GRK5-mediated exacerbation of pathological cardiac hypertrophy involves facilitation of nuclear NFAT activity.
ABSTRACT: G protein-coupled receptor kinases (GRKs) acting in the cardiomyocyte regulate important signaling events that control cardiac function. Both GRK2 and GRK5, the predominant GRKs expressed in the heart, have been shown to be upregulated in failing human myocardium. Although the canonical role of GRKs is to desensitize G protein-coupled receptors via phosphorylation, it has been demonstrated that GRK5, unlike GRK2, can reside in the nucleus of myocytes and exert G protein-coupled receptor-independent effects that promote maladaptive cardiac hypertrophy and heart failure.To explore novel mechanisms by which GRK5 acting in the nucleus of cardiomyocytes participates in pathological cardiac hypertrophy.In this study, we have found that GRK5-mediated