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. 2004 Dec;24(24):10611-20.
doi: 10.1128/MCB.24.24.10611-10620.2004.

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload

Kazuhiko Nishida  1 , Osamu Yamaguchi, Shinichi Hirotani, Shungo Hikoso, Yoshiharu Higuchi, Tetsuya Watanabe, Toshihiro Takeda, Soh Osuka, Takashi Morita, Gen Kondoh, Yoshihiro Uno, Kazunori Kashiwase, Masayuki Taniike, Atsuko Nakai, Yasushi Matsumura, Jun-ichi Miyazaki, Tatsuhiko Sudo, Kenichi Hongo, Yoichiro Kusakari, Satoshi Kurihara, Kenneth R Chien, Junji Takeda, Masatsugu Hori, Kinya Otsu
Affiliations

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload

Kazuhiko Nishida et al. Mol Cell Biol. 2004 Dec.

Abstract

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.

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Figures

FIG. 1.
FIG. 1.
Targeted modification of the p38α gene. (A) Schematic structures of genomic p38α sequences, the targeting construct, and targeted allele. The closed boxes and triangles represent the floxed p38α exon and loxP site, respectively. The targeting construct includes the PGK-neomycin resistance gene cassette (Neo) flanked by loxP sites and the diphtheria toxin (DTA). The small arrows correspond to the primer sequences for PCR, and the open boxes (5′ probe) correspond to the sequence used for Southern blotting analysis in panel B. Restriction sites: N, NcoI; X, XhoI; H, HindIII; Bg, BglII; Sm, SmaI; Ba, BamHI; Sc, SacII; Sl, SalI. (B) Genomic analysis of ES cells. Genomic DNA was isolated from five ES cell clones identified to be homologous recombinants, digested with XhoI, and analyzed by Southern blotting with the 5′ probe. (C) Protein expression of the MAP kinase family in p38α CKO heart. Total cell lysates from p38α CKO and CTL hearts were examined for p38 isoforms, MKK3/6, ERK, and JNK with immunoblotting. The expression of p38α protein in isolated cardiomyocytes (CM) was also examined. (D) p38α activity in vitro kinase assay from phosphate-buffered saline- and phenylephrine (PE)-injected p38α CKO or CTL mice. Upper panel, phosphorylation of MBP was monitored by in vitro kinase assay with p38α-specific antibody. Lower panel, quantification of phospho-MBP protein levels by densitometric analysis. Open and closed bars represent phosphate-buffered saline- and PE-injected mice, respectively (n = 3). *, P < 0.05 versus corresponding phosphate-buffered saline-injected mice. #, P < 0.05 versus PE-injected CTL. Error bars indicate standard errors of the means.
FIG. 2.
FIG. 2.
Morphological and functional consequences of pressure overload in p38α CKO heart. (A) Macroscopic hematoxylin-eosin-stained histological sections of hearts from CTL and p38α CKO mice before and 7 days after TAC or sham operation. (B) Transthoracic M-mode echocardiographic tracings from a p38α CKO mouse and a CTL mouse before and 1 week after TAC or sham operation. (C) Changes in the echocardiographic parameters end-diastolic (LVDd) and end-systolic (LVDs) LV diameters and FS by TAC. Echocardiography was sequentially performed on mice 2 days before operation (Pre) and 7 days after operation (Post). Closed boxes, closed circles, open boxes, and open circles represent TAC-operated p38α CKO (n = 9), TAC-operated CTL (n = 10), sham-operated p38α CKO (n = 5) and sham-operated CTL (n = 6) mice, respectively. *, P < 0.05 versus corresponding preoperation. Error bars indicate standard errors of the means.
FIG. 3.
FIG. 3.
Histological analysis of p38α CKO mice after TAC. (A) Microscopic Masson-trichrome-stained histological sections (magnification, ×200) of hearts from CTL and p38α CKO mice before and 1 week after TAC or sham operation. (B) The fibrotic lesions in LV myocardium were measured by image analysis software. Open and closed bars represent sham-operated and TAC-operated mice, respectively (n = 3). *, P < 0.05 versus all other groups, including corresponding sham-operated mice. Error bars indicate standard errors of the means. (C) mRNA expression of collagen I or III was evaluated 1 week after TAC by dot blot analysis.
FIG. 4.
FIG. 4.
Cardiac hypertrophy by pressure overload in p38α CKO mice. TAC or sham operation was applied in either CTL or p38α CKO mice. Open and closed bars represent sham-operated and TAC-operated mice, respectively. *, P < 0.05 versus corresponding sham-operated mice. #, P < 0.05 versus TAC-operated CTL. Error bars indicate standard errors of the means. (A) The LV weight (LVW) (milligrams)/body weight (BW) (grams) ratio (left panel) or the LVW (milligrams)/tibia length (TL) (millimeters) ratio (right panel) was obtained 1 week after TAC. TAC-operated p38α CKO. n = 9; TAC-operated CTL, n = 10; sham-operated p38α CKO, n = 5; and sham-operated CTL, n = 6. (B) Cardiomyocyte cross-sectional areas. Myocyte cross-sectional area was measured by tracing the outline of 100 to 200 myocytes in each section (n = 3). Longitudinal cell length was estimated as the distance between cell edges identified by connexin 43 staining. (C) Cell surface area (left panel) and cell length (right panel). Cardiomyocytes were isolated from mouse hearts (n = 3) 1 week after TAC. Myocyte surface area was measured by tracing the outline of 100 to 200 myocytes. (D) mRNA expression of ANF, BNP, α-skeletal actin (αSkA), or α-myosin heavy chain (αMHC) 1 week after TAC was evaluated by dot blot analysis (n = 4). Densitometric analyses were performed. The mean of the ANF/GAPDH, BNP/GAPDH, αSkA/GAPDH, or αMHC/GAPDH ratio in CTL mice subjected to sham operation was expressed as 1.
FIG. 5.
FIG. 5.
Apoptosis in p38α CKO heart after TAC. (A) Confocal analysis of p38α CKO ventricular myocardium 1 week after TAC. Triple staining (propidium iodide, TUNEL, and anti-α-sarcomeric actin antibody) was performed. Staining for propidium iodide and anti-α-sarcomeric actin antibody is shown in red, and that for TUNEL is in green. In the overlay image, a nucleus stained by both TUNEL and propidium iodide is shown in yellow. (B) Morphology of nuclei in TUNEL-positive cells. (C) Number of TUNEL-positive cells in p38α CKO hearts compared to that in CTL 7 days after TAC. Open and closed bars represent sham (n = 3)- and TAC (n = 6)-operated mice, respectively. *, P < 0.05 versus all other groups. Error bars indicate standard errors of the means. (D) Analysis of cleaved caspase-3 protein levels in cardiac tissue 7 days after TAC or sham operation. Immunoblotting was performed with anti-cleaved caspase-3 antibody. (E) Analysis of cytochrome (Cyt) c protein levels in the cytosolic fraction of LV 7 days after TAC or sham operation. Immunoblotting was performed with anti-Cyt c antibody. Densitometric analyses were performed (right panel). TAC-operated p38α CKO, n = 6; TAC-operated CTL, n = 6; sham-operated p38α CKO, n = 4; and sham-operated CTL, n = 4. *, P < 0.05 versus all other groups. (F) Immunoblot analysis of apoptosis-related proteins in the mitochondrial fraction from LV myocardium after TAC or sham operation. Densitometric analyses of TAC-operated mice were performed, and the ratio of Bax to Bcl-2 is shown (right panel). TAC-operated p38α CKO, n = 4; TAC-operated CTL, n = 3. *, P < 0.05 versus TAC-operated CTL.
FIG. 6.
FIG. 6.
Western blot analysis of JNK, ERK, or MKK3/6 phosphorylation and phospho-JNK staining in the hearts of p38α CKO or CTL mice. Ventricular protein lysates were obtained 7 days after TAC or sham operation. (A) For each group, the phosphorylation level of the protein was estimated by using antiphospho (phos) antibody. Total protein levels were also examined with their specific antibodies. n.s., nonspecific bands. (B) Densitometric analyses were performed. Open and closed bars represent sham-operated and TAC-operated mice, respectively; n = 4 for each group. *, P < 0.05 versus corresponding sham-operated mice. #, P < 0.05 versus TAC-operated CTL. Error bars indicate standard errors of the means. (C) Phospho-JNK staining of p38α CKO or CTL hearts 7 days after TAC.
FIG. 7.
FIG. 7.
Isoproterenol-induced cardiomyocyte death. (A) Representative transthoracic M-mode echocardiographic tracings from isoproterenol-treated p38α CKO (p38α CKO-ISO) and CTL (CTL-ISO) mice. Mice were treated with isoproterenol for 2 days. (B) Changes in the echocardiographic parameters LVDd and FS after isoproterenol treatment. Open and closed bars represent saline-treated and isoproterenol-treated mice, respectively. Saline-treated p38α CKO, n = 4; isoproterenol-treated p38α CKO, n = 3; saline-treated CTL, n = 3; isoproterenol-treated CTL, n = 3. *, P < 0.05 versus all other groups, including saline-treated CTL. Error bars indicate standard errors of the means. (C) Relative number of TUNEL-positive cells in p38α CKO hearts compared to that in CTL hearts. Open and closed bars represent saline-treated and isoproterenol-treated mice, respectively. Saline-treated p38α CKO, n = 3; isoproterenol-treated p38α CKO, n = 5; saline-treated CTL, n = 3; isoproterenol-treated CTL, n = 3. *, P < 0.05 versus all other groups, including saline-treated CTL. (D) Cell viability was assessed by using a Cell Counting Kit-8 (n = 3). Viability of cells is expressed as the percentage of viability of cells in the absence of isoproterenol. Mouse neonatal cardiomyocytes isolated from p38α CKO (closed bars) or CTL (open bars) mice were incubated with various concentrations of isoproterenol for 48 h. *, P < 0.05 versus corresponding CTL.
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References

