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. 2000 Dec 1;19(23):6341-50.
doi: 10.1093/emboj/19.23.6341.

The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice

O F Bueno  1 , L J De Windt, K M Tymitz, S A Witt, T R Kimball, R Klevitsky, T E Hewett, S P Jones, D J Lefer, C F Peng, R N Kitsis, J D Molkentin
Affiliations

The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice

O F Bueno et al. EMBO J. .

Abstract

Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.

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Figures

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Fig. 1. Cardiac histological analysis of MEK1 transgenic mice. (A) Macroscopic hematoxylin–eosin-stained histological section of hearts from a low, intermediate (int) and high transgene copy number MEK1 line at 8 weeks of age. ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle. (B) Microscopic histological analysis revealed no histopathology in hematoxylin–eosin- or trichrome-stained heart sections from high-copy-number MEK1 transgenic mice. Wheat germ agglutinin (WGA)–TRITC-stained histological sections demonstrated noticeably larger myofibrils in high-copy-number MEK1 transgenic hearts. Identical histological observations were made in intermediate- and low-copy-number MEK1 lines (data not shown). (C) Myofibrillar cross-sectional areas were quantified from WGA–TRITC-stained histological sections. At least 150 fibers were measured each in two wild-type (wt) hearts and a high and intermediate MEK1 transgenic heart. *P <0.05 compared with wild-type hearts.
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Fig. 2. Quantitation of MEK1/2 protein levels and ERK1/2 activation in MEK1 transgenic hearts. (A) Western blotting was performed on protein extracts from two non-transgenic (NTG) hearts and five high-copy MEK1 transgenic hearts at 8 weeks. Quantitative analysis revealed a significant increase in MEK1/2 protein, ERK1/2 protein (*) and phosphorylated ERK1/2 (*) analyzed with a phospho-specific antibody. However, JNK1/2 and p38 phosphorylation and total protein levels were unaffected. (B) Western blotting for MEK1/2 and ERK1/2 protein levels across low-, intermediate (int)- and high-copy-number MEK1 transgenic lines revealed an increase in MEK1/2 protein with increasing copy number (two separate hearts each). However, ERK1/2 protein levels remained elevated and did not increase significantly as copy number increased. GAPDH protein levels were invariant.
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Fig. 3. RNA dot blotting of hypertrophy-associated genes in MEK1 hearts. Hearts from high expressing MEK1 transgenic mice were harvested at 8 weeks of age and total ventricular RNA was isolated and subjected to dot-blot hybridization to measure levels of ANF, BNP, skeletal α-actin, α-MHC, β-MHC, PLB and GAPDH as a loading control. The quantitation of these data is shown as a bar graph. Error bars represent the SEM.
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Fig. 4. Adenoviral MEK1 infection induces hypertrophy in cultured neonatal cardiomyocytes. (A and B) AdMEK1 infection dramatically augmented MEK1/2 protein levels (MEK1/2 antibody in green) in neonatal cardiomyocytes compared with Adβgal-infected cells. (C and D) Cardiomyocytes were co-immunostained for α-actinin (red/orange) to show both sarcomeric organization and myocyte surface area in response to AdMEK1 or Adβgal infection (after 24 h). (E) Western blot analysis from AdMEK1-infected neonatal cardiomyocytes revealed a significant increase in MEK1/2 protein and ERK1/2 phosphorylation. The MEK1 inhibitor U0126 (20 µM) blocked ERK1/2 phosphorylation. AdMEK1 infection did not result in p38 or JNK1/2 (p46 and p54) phosphorylation, nor were ERK1/2, p38 or JNK1/2 protein levels changed after 24 h of infection. Identical results were obtained in three independent experiments.
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Fig. 5. Quantitation of the hypertrophic phenotype of AdMEK1-infected cardiomyocytes. (A) Cell surface area was measured in α-actinin-stained cardiomyocyte cultures using confocal microscopy and digitized imaging (24 h after infection). Cultures were left in serum-free medium with no stimulation or were infected with Adβgal or AdMEK1, or were treated with U0126. (B) Cardiomyocytes were also stained with an ANF-specific antibody to quantify the percentage of cells expressing ANF. The data in (A) and (B) were obtained in two independent experiments. (C) Total ANF mRNA levels were also quantified by RT–PCR, showing similar induction of ANF mRNA between 1% FBS and AdMEK1 infection. (D) Protein synthesis rates were monitored by the incorporation of [3H]leucine in Adβgal-, AdMEK1- and 1% FBS-stimulated cardiomyocytes. *P <0.05 compared with Adβgal; †P <0.05 compared with AdMEK1 infection alone.
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Fig. 6. MEK1 confers protection from TUNEL, caspase 3 activation and DNA laddering. (A) Cultured neonatal cardiomyocytes were infected with Adβgal, AdAkt or AdMEK1 and 24 h later placed in glucose-free and serum-free media in the presence of 2-deoxyglucose to induce apoptosis. The data demonstrate that culturing for 12 or 18 h in the presence of 2-deoxyglucose induced significant TUNEL in Adβgal-infected myocytes, while AdAkt or AdMEK1 infection conferred resistance. (B) AdMEK1 and AdAKT also provided partial protection from caspase 3 activation induced by 2-deoxyglucose. (C) In vivo, MEK1 transgenic and wild-type mice were subjected to 45 min of cardiac ischemia followed by 4 h of reperfusion (I/R). Forty micrograms of genomic DNA were size fractionated on a 1.4% agarose gel containing ethidium bromide to qualitatively analyze DNA laddering in the heart. MEK1 transgenic hearts were partially resistant to DNA laddering following the I/R procedure compared with sham hearts. Wild-type–sham, n = 6; wild-type–I/R, n = 11; MEK1-TG–sham, n = 8; MEK1-TG–I/R, n = 13. *P <0.05 compared with Adβgal.
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References

    1. Adderley S.R. and Fitzgerald,D.J. (1999) Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J. Biol. Chem., 274, 5038–5046. - PubMed
    1. Aikawa R., Komuro,I., Yamazaki,T., Zou,Y., Kudoh,S., Tanaka,M., Shiojima,I., Hiroi,Y. and Yazaki,Y. (1997) Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J. Clin. Invest., 100, 1813–1821. - PMC - PubMed
    1. Bialik S., Geenen,D.L., Sasson,I.E., Cheng,R., Horner,J.W., Evans,S.M., Lord,E.M., Koch,C.J. and Kitsis,R.N. (1997) Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53. J. Clin. Invest., 100, 1363–1372. - PMC - PubMed
    1. Bialik S., Cryns,V.L., Drincic,A., Miyata,S., Wollowick,A.L., Srinivasan,A. and Kitsis,R.N. (1999) The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes. Circ. Res., 85, 403–414. - PubMed
    1. Bogoyevitch M.A., Glennon,P.E. and Sugden,P.H. (1993) Endothelin-1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes. FEBS Lett., 317, 271–275. - PubMed

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