Loss of muscle-specific RING-finger 3 predisposes the heart to cardiac rupture after myocardial infarction - PubMed (original) (raw)

Loss of muscle-specific RING-finger 3 predisposes the heart to cardiac rupture after myocardial infarction

Jens Fielitz et al. Proc Natl Acad Sci U S A. 2007.

Abstract

RING-finger proteins commonly function as ubiquitin ligases that mediate protein degradation by the ubiquitin-proteasome pathway. Muscle-specific RING-finger (MuRF) proteins are striated muscle-restricted components of the sarcomere that are thought to possess ubiquitin ligase activity. We show that mice lacking MuRF3 display normal cardiac function but are prone to cardiac rupture after acute myocardial infarction. Cardiac rupture is preceded by left ventricular dilation and a severe decrease in cardiac contractility accompanied by myocyte degeneration. Yeast two-hybrid assays revealed four-and-a-half LIM domain (FHL2) and gamma-filamin proteins as MuRF3 interaction partners, and biochemical analyses showed these proteins to be targets for degradation by MuRF3. Accordingly, FHL2 and gamma-filamin accumulated to abnormal levels in the hearts of mice lacking MuRF3. These findings reveal an important role of MuRF3 in maintaining cardiac integrity and function after acute myocardial infarction and suggest that turnover of FHL2 and gamma-filamin contributes to this cardioprotective function of MuRF3.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Gene targeting and generation of MuRF3 knockout mice. (A) Domains of mouse MuRF3 protein are shown at the top. RF, RING finger; MFC, MuRF family conserved domain; cc, coiled-coil; ARR, acidic-rich region. Amino acids are shown above the protein structure. The targeting vector contained a 4.5-kb 5′ arm, a 1.7-kb 3′ arm, a neomycin cassette (Neo), and a thymidine kinase gene (tk). Exons 1–9 are shown in boxes. Positions of 5′ and 3′ probes and PCR primers are indicated. (B) H&E-stained sections of 8-week-old WT and _MuRF3_−/− (KO) hearts. (Right) Representative sarcomeric structures visualized by transmission electron microscopy. (×6,000 magnification; scale bar, 2 μm.) (C) The distance between sarcomeric Z-lines in cardiomyocytes from WT and _MuRF3_−/− (KO) hearts as visualized by electron microscopy. Arrows indicate Z-lines. ∗, P < 0.01 vs. WT.

Fig. 2.

Fig. 2.

Analyses of _MuRF3_−/− hearts. (A) Echocardiographic measurements of WT sham (n = 10), WT MI (n = 12), _MuRF3_−/− (KO) sham (n = 7), and KO MI (n = 12) mice. LVESD, left ventricular end-systolic dimension; LVEDD, left ventricular end-diastolic dimension. (B) Representative _MuRF3_−/− ruptured hearts 3 days after MI. Arrow indicates site of rupture. (C) H&E-stained sections of WT and _MuRF3_−/− (KO) hearts after sham operation or MI. Arrow indicates site of rupture. (Right) High-magnification images of the periinfarct zone (marked by box in Center). ∗, P < 0.01 vs. sham; †, P < 0.01 vs. WT MI.

Fig. 3.

Fig. 3.

Expression of cardiac stress markers. Real-time RT-PCR was used to measure the expression of transcripts encoding ANF, BNP, α-MHC, β-MHC, and c-fos in the hearts of WT sham (n = 4), WT MI (n = 5), _MuRF3_−/− (KO) sham (n = 6), and KO MI (n = 6) mice. ∗, P < 0.01 vs. sham; †, P < 0.01 vs. WT MI.

Fig. 4.

Fig. 4.

Treatment of _MuRF3_−/− mice with isoproterenol reduces cardiac performance. (A) Representative H&E-stained sections of hearts from WT and _MuRF3_−/− (KO) mice treated with vehicle or isoproterenol (Iso) for 7 days. (B) Fractional shortening measured by echocardiography from WT vehicle-treated (n = 5), WT Iso-treated (n = 8), _MuRF3_−/− (KO) vehicle-treated (n = 4), and KO Iso-treated (n = 8) mice. (C) Numbers of TUNEL-positive nuclei per left ventricular section of WT vehicle-treated (n = 2), WT Iso-treated (n = 3), _MuRF3_−/− (KO) vehicle-treated (n = 2), and KO Iso-treated (n = 3) mice. n.d., none detected. ∗, P < 0.01 vs. sham; †, P < 0.01 vs. WT Iso.

Fig. 5.

Fig. 5.

MuRF3 interacts with and degrades FHL2 and γ-filamin. C2C12 cells were cotransfected with expression plasmids encoding Myc-MuRF3 or Myc-MuRF3Δcc1 and HA-FHL2 (A) or Flag-filamin A (Flag-FLNA) or Flag-γ-filamin (Flag-FLNC) (B) and coimmunoprecipitated 24 h posttransfection by using Myc or Flag antibody and immunoblotted with HA or Myc antibody. IP, immunoprecipitated; IB, immunoblotted. (C) C2C12 cells were cotransfected with expression plasmids encoding HA-FHL2 or Flag-γ-filamin (Flag-FLNC) with or without Myc-tagged or nontagged MuRF3. Protein lysates were used in Western blot analyses with HA or Flag antibody. α-Tubulin was used as control. (D) Endogenous FHL2 and γ-filamin (FLNC) proteins were measured in extracts from hearts of three individual WT and _MuRF3_−/− (KO) mice by using FHL2 and γ-filamin-specific antibodies. GAPDH was used as a control. (E) FHL2 and γ-filamin mRNA were measured by quantitative real-time PCR using ABI TaqMan probes and normalized to GAPDH. No change in mRNA expression of FHL2 and γ-filamin was found between WT (n = 4) and _MuRF3_−/− (KO, n = 4) mice hearts.

Similar articles

Cited by

References

    1. Wang X, Robbins J. Circ Res. 2006;99:1315–1328. - PubMed
    1. Ciechanover A. Neurology. 2006;66:S7–S19. - PubMed
    1. Razeghi P, Sharma S, Ying J, Li YP, Stepkowski S, Reid MB, Taegtmeyer H. Circulation. 2003;108:2536–2541. - PubMed
    1. Glass DJ. Nat Cell Biol. 2003;5:87–90. - PubMed
    1. Depre C, Wang Q, Yan L, Hedhli N, Peter P, Chen L, Hong C, Hittinger L, Ghaleh B, Sadoshima J, et al. Circulation. 2006;114:1821–1828. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources