consist of three distinct groups of kinases; ERKs, c-Jun N-terminal kinase, and p38 MAPKs. The ERKs are generally associated with cell growth and differentiation and are activated through phosphorylation by upstream MEK1. Specific activation of ERK in the heart by overexpression of activated MEK1 resulted in concentric LV hypertrophy (i.e. enhanced cardiac growth) (22). Our results show that activation of GSK-3{alpha} in the heart in vivo decreased ERK phosphorylation and attenuated cardiac physiological growth. Furthermore, our in vitro data demonstrated that the MEK1 inhibitor abolished the effect of GSK-3{alpha} silencing on protein synthesis. These results suggest that negative regulation of cardiac growth by GSK-3{alpha} is primarily mediated by the MEK1-ERK pathway, although GSK-3{alpha} could have many other potential down-stream signaling mechanisms (Fig. 12). The results of the present study suggest that inhibition of ERK by GSK-3{alpha} may lead to inhibition of cardiac growth. Abundant in vitro evidence shows that inhibition of MEK/ERK signaling attenuates cardiac myocyte hypertrophy induced by many agonists (23-27), suggesting that ERK activation is intimately involved in the development of cardiac hypertrophy. After aortic banding, an increase in ERK phosphorylation was associated with an increase in left ventricular mass (28). In response to pressure overload, in dominant negative Raf1 transgenic mice, ERK activity was reduced, and cardiac hypertrophy was attenuated (29). These observations support the notion that MEK/ERK signaling is required for the development of cardiac hypertrophy under pressure overload. Therefore, inhibition of ERK by GSK-3{alpha} may also, at least in part, account for decreased cardiac hypertrophy in Tg-GSK-3{alpha} mice in response to pressure overload. Because GSK-3{alpha} is up-regulated by TAC, GSK-3{alpha} serves as a negative feedback regulator of ERKs and hypertrophy in response to TAC.

The inhibition of ERK may be induced by GSK-3{alpha} but not by GSK-3beta. Overexpression of GSK-3beta in the heart increased ERK phosphorylation in transgenic mice (17). In our GSK-3{alpha} transgenic mouse heart, ERK phosphorylation was inhibited. Furthermore, our results show that GSK-3beta did not decrease the phosphorylation of ERK in cardiac myocytes but that GSK-3{alpha} did. It is speculated that the presence of ERK inactivation makes GSK-3{alpha} overexpression mice more prone to LV dysfunction in response to pressure overload compared with GSK-3beta (S9A) overexpression mice, although this notion needs to be tested experimentally.


Figure 10
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  		FIGURE 10.
A, protein content of cardiac myocytes transduced with indicated adenovirus (30 m.o.i.) with or without PD98059 (5 µm). The protein content of LacZ control is designated as 1. *, p < 0.05 versus LacZ. #, p < 0.01 versus corresponding control. n = 6. B, protein content of cardiac myocytes transduced with the indicated adenovirus (30 m.o.i.) with or without rottlerin (2 µM) or PKC {epsilon} inhibitor peptide (1 µM). The protein content of LacZ control is designated as 1. *, p < 0.05 versus LacZ control. #, p < 0.01 versus corresponding control; n = 6. C, apoptosis detected by Cell Death ELISA Plus in cardiac myocytes transduced with indicated adenovirus (30 m.o.i.). SF, serum free. *, p < 0.01 versus LacZ SF. #, p < 0.001 versus corresponding sera control. $, p < 0.01 versus LacZ SF; &, p < 0.001 versus shRNA SF; n = 6.

 


Figure 11
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  		FIGURE 11.
A, immunoblotting of phospho-Akt (pAkt, S473), Akt, phospho-p70 S6 kinase (pS6K, T389), and S6K in mouse heart (line 28) homogenates. B, immunoblotting of pAkt, Akt, pS6K, and S6K in lysates of cardiac myocytes transduced with LacZ (30 m.o.i.), shRNA-GSK-3{alpha} (shRNA, 30 m.o.i.), or GSK-3{alpha} (30 m.o.i.). C, immunoblotting of pS6K and S6K in lysates of cardiac myocytes transduced with LacZ (30 m.o.i.) or shRNA-GSK-3{alpha} (shRNA, 30 m.o.i.) with or without PD98059 (5 µM).

 


Figure 12
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  		FIGURE 12.
Schematic demonstration of the mechanisms of action of GSK-3{alpha}on cardiac hypertrophy.

 
How, then, does GSK-3{alpha} inhibit the phosphorylation of ERK? Overexpression of PKC{epsilon} in adult cardiac myocytes significantly increased PKC{epsilon} activity and elevated ERK activity (30). In the PKC{epsilon} knock out mouse left ventricle, ERK phosphorylation was attenuated both at baseline and after TAC operation (31). These data point to a possibility that PKC{epsilon} activity is required for ERK phosphorylation. In our GSK-3{alpha} transgenic mouse hearts, PKC{epsilon} activation was attenuated (data not shown). GSK-3{alpha} may inhibit ERK activation through down-regulation of PKC{epsilon} activity. Our in vitro data showed that ERK phosphorylation caused by GSK-3{alpha} knockdown was inhibited by both PKC{delta} and PKC{epsilon} inhibitors, suggesting that endogenous GSK-3{alpha} inhibits ERK phosphorylation through PKC{delta}/{epsilon}-dependent mechanisms. It has been reported that PKC{epsilon} activation is mediated through PKC{delta} in ethanol-induced cardiac protection from ischemia (32). It is, therefore, possible that PKC{delta} and PKC{epsilon} are in the same pathway mediating ERK phosphorylation caused by GSK-3{alpha} silencing.