    1. Adams, R. H., A. Porras, G. Alonso, M. Jones, K. Vintersten, S. Paneill, A. Valladares, l. Perez, R. Kein, and A. R. Nebreda. 2000. Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development. Mol. Cell 6:109-116. - PubMed
    1. Agah, R., P. A. Frenkel, B. A. French, L. H. Michael, P. A. Overbeek, and M. D. Schneider. 1997. Gene recombination in postmitotic cells: targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J. Clin. Investig. 100:169-179. - PMC - PubMed
    1. Braz, J. C., O. F. Bueno, Q. Liang, B. J. Wilkins, Y. S. Dai, S. Parsons, J. Braunwart, B. J. Glascock, R. Klevitsky, T. F. Kimball, T. E. Hewett, and J. D. Molkentin. 2003. Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling. J. Clin. Investig. 111:1475-1486. - PMC - PubMed
    1. Chen, J., S. Kubalak, and K. Chien. 1998. Ventricular muscle-restricted targeting of the RXRalpha gene reveals a non-cell-autonomous requirement in cardiac chamber morphogenesis. Development 125:1943-1949. - PubMed
    1. Chevalier, D., and B. G. Allen. 2000. Two distinct forms of MAPKAP kinase-2 in adult cardiac ventricular myocytes. Biochemistry 39:6145-6156. - PubMed

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