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

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.

Figure 1.

MURF1 interacts with RACK1 in vivo and in vitro. (A) In a yeast two-hybrid screen the NH₂ terminus of MURF1 was used as bait to screen an adult heart library. (B) To confirm the yeast two-hybrid interaction, COS 7 cells were transfected with RACK1 and/or Myc-MURF1 as indicated. The cell lysates were immunoprecipitated with anti-Myc antibody followed by Western blotting with RACK1 antibody. (C) To test the interaction between MURF1 and RACK1 in vitro, GST fusion proteins (GST alone, GST-RACK1, or GST-DB1 as a negative control) were incubated with COS 7 cell lysates that express Myc-MURF1. The protein complexes were immunoblotted with anti-Myc or anti-RACK1 antibody. (D) GST fusion proteins (GST alone or GST-MURF1) were incubated with COS 7 cell lysates expressing the indicated HA-tagged RACK1 deletions (The rapidly migrating HA-immunoreactive bands in cell lysates probably represent RACK1 degradation products). The protein complexes were immunoblotted with anti-HA, -PKCε, or -GST antibody. (E) NRVM were infected with Ad.GFP or Ad.MURF1 at the indicated multiplicities of infection (MOI), and lysates were immunoprecipitated with an antibody against PKCε. Immunoprecipitates were then blotted for the presence of RACK1 and MURF1 (using an anti-Myc antibody).

Figure 2.

MURF1 colocalizes with RACK1 in cultured cardiac myocytes. Rat cardiac myocytes were infected with recombinant adenovirus Ad.GFP (A and B) or Ad.MURF1 (C and D) for 24 h in serum free medium followed by induction with PE (B and D) for 48 h. Immunostaining was done with anti-Myc and anti-RACK1 antibodies. This was followed by secondary antibody incubation with anti–rabbit Alexa 568 (red) and anti–mouse AMCA (blue). The green color represents GFP expression.

Figure 3.

MURF1 does not alter PE- and PMA-induced translocation of PKCβII from cytosol to the perinuclear region in NRVM. NRVM were infected with Ad.GFP (A–C) or Ad.MURF1 (D–F) for 24 h in serum-free medium followed by induction with PE or PMA for 15 min. The cells were fixed and incubated with anti-RACK1 (blue) and anti-PKCβII (red) for 2 h. (G) Quantitative determination of perinuclear PKCβII staining under the indicated conditions.

Figure 4.

MURF1 inhibits PMA-induced translocation of PKCε from the perinuclear region to focal adhesion complexes in NRVM. NRVM were infected with Ad.GFP (A and B) or Ad.MURF1 (C and D) for 24 h in serum-free medium followed by induction with PMA (B and D) for 15 min. The cells were fixed and incubated with anti-vinculin (blue) and anti-PKCε (red) for 2 h. Arrows indicate focal adhesions.

Figure 5.

MURF1 inhibits adrenergic agonist-induced PKCε activity (but not PKCβII activity) in the particulate fraction of NRVM. (A–C) After infection with Ad.GFP or Ad.MURF1 for 24 h in serum-free medium, NRVM were induced with PE or PMA for 15 min and subjected to subcellular fractionation. The detergent-soluble (S) and particulate (P) fractions were immunoprecipitated with anti-PKCβII (A) or anti-PKCε antibody (B and C) and were subjected to in vitro kinase assays using histone H1 and γ[³²P]ATP. (D and E) The results from densitometric scanning of kinase assays from three independent experiments are presented as means ± SEM of PKCε (D) or PKCβII (E) activity in the particulate fraction compared with soluble fraction. *, P < 0.05 compared with control.

Figure 6.

MURF1 inhibits focal adhesion formation in NRVM. NRVM were infected with Ad.GFP (A, B, and E) or Ad.MURF1 (C, D, and F) for 24 h in serum-free medium followed by induction with PE for 48 h (B and D). The cells were fixed in 3.7% formaldehyde followed by incubation with anti-paxillin (A–D) or anti-vinculin (E and F) antibodies. Arrows indicate focal adhesions. (G) Quantitative determination of paxillin-positive focal adhesions per cell under the indicated conditions.

Figure 7.

Effects of MURF1 on components of the FAK signaling pathway. (A) NRVM were infected with Ad.GFP or Ad.MURF1 for 24 h followed by induction with PE or PMA. NRVM lysates were separated by SDS-PAGE. Blots were incubated with anti-pFAK (397Y) antibody and anti-FAK antibody (loading control). (B) NRVM cell lysates treated as in A were immunoprecipitated with anti-paxillin antibody followed by immunoblotting with anti-phosphotyrosine (PY) antibody. (C) To test for activation of MAP kinase signaling, NRVM cell lysates were analyzed by Western blotting with anti-pERK antibody and anti-ERK antibody (loading control). The cumulative results are presented as fold increase in ERK activity from basal level ± SEM.

Figure 8.

MURF1 inhibits adrenergic agonist-induced cardiac gene expression. (A) NRVM were infected with Ad.GFP or Ad.MURF1 for 24 h followed by induction with PE for 48 h. The cells were followed by immunostaining with rabbit anti-ANF antibody (red). (B) After adenoviral infection for 24 h in serum-free medium, cells were induced with PE, Ang II, IGF-1, endothelin-1 (ET-1), and serum for 24 h. mRNA was isolated and subjected to RT-PCR using primers specific to skeletal α-actin, ANF, β-MHC, and GAPDH.

Figure 9.

Effects of MURF1 on cardiac cell size and sarcomere organization. (A) NRVM were infected with Ad.GFP or Ad.MURF1 for 24 h followed by induction with PE or PMA for 48 h. The cells were observed under a fluorescent microscope with a 20× objective lens. (B) Approximately 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 mouse anti-α-actinin antibody (blue). (D) Insets reveal sarcomeric organization in myocytes.

Figure 10.

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.