mTOR and its downstream targets, including p70 S6 kinase, play an important role in mediating increases in protein synthesis during cardiac hypertrophy (33, 34). Accumulating lines of evidence have suggested that the MEK/ERK pathway is involved in activation of mTOR signaling. For example, activation of protein synthesis in cardiac myocytes by the hypertrophic agonist phenylephrine requires ERK-dependent activation of mTOR signaling (24). The MEK/ERK pathway plays a major role in mediating activation of p70 S6 kinase during hypertrophic cardiac growth (35). The present study demonstrated a role of GSK-3{alpha} in inhibiting ERK-dependent p70 S6 kinase activation. MEK/ERK phosphorylates tuberous sclerosis complex 2 (TSC2) either directly or through p90RSK, thereby inducing dissociation of the TSC1-TSC2 complex, impairment of the ability of TSC2 to inhibit mTOR signaling, and subsequent increases in protein synthesis (36, 37). The precise mechanism by which the MEK/ERK pathway promotes mTOR signaling in cardiac myocytes, however, remains to be elucidated.

Cardiac hypertrophy is usually considered to be a compensatory mechanism, although prolonged presentation of hypertrophy often leads to heart failure. According to Laplace's Law, an increase in wall thickness of the left ventricle leads to a decrease in wall stress and, consequently, in oxygen consumption. In this sense, cardiac hypertrophy may be an adaptive response to mechanical overload. In agreement with this notion, impairment of this compensatory process results in cardiac dysfunction in some animal models of cardiac hypertrophy. For example, cyclosporine A, an inhibitor of calcineurin, attenuated cardiac hypertrophy but enhanced cardiac dysfunction and heart failure due to pressure overload (38). RGS4, a promoter of heterotrimeric G protein deactivation, overexpressed in the heart reduced cardiac hypertrophy in response to pressure overload but increased mortality due to heart failure (39). On the other hand, attenuation of pressure overload-induced cardiac hypertrophy by the C-terminal peptide of G{alpha}q, thioredoxin, and GSK-3beta (S9A) did not induce heart failure despite elevated wall stress (15, 40, 41). It is probable that the underlying signaling molecules altered by cardiac hypertrophic stimuli rather than the presence of hypertrophy alone are more important predictors of the prognosis of cardiac hypertrophy. In the case of GSK-3{alpha} activation, the present study shows that Tg-GSK-3{alpha} mice developed less cardiac hypertrophy but more apoptosis and interstitial fibrosis and more severe cardiac dysfunction in response to pressure overload. In the presence of GSK-3{alpha} overexpression, increased wall stress caused by lack of sufficient hypertrophy and increased apoptosis may stimulate one another, thereby facilitating heart failure.

In conclusion, the data presented here show that GSK-3{alpha} inhibits cardiac physiological growth, at least in part through inhibition of ERK activation. In response to pressure overload, GSK-3{alpha} transgenic mice developed less cardiac hypertrophy but more fibrosis and apoptosis along with more severe cardiac dysfunction. We propose that GSK-3{alpha} is a negative regulator of compensatory hypertrophy and a promoter of apoptosis. Thus, it is likely that stimulation of GSK-3{alpha} is detrimental during pressure overload-induced cardiac hypertrophy.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL33107, HL59139, HL67724, HL67727, HL69020, and HL73048 and by American Heart Association Grant 0340123N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. I-V. Back

1 Supported by National Institutes of Health Grant 1 F32 HL080861. Back

2 To whom correspondence should be addressed: 185 South Orange Ave., MSB G609, Newark, NJ 07103. Tel.: 973-972-8619; Fax: 973-972-8919; E-mail: sadoshju{at}umdnj.edu.

3 The abbreviations used are: GSK-3, glycogen synthase kinase-3; PKC, protein kinase C; LV, left ventricular; LVW, LV weight; BW, body weight; LVEDP, LV end-diastolic pressure; TL, tibia length; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; NRCM, neonatal rat cardiac myocyte; shRNA, short hairpin RNA; TUNEL, terminal dUTP nick-end labeling; TAC, transverse aortic constriction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tg, transgenic mice; NTg, non-Tg; m.o.i., multiplicity of infection; ELISA, enzyme-linked immunosorbent assay; EDD, end-diastolic dimension. Back

4 Hirotani, S., Zhai, P., Tomita, H., Galeotti, J., Marquez, J.P., Gao, S., Hong, C., Yatani, A., Avila, J., and Sadoshima, J. Circ. Res. in press. Back


   ACKNOWLEDGMENTS
 
We thank Daniela Zablocki for critical reading of the manuscript.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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P. Zhai and J. Sadoshima
Overcoming an Energy Crisis?: An Adaptive Role of Glycogen Synthase Kinase-3 Inhibition in Ischemia/Reperfusion
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B.-C. Yu, C.-K. Chang, H.-Y. Ou, K.-C. Cheng, and J.-T. Cheng
Decrease of peroxisome proliferator-activated receptor delta expression in cardiomyopathy of streptozotocin-induced diabetic rats
Cardiovasc Res, October 1, 2008; 80(1): 78 - 87.
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Y. Nishino, I. G. Webb, S. M. Davidson, A. I. Ahmed, J. E. Clark, S. Jacquet, A. M. Shah, T. Miura, D. M. Yellon, M. Avkiran, et al.
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Diacylglycerol kinase-{varepsilon} restores cardiac dysfunction under chronic pressure overload: a new specific regulator of G{alpha}q signaling cascade
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