Research ArticleCardiovascular Biology

A peptide of the RGS domain of GRK2 binds and inhibits Gαq to suppress pathological cardiac hypertrophy and dysfunction

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Science Signaling  22 Mar 2016:
Vol. 9, Issue 420, pp. ra30
DOI: 10.1126/scisignal.aae0549
  • Fig. 1 Cardiac-specific βARKrgs expression does not alter hemodynamic function.

    (A) Representative Western blot of TgβARKrgs (Tg) and nontransgenic littermate control (NLC or N) ventricular lysates probed for Flag-tagged βARKrgs and the loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B to E) Hemodynamics were recorded from 10- to 12-week-old TgβARKrgs and nontransgenic mice. Quantification of mean systemic pressure (B), heart rate (C), left ventricular +dP/dt average maximum (D), and left ventricular −dP/dt average minimum (E) at baseline and with increasing doses of isoproterenol. Statistics are relative to nontransgenic littermate control mice by nonparametric Student’s t test for (B) and two-way analysis of variance (ANOVA) with repeated measures and Bonferroni post hoc test for (C) to (E) as appropriate. The genotype of all animals was confirmed by Western blot before unblinding the analysis and results, meaning n > 10 independent experiments for (A); n = 12 mice per group for (B) to (E).

  • Fig. 2 TgβARKrgs mice exhibit a similar decrease in cardiac hypertrophy as TgGqI mice after TAC.

    (A to F) TgβARKrgs and nontransgenic littermate control sham (Sh) and post-TAC animals were analyzed at baseline and at 2 and/or 4 weeks after surgery. Percent left ventricular ejection fraction (A). Serial measures for left ventricular (LV) posterior wall thickness during systole (LVPWs) (B), diastole (LVPWd) (C), and left ventricular mass (D). Measures of heart weight (HW) normalized to tibia length (TL) (HW/TL) (E) and left atrial weight (F) 4 weeks after surgery. (G and H) TgGqI and nontransgenic sham and post-TAC animals were analyzed at baseline and at 2 and/or 4 weeks after surgery. Serial measures for LVPWs (G) and LV mass (H). (I) Measures of HW/TL. *P = 0.01; **P = 0.001; ***P < 0.0001 as determined by two-way ANOVA with repeated measures and Bonferroni post hoc test for (A) to (D) and (G) to (H) and by one-way ANOVA with Tukey post hoc test for (E), (F), and (I) relative to nontransgenic sham mice. τP = 0.01; τττP < 0.0001 by two-way ANOVA with repeated measures and Bonferroni post hoc test relative to nontransgenic TAC mice. tP = 0.01 by one-way ANOVA with Tukey post hoc test relative to nontransgenic TAC mice. n = 10 to 16 mice per group for (A) to (I).

  • Fig. 3 Hypertrophy is unaltered in TgβARKct and TgGRK2 mice after TAC.

    (A and B) Serial measures of TgβARKct and nontransgenic littermate control sham and post-TAC animals for LVPWs (A) and LV mass (B). (C and D) Serial measures of TgGRK2 and nontransgenic sham and post-TAC animals for LVPWs (C) and LV mass (D). (E and F) Measures of HW/TL (E) and left atrial weight (F) in TgβARKct and nontransgenic sham and post-TAC animals 4 weeks after surgery. (G and H) Measures of HW/TL (G) and left atrial weight (H) in TgGRK2 and nontransgenic sham and post-TAC animals 4 weeks after surgery. ***P < 0.0001 by two-way ANOVA with repeated measures and Bonferroni post hoc test for (A) to (D). *P = 0.01; **P = 0.001; ***P < 0.0001 by one-way ANOVA with Tukey post hoc test (E to H) relative to corresponding nontransgenic sham mice. n = 8 to 14 mice per group for (A) to (H).

  • Fig. 4 Chronic pressure overload–induced heart failure is inhibited by cardiac βARKrgs expression.

    (A to E) Serial measures of nontransgenic littermate control and TgβARKrgs sham and post-TAC animals for percent LV ejection fraction (A), LV mass (B), and LVPWs (C). Lung weight normalized to TL (LW/TL) (D) and left atrial weight (E) 14 weeks after surgery. *P = 0.01; **P = 0.001; ***P < 0.0001 by two-way ANOVA with repeated measures and Bonferroni post hoc test for (A) to (C). ***P < 0.0001 by one-way ANOVA with Tukey post hoc test for (D) to (E) relative to nontransgenic sham mice. τττP < 0.0001 by two-way ANOVA with repeated measures and Bonferroni post hoc test relative to nontransgenic TAC mice. n = 8 to 15 mice per group for (A) to (E).

  • Fig. 5 Molecular markers of left ventricular dysfunction are reduced in TgβARKrgs and TgGqI mice but not in TgβARKct mice.

    (A to F) Quantification of RT-PCR data in nontransgenic littermate control and transgenic sham and post-TAC mice 4 weeks after surgery showing fold change in mRNA expression of atrial natriuretic factor (ANF) in TgβARKrgs (A) and TgGqI (B) mice, brain natriuretic peptide (BNP) in TgβARKrgs (C) and TgGqI (D) mice, and β-myosin heavy chain (βMHC) in TgβARKrgs (E) and TgGqI (F) mice. **P = 0.001; ***P < 0.0001 by one-way ANOVA with Tukey post hoc test relative to nontransgenic sham mice. tP = 0.001; ttP < 0.0001 by one-way ANOVA with Tukey post hoc test relative to nontransgenic TAC mice. n = 8 to 10 mice per group for (A), (C), and (E), and n = 12 to 13 mice per group for (B), (D), and (F). (G and H) Quantification of GRK2 protein abundance normalized to GAPDH from Western blots in TgβARKrgs (G) and TgβARKct (H) sham and post-TAC animals 4 weeks after surgery. See fig. S3, D and E, for representative Western blots. *P = 0.01; **P = 0.001 by nonparametric one-way ANOVA with Dunn post hoc test relative to nontransgenic sham mice. n = 5 to 10 mice per group and 4 blots for (G) and 3 blots for (H).

  • Fig. 6 Left ventricular structural remodeling is inhibited in TgβARKrgs mice.

    (A) Representative images of WGA and 4′,6-diamidino-2-phenylindole (DAPI)–stained murine heart sections from nontransgenic littermate control and TgβARKrgs hearts 4 weeks after sham or TAC surgery. Scale bar, 50 μm. (B) Quantification of cardiomyocyte CSA in these animals. */tP = 0.0001 by one-way ANOVA with repeated measures and Tukey post hoc test relative to nontransgenic sham and TAC animals, respectively. n = 5 to 9 hearts per group, 40 images per heart. (C) Representative images of Masson trichrome–stained murine heart sections from nontransgenic and TgβARKrgs hearts 4 weeks after sham or TAC surgery. Scale bar, 2000 μm. (D) Representative higher magnification (×40) images of Masson trichrome–stained post-TAC murine heart sections from nontransgenic and TgβARKrgs mice demonstrating interstitial fibrosis. Scale bar, 200 μm. (E) Quantification of the percent of fibrotic tissue in the left ventricular area in nontransgenic and TgβARKrgs hearts 4 weeks after TAC. ***P < 0.0001 by Student’s t test relative to nontransgenic TAC mice. n = 108 images from nine hearts for nontransgenic mice (~11 images per heart) and 78 images from seven hearts for TgβARKrgs mice.

  • Fig. 7 The βARKrgs peptide interacts with endogenous Gαq in the heart.

    (A) Representative image of a monoclonal Flag immunoprecipitation (IP) followed by Western blotting probing for rabbit polyclonal Gαq and rabbit polyclonal Flag-tagged βARKrgs in cardiac lysates from nontransgenic littermate control and TgβARKrgs mice. (B and C) Quantification of (B) Gαq normalized to βARKrgs-Flag and (C) total βARKrgs in TgβARKrgs lysates after monoclonal Flag IP 4 weeks after sham or TAC (T) surgery. (D) Representative image of a polyclonal GRK2 IP followed by Western blotting probing with goat polyclonal Gαq/11 and mouse monoclonal GRK2 in cardiac lysates from nontransgenic and TgβARKrgs mice. **P < 0.001 by nonparametric Student’s t test relative to TgβARKrgs TAC mice. n = 6 sham hearts and 8 TAC hearts and 4 Western blots each with sham and TAC hearts run side by side. (E) Quantification of IP3 concentrations from nontransgenic and TgβARKrgs lysates 4 weeks after sham or TAC surgery. *P < 0.01 by nonparametric one-way ANOVA with Dunn post hoc test relative to nontransgenic sham mice. n = 6 to 8 mice per group.

  • Fig. 8 Model for GPCR signaling in the heart.

    Agonist binding to GPCRs in the heart leads to activation and dissociation of the heterotrimeric G protein complex. Gα and Gβγ subunits then bind to downstream effector proteins triggering signaling cascades that control cardiac function. Catecholamine binding to β1-adrenergic receptors (β1ARs) leads to activation of Gαs, which directly stimulates adenylyl cyclase at the membrane, altering heart rate and contractility. Concomitantly, GRK2 associates with dissociated Gβγ subunits and translocates to the membrane where it phosphorylates the agonist-bound receptor, leading to receptor desensitization and internalization. Agonist binding to Gαq-coupled receptors leads to activation of multiple Gαq effector proteins involved in cardiomyocyte hypertrophy and vascular function. This study suggests that a direct interaction of the βARKrgs peptide, but not the RGS domain within endogenous GRK2, with Gαq competes for binding to other Gαq effector proteins, inhibiting downstream signaling and cardiac hypertrophy.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/420/ra30/DC1

    Fig. S1. TgβARKrgs mice exhibit a similar decrease in cardiac hypertrophy as TgGqI mice after TAC.

    Fig. S2. Chronic pressure overload–induced heart failure is inhibited by cardiac βARKrgs expression.

    Fig. S3. Molecular markers of left ventricular dysfunction are reduced in TgβARKrgs and TgGqI mice but not in TgβARKct mice.

    Fig. S4. Left ventricular structural remodeling is inhibited in TgβARKrgs mice.

    Fig. S5. The βARKrgs peptide interacts with endogenous Gαq in the heart.

  • Supplementary Materials for:

    A peptide of the RGS domain of GRK2 binds and inhibits Gαq to suppress pathological cardiac hypertrophy and dysfunction

    Sarah M. Schumacher, Erhe Gao, Maya Cohen, Melissa Lieu, J. Kurt Chuprun, Walter J. Koch*

    *Corresponding author. E-mail: walter.koch{at}temple.edu

    This PDF file includes:

    • Fig. S1. TgβARKrgs mice exhibit a similar decrease in cardiac hypertrophy as TgGqI mice after TAC.
    • Fig. S2. Chronic pressure overload–induced heart failure is inhibited by cardiac βARKrgs expression.
    • Fig. S3. Molecular markers of left ventricular dysfunction are reduced in TgβARKrgs and TgGqI mice but not in TgβARKct mice.
    • Fig. S4. Left ventricular structural remodeling is inhibited in TgβARKrgs mice.
    • Fig. S5. The βARKrgs peptide interacts with endogenous Gαq in the heart.

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    Citation: S. M. Schumacher, E. Gao, M. Cohen, M. Lieu, J. K. Chuprun, W. J. Koch, A peptide of the RGS domain of GRK2 binds and inhibits Gαq to suppress pathological cardiac hypertrophy and dysfunction. Sci. Signal. 9, ra30 (2016).

    © 2016 American Association for the Advancement of Science

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