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Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.

Arya R, Kedar V, Hwang JR, McDonough H, Li HH, Taylor J, Patterson C - J. Cell Biol. (2004)

Bottom Line: Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy.MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1.MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size.

View Article: PubMed Central - PubMed

Affiliation: Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCepsilon translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.

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Effect of endogenous MURF1 on cardiac cell size and ANF expression. (A) COS 7 cells were transiently transfected with pCMV-Myc-MURF1 and pSIREN-MURF1 siRNA or with control vectors as indicated. The cell lysates were subjected to SDS-PAGE analysis followed by Western blotting with anti-Myc antibody. (B) NRVM cells were transfected with GFP and MURF1 siRNA for 36 h followed by induction with PE or PMA for 48 h. The cells were observed with a fluorescent microscope with a 60× objective lens. 150–200 cells per condition were measured for cell surface area using the ImageJ program. The data are presented as means ± SEM. *, P < 0.05 compared with control. (C) After induction with PE or PMA, cells were fixed and immunostained with rabbit anti-ANF antibody. 200 cells per condition were counted for ANF expression. The data are presented as means ± SEM. *, P < 0.05 compared with control.
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fig10: Effect of endogenous MURF1 on cardiac cell size and ANF expression. (A) COS 7 cells were transiently transfected with pCMV-Myc-MURF1 and pSIREN-MURF1 siRNA or with control vectors as indicated. The cell lysates were subjected to SDS-PAGE analysis followed by Western blotting with anti-Myc antibody. (B) NRVM cells were transfected with GFP and MURF1 siRNA for 36 h followed by induction with PE or PMA for 48 h. The cells were observed with a fluorescent microscope with a 60× objective lens. 150–200 cells per condition were measured for cell surface area using the ImageJ program. The data are presented as means ± SEM. *, P < 0.05 compared with control. (C) After induction with PE or PMA, cells were fixed and immunostained with rabbit anti-ANF antibody. 200 cells per condition were counted for ANF expression. The data are presented as means ± SEM. *, P < 0.05 compared with control.

Mentions: To demonstrate that endogenous expression of MURF1 has a role in the regulation of hypertrophy, we knocked down MURF1 expression in NRVM by generating small interfering RNA (siRNA) specific to MURF1. MURF1 siRNA inhibited expression of MURF1 in COS 7 cells by >70% (Fig. 10 A). MURF1 siRNA was cotransfected in NRVM along with EGFP to mark transfected cells. A total of 150–200 cells per condition were scored for their cell surface areas. When MURF1 expression was knocked down, cells were 2.5-fold larger than the control cells under quiescent conditions and 1.5-fold higher after treatment with PE or PMA (Fig. 10 B). To further confirm the affect of endogenous MURF1 on hypertrophy, we examined the expression of ANF in cells transfected with MURF1 siRNA and counted the number of cells expressing ANF. We observed a 20-fold increase in cells expressing ANF under basal conditions and enhancement of PE- and PMA-induced ANF expression (Fig. 10 C). These data indicate that endogenous MURF1 regulates NRVM cell size tonically and also after stimulation with hypertrophic agonists.


Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.

Arya R, Kedar V, Hwang JR, McDonough H, Li HH, Taylor J, Patterson C - J. Cell Biol. (2004)

Effect of endogenous MURF1 on cardiac cell size and ANF expression. (A) COS 7 cells were transiently transfected with pCMV-Myc-MURF1 and pSIREN-MURF1 siRNA or with control vectors as indicated. The cell lysates were subjected to SDS-PAGE analysis followed by Western blotting with anti-Myc antibody. (B) NRVM cells were transfected with GFP and MURF1 siRNA for 36 h followed by induction with PE or PMA for 48 h. The cells were observed with a fluorescent microscope with a 60× objective lens. 150–200 cells per condition were measured for cell surface area using the ImageJ program. The data are presented as means ± SEM. *, P < 0.05 compared with control. (C) After induction with PE or PMA, cells were fixed and immunostained with rabbit anti-ANF antibody. 200 cells per condition were counted for ANF expression. The data are presented as means ± SEM. *, P < 0.05 compared with control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172633&req=5

fig10: Effect of endogenous MURF1 on cardiac cell size and ANF expression. (A) COS 7 cells were transiently transfected with pCMV-Myc-MURF1 and pSIREN-MURF1 siRNA or with control vectors as indicated. The cell lysates were subjected to SDS-PAGE analysis followed by Western blotting with anti-Myc antibody. (B) NRVM cells were transfected with GFP and MURF1 siRNA for 36 h followed by induction with PE or PMA for 48 h. The cells were observed with a fluorescent microscope with a 60× objective lens. 150–200 cells per condition were measured for cell surface area using the ImageJ program. The data are presented as means ± SEM. *, P < 0.05 compared with control. (C) After induction with PE or PMA, cells were fixed and immunostained with rabbit anti-ANF antibody. 200 cells per condition were counted for ANF expression. The data are presented as means ± SEM. *, P < 0.05 compared with control.
Mentions: To demonstrate that endogenous expression of MURF1 has a role in the regulation of hypertrophy, we knocked down MURF1 expression in NRVM by generating small interfering RNA (siRNA) specific to MURF1. MURF1 siRNA inhibited expression of MURF1 in COS 7 cells by >70% (Fig. 10 A). MURF1 siRNA was cotransfected in NRVM along with EGFP to mark transfected cells. A total of 150–200 cells per condition were scored for their cell surface areas. When MURF1 expression was knocked down, cells were 2.5-fold larger than the control cells under quiescent conditions and 1.5-fold higher after treatment with PE or PMA (Fig. 10 B). To further confirm the affect of endogenous MURF1 on hypertrophy, we examined the expression of ANF in cells transfected with MURF1 siRNA and counted the number of cells expressing ANF. We observed a 20-fold increase in cells expressing ANF under basal conditions and enhancement of PE- and PMA-induced ANF expression (Fig. 10 C). These data indicate that endogenous MURF1 regulates NRVM cell size tonically and also after stimulation with hypertrophic agonists.

Bottom Line: Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy.MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1.MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size.

View Article: PubMed Central - PubMed

Affiliation: Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCepsilon translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.

Show MeSH
Related in: MedlinePlus