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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.

SUBMITTER: Jean-Charles PY

PROVIDER: S-EPMC5621915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimsTo determine the mechanisms by which the ?1A-adrenergic receptor (AR) regulates cardiac contractility.BackgroundWe reported previously that transgenic mice with cardiac-restricted ?1A-AR overexpression (?1A-TG) exhibit enhanced contractility but not hypertrophy, despite evidence implicating this G?q/11-coupled receptor in hypertrophy.MethodsContractility, calcium (Ca(2+)) kinetics and sensitivity, and contractile proteins were examined in cardiomyocytes, isolated hearts and skinned fibers from ?1A-TG mice (170-fold overexpression) and their non-TG littermates (NTL) before and after ?1A-AR agonist stimulation and blockade, angiotensin II (AngII), and Rho kinase (ROCK) inhibition.ResultsHypercontractility without hypertrophy with ?1A-AR overexpression is shown to result from increased intracellular Ca(2+) release in response to agonist, augmenting the systolic amplitude of the intracellular Ca(2+) concentration [Ca(2+)]i transient without changing resting [Ca(2+)]i. In the absence of agonist, however, ?1A-AR overexpression reduced contractility despite unchanged [Ca(2+)]i. This hypocontractility is not due to heterologous desensitization: the contractile response to AngII, acting via its G?q/11-coupled receptor, was unaltered. Rather, the hypocontractility is a pleiotropic signaling effect of the ?1A-AR in the absence of agonist, inhibiting RhoA/ROCK activity, resulting in hypophosphorylation of both myosin phosphatase targeting subunit 1 (MYPT1) and cardiac myosin light chain 2 (cMLC2), reducing the Ca(2+) sensitivity of the contractile machinery: all these effects were rapidly reversed by selective ?1A-AR blockade. Critically, ROCK inhibition in normal hearts of NTLs without ?1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility.ConclusionsWe report for the first time pleiotropic ?1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.

Project description:BackgroundAltered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and β-adrenergic hyporesponsiveness.Methods and resultsWe simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT.ConclusionsHypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism.

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

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