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Methods and compositions relating to mek5 and cardiac hypertrophy and dilated cardiomyopathyRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain StructureMethods and compositions relating to mek5 and cardiac hypertrophy and dilated cardiomyopathy description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050261187, Methods and compositions relating to mek5 and cardiac hypertrophy and dilated cardiomyopathy. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to the fields of developmental biology and molecular biology. More particularly, it concerns gene regulation and cellular physiology in cardiomyocytes. [0004] 2. Description of Related Art [0005] Cardiac cells do not divide after birth, so both normal growth of the myocardium as well as stress-induced myocardial remodeling must take place through hypertrophic growth without cell division (MacLellan and Schneider, 2000). Cardiac hypertrophy can occur by an increase in width of myofibrils, resulting in a thickening of the myocardial wall or "concentric hypertrophy," or by an increase in myofibril length, producing chamber dilation or "eccentric hypertrophy." These contrasting forms of hypertrophy are coupled to parallel versus serial assembly of sarcomeres, respectively. [0006] In the case of normal physiological growth or exercise-induced hypertrophy, concentric and eccentric hypertrophy occur simultaneously and in a balanced manner, enabling the heart to increase pumping capacity in response to increased demand. Disease states that put stress on the heart can also induce hypertrophy. Depending on the stimulus, however, either concentric or eccentric hypertrophy may predominate. Although hypertrophy may initially compensate for the additional demands placed on the heart by disease, almost inevitably continued stress results in decompensation and the development of hypertrophic or dilated cardiomyopathy. In order for any form of hypertrophic remodeling to occur, stress stimuli must activate signaling pathways that regulate protein synthesis, sarcomeric assembly and organization, and gene expression (Chien, 1999; Nicol et al., 2000; Sugden and Clerk, 1998). [0007] Mitogen-activated protein kinase (MAPK) pathways provide an important connection between external stimuli that activate a wide variety of cell-signaling systems and the nucleus. At the core of each MAPK cascade is a three-kinase module in which the most downstream member, the MAPK, is activated by a MAPK kinase (MAPKK or MEK), which is in turn activated by a MAPKK kinase (MAPKKK or MEKK) (English et al., 1999a). MAPKs can be divided into three major subfamilies based on sequence homology: the extracellularly-responsive kinases (ERKs), the c-Jun NH.sub.2-terminal kinases (JNKs), also known as stress-activated protein kinases (SAPKs), and the p38-MAPKs. In the heart, all three classes of MAP kinases are activated by G-protein coupled receptor (GPCR) agonists, stretch, and certain types of stress, including ischemia (Abe et al., 2000; Ruwhof and van der Laarse, 2000; Sugden and Clerk, 1998). A critical role for MAPK pathways in the development of hypertrophy in vivo has been demonstrated by the finding that transgenic expression of a MAP kinase phosphatase in the mouse heart can attenuate hypertrophy induced by aortic banding and catecholamine infusion (Bueno et al., 2001). The role of individual MAPK pathways in various aspects of the hypertrophic response is more controversial (Sugden and Clerk, 1998). [0008] ERK5, also known as big MAPK 1 (BMK1), has an amino terminal domain that is homologous to ERKs 1 and 2, but has unique carboxyl-terminal and loop-12 domains (Lee et al., 1995; Zhou et al., 1995). MEK5, the activating MAPKK for ERK5, is a highly specific ERK5 kinase and does not activate other MAPKs even when overexpressed in cultured cells (English et al., 1995; Zhou et al., 1995). MEK5-ERK5 signaling has been shown to be activated by growth stimuli including serum and ligands for tyrosine kinase and GPCRs (Fukuhara et al., 2000; Kamakura et al., 1999; Kato et al., 1997), as well as by oxidative and osmotic stress (Abe et al., 1996). Signaling by this MAPK module has not been studied in detail in cardiac cells, but one report suggests that ERK5 may be regulated differently from ERK1/2 in these cells (Takeishi et al., 1999). Interestingly, the MEK1 inhibitors PD098059 and U0126 also inhibit activation of ERK5 (Kamakura et al., 1999), suggesting that functions previously attributed to ERK1/2 may also be mediated by ERK5. SUMMARY OF THE INVENTION [0009] Thus, in accordance with the present invention, there is provided a method for inhibiting cardiac hypertrophy in a subject comprising administering to said subject an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity inhibits cardiac hypertrophy. In another embodiment, there is provided a method for inhibiting dilated cardiomyopathy in a subject comprising administering to said subject an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity inhibits dilated cardiomyopathy. In yet another embodiment, there is provided a method for inhibiting heart failure in a subject comprising administering to said subject an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity inhibits heart failure. [0010] The method may further comprise administering to said subject a second anti-hypertrophic composition, for example, "beta blockers," anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators, hormone antagonists, endothelin antagonists, calcium channel blockers, phosphodiesterase inhibitors, angiotensin type 2 antagonists or cytokine blockers/inhibitors. The composition may be a MEK5 antisense molecule, an anti-MEK5 antibody, or a MEK5 ribozyme. The composition may inhibit or block MEK5 function, inhibit or block MEK5 transcription, inhibit or block MEK5 translation, inhibit or block MEK5 processing, or decrease MEK5 half-life. [0011] Alternatively, the composition may be a nucleic acid encoding a dominant negative MEK5 polypeptide, for example, a dominant-negative MEK5 polypeptide that contains at least one mutation in the ATP binding site, under the control of a promoter active in cardiac cells of said subject. The promoter may be myosin light chain-2 promoter, the .alpha. actin promoter, the troponin 1 promoter, the Na.sup.+/Ca.sup.2+ exchanger promoter, the dystrophin promoter, the creatine kinase promoter, the alpha7 integrin promoter, the brain natriuretic peptide promoter, the .alpha. B-crystallin/small heat shock protein promoter, .alpha. myosin heavy chain promoter or the ANF promoter. The nucleic acid may further comprise a polyadenylation signal, and may be comprised within an expression vector, which can include an origin of replication and a selectable marker gene. Expression vectors include both plasmids and viral vectors, for example, adenovirus, retrovirus, adeno-associated virus, vaccinia virus, herpesvirus or polyoma virus. Vectors may be comprised with a liposome. The viral vector may be comprised within a viral particle. The viral vector may be replication defective. [0012] In yet another embodiment, there is provided a method of inhibiting cardiac myocyte elongation in a cell comprising administering to said cell an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity inhibits cardiac myocyte elongation. In still yet another embodiment, there is provided a method of restoring balance between serial and parallel sarcomere assembly in a cell comprising administering to cell an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity restores the balance between serial and parallel sarcomere assembly. In yet a further embodiment, there is provided a method of inhibiting ventricular wall thinning in a subject comprising administering to said subject an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity inhibits ventricular wall thinning. In still yet a further embodiment, there is provided a method of reducing sensitivity of MEK5 to G-protein coupled receptor ("GPCR") agonists in a cell comprising administering to said cell an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity reduces sensitivity of MEK5 to GPCR agonists. In yet even a further embodiment, there is provided a method for inhibiting MEK5-induced hypertrophic signaling in a cell comprising administering to said cell an amount of a composition effective to inhibit MEK5 activity, whereby inhibition of MEK5 activity inhibits MEK5-induced cardiac hypertrophic signalling. [0013] In an additional embodiment, there is provided a non-human transgenic mammal, cells of which comprise a constitutively-activated MEK5 coding region under the control of a heterologous promoter, wherein said constitutively activated MEK5 is expressed in said cells. The promoter may be an inducible promoter, a tissue specific promoter, or a constitutive promoter. The activated MEK5 may contain phosporylation sites substituted with acidic residues. In a similar embodiment, there is provided a MEK5 coding region under the control of a promoter, wherein said MEK5 is expressed in said cells. In yet another related embodiment, there is provide a non-human transgenic mammal, cells of which comprise a dominant-negative MEK5 coding region under the control of a promoter, wherein said dominant-negative MEK5 is expressed in said cells. The dominant-negative MEK5 coding region may contain at least one mutation in the ATP binding site. [0014] In a further embodiment, there is provided a method of screening for an inhibitor of cardiac hypertrophy comprising (a) providing a cell comprising a MEK5 coding region under the control of a promoter, wherein MEK5 is expressed therefrom; (b) contacting said cell with a candidate inhibitor substance; and (c) determining MEK5 activity of said cell; wherein a reduction in MEK5 activity in the presence of said candidate inhibitor substance, as compared to the MEK5 activity in the absence of said candidate inhibitor substance, indicates that said candidate inhibitor substance is an inhibitor of MEK5 activity, and hence, an inhibitor of cardiac hypertrophy. The promoter may be heterologous to said MEK5 coding region. The transgenic cell may be cardiomyocyte located in a non-human transgenic animal. The assay may further comprise determining the activity of MEK5 in a comparable cell in the absence of said candidate inhibitor substance. The candidate inhibitor substance may be a nucleic acid or a small molecule. The step of determining may comprise measuring MEK5 kinase activity, measuring MEK5-induced cardiac hypertrophy signaling, measuring one or more aspects of cellular morphology (e.g., cell elongation, cell size and cell contractility) or measuring cardiac hypertrophy, or a symptom thereof (e.g., hypertrophic or fetal gene expression (ANF, alpha skeletal actin, myosin heavy chain gene switch or BNF), fibrosis, reduced cardiac contractility (measured by LV dp/dt, LV ejection fraction, RV ejection fraction, or altered LV pressure/volume loops), or increased heart/body, heart/brain weight or heart/tibia weight ratios). The MEK5 coding region may encode a constitutively-activated MEK5, for example, an activated MEK5 containing phosporylation sites substituted with acidic residues. The method may further comprise contacting said cell is a G-protein coupled receptor (GPCR) agonist, for example, an IL-6 family cytokine, leukemia inhibitory factor, or cardiotrophin-1. BRIEF DESCRIPTION OF THE DRAWINGS [0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0016] FIGS. 1A-D. Activation of endogenous ERK5 by hypertrophic and stress agents. Serum-deprived neonatal rat cardiomyoctyes were treated with (FIG. 1A) 100 .mu.M PE, (FIG. 1B) 1000 units/ml LIF, (FIG. 1C) 200 .mu.M H.sub.2O.sub.2, and (FIG. 1D) 0.3M sorbitol for the indicated times, harvested, and ERK5 kinase activity was measured. Top, ERK5 was immunoprecipitated from 200 .mu.g of cellular lysate with an antibody specific for the carboxyl-terminal 20 amino acids. Kinase assays were performed with immunoprecipitated ERK5 using GST-MEF2C substrate in the presence of [.gamma.-.sup.32P]-ATP. GST-MEF2C phosphorylation was detected by autoradiography after SDS-PAGE. Middle, immunoblotting was performed on immunoprecipitated material using rabbit anti-ERK5 antibody. Bottom, levels of .sup.32P-phosphorylated GST-MEF2C were quantitated with a Phosphor Imager. The averaged result .+-.standard deviation (SD) of three independent experiments is shown. [0017] FIGS. 2A-H. Activated MEK5 induces elongation of cultured neonatal rat cardiomyocytes. Adenoviruses expressing HA-tagged MEK5KM, MEK5WT, and MEK5DD were used to infect COS cells at an moi of 100. (FIG. 2A) Lysates were prepared 48 hrs post-infection and 5 .mu.g of protein was separated by SDS-PAGE and immunoblotted with anti-HA antibody. (FIG. 2B) Immunoprecipitations were performed on 100 .mu.g of protein with anti-HA antibody. Kinase assays were performed with immunoprecipitated HA-MEK5 using GST-ERK5KM.DELTA. substrate in the presence of [.gamma.-.sup.32P]-ATP. GST-ERK5KM.DELTA. phosphorylation was detected after SDS-PAGE by autoradiography. Serum-deprived cardiomyocytes were infected at an moi of 100 with adenovirus expressing (FIG. 2C) .beta.-galactosidase, (FIG. 2D) MEK1CA and (FIG. 2E and FIG. 2G) MEK5DD or not infected and treated with (FIG. 2F and FIG. 2H) PE (50 .mu.M). Cells were fixed 72 hours post-infection and immunostained with anti-sarcomeric .alpha.-actinin antibody. Note that cells in FIGS. 2G and H are shown at higher magnification than cells in FIGS. 2C-F. Bar=20 .mu.m. [0018] FIGS. 3A-F. Dominant negative MEK5 blocks LIF-induced elongation of neonatal rat cardiomyocytes. Cardiomyocytes were either not infected or infected with adenovirus at an moi of 100, serum-deprived, and 24 hours post-infection treated either with LIF (1000 units/ml) or PE (50 .mu.M) for an additional 48 hrs prior to fixation and immunostaining with anti-sarcomeric .alpha.-actinin. (FIG. 3A) uninfected cells treated with LIF (FIG. 3B) AdMEK5KM-infected cells treated with LIF (FIG. 3C) Ad.beta.-gal-infected cells treated with LIF (FIG. 3D) uninfected cells treated with PE (FIG. 3E) AdMEK5KM-infected cells treated with PE (FIG. 3F) Ad.beta.-gal-infected cells treated with PE. Bar=20 .mu.m. [0019] FIGS. 4A-B. MEK5 signaling contributes to the regulation of cardiomyocyte fetal gene expression by PE and LIF. (FIG. 4A) Cardiomyocytes were either not infected (-) or infected with MEK5WT, MEK5KM or .beta.-gal adenoviruses at an moi of 20 and serum-deprived. Thirty-six hours post-infection, cells were either not treated or treated with 50 .mu.M PE (black bar) or 1000 units/ml LIF (white bar) for an additional 24 hours. RNA was prepared and used for dot blots with oligonucleotide probes specific for skeletal .alpha.-actin, ANF or BNP. Signal intensity was quantitated using a Phosphor Imager. The average fold induction .+-.SD of three independent experiments is shown. "-fold" induction is relative to uninfected cells without PE or LIF treatment. (FIG. 4B) Cardiomyocytes were either not infected or infected with MEK5DD or .beta.-gal adenoviruses at an moi of 20 and serum-deprived. Forty-eight hours post-infection, the cells were harvested and RNA was prepared. Transcript levels for .alpha.-skeletal actin, ANF or BNP were determined as described in FIG. 4A. "-fold" induction is relative to uninfected cells. [0020] FIGS. 5A-C. Expression of MEK5 and ERK5 in wild-type and MEK5DD-transgenic mice. Lysates were prepared from wild-type (WT) and transgenic (TG) hearts, and 20 ug of protein was separated by SDS-PAGE. (FIG. 5A) Expression of HA-tagged MEK5DD was analyzed in different lines of transgenic mice by immunoblotting with anti-HA antibody. Lines of MEK5DD-transgenic mice are indicated by identifying numbers. For each line, lysate was prepared from two hearts and loaded in adjacent lanes. Expression of (FIG. 5B) MEK5 and (FIG. 5C) ERK5 was analyzed in wild-type and line 367 MEK5DD-transgenic mice by immunoblotting with L610 rabbit anti-MEK5 antiserum and rabbit anti-ERK5. Bands which are either nonspecific (asterisk) or degradation products (arrowhead) are indicated. Note reduced mobility of ERK5 in transgenic animals relative to wild-type. [0021] FIG. 6. Survival curve for wild-type and MEK5DD transgenic mice. F.sub.1 hemizygous transgenic mice were generated by backcrossing the transgenic founder mouse with C57B6 mice. The open circles represent percent survival of nontransgenic (NTG) F.sub.1 mice (n=24); the closed circles represent percent survival of transgenic (TG) F.sub.1 mice (n=24). [0022] FIGS. 7A-B. MEK5DD-transgenic hearts show progressive dilation and thinning of ventricular walls with age. (FIG. 7A) Hearts were removed from wild-type and MEK5DD-transgenic mice at 3 weeks, 6 weeks, and 12 weeks of age. Hearts were fixed in 10% PBS-buffered formalin and photographed. (FIG. 7B) Hearts from 12 week-old MEK5DD-transgenic and wild-type mice were fixed and sectioned longitudinally or at the midsagittal level parallel to the base and stained with hematoxylin-eosin. ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle. Continue reading about Methods and compositions relating to mek5 and cardiac hypertrophy and dilated cardiomyopathy... 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