Mdm2 regulates cardiac contractility by inhibiting GRK2-mediated desensitization of β-adrenergic receptor signaling
Pierre-Yves Jean-Charles, … , Howard A. Rockman, Sudha K. Shenoy
Pierre-Yves Jean-Charles, … , Howard A. Rockman, Sudha K. Shenoy
Published September 7, 2017
Citation Information: JCI Insight. 2017;2(17):e95998. https://doi.org/10.1172/jci.insight.95998.
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Research Article Cardiology Oncology

Mdm2 regulates cardiac contractility by inhibiting GRK2-mediated desensitization of β-adrenergic receptor signaling

Abstract

The oncoprotein Mdm2 is a RING domain–containing E3 ubiquitin ligase that ubiquitinates G protein–coupled receptor kinase 2 (GRK2) and β-arrestin2, thereby regulating β-adrenergic receptor (βAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects βAR signaling and cardiac function in adult mice. Using Mdm2/p53–KO mice, which survive for 9–12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac β1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53–KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53–KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53–KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53–KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53–KO was impaired. Mdm2/p53–KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53–KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable βAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued β1AR-induced cardiac contractility in Mdm2/p53–KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of βAR signaling.

Authors

Pierre-Yves Jean-Charles, Samuel Mon-Wei Yu, Dennis Abraham, Reddy Peera Kommaddi, Lan Mao, Ryan T. Strachan, Zhu-Shan Zhang, Dawn E. Bowles, Leigh Brian, Jonathan A. Stiber, Stephen N. Jones, Walter J. Koch, Howard A. Rockman, Sudha K. Shenoy

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Figure 1

Cardiac morphology, histopathology, and morphometry of WT and Mdm2/p53–KO mice.

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Cardiac morphology, histopathology, and morphometry of WT and Mdm2/p53–K...
(A) Immunoblot of cardiac lysates from 3- to 4-month-old WT and Mdm2/p53–KO mice with Mdm2 and GAPDH antibodies. Blot is representative of similar results obtained from greater than 10 mice of each genotype. (B) Representative whole hearts of 3- to 4-month-old WT and Mdm2/p53–KO mice (n = 3). Scale bars: 1 mm. (C) Heart weights (mg) of 3- to 4-month-old WT (n = 9) and Mdm2/p53–KO (n = 12) mice. Error bars indicate average ± SEM. *P < 0.01 versus WT; Student’s t test. (D) Body weights (g) of 3- to 4-month-old WT (n = 9) and Mdm2/p53–KO (n = 12) mice. Error bars indicate average ± SEM. *P < 0.01 versus WT; Student’s t test. (E) Heart weight/body weight (HW/BW) ratio calculated from data shown in C and D. P = 0.17, Student’s t test. (F) Representative Masson’s trichrome staining of cardiac sections from of 3- to 4-month-old WT and Mdm2/p53–KO (n = 5). Scale bars: 1 mm. (G) Representative baseline echocardiograms via M-mode, short-axis imaging, of WT and Mdm2/p53–KO mice (see Table 1 for ECHO analyses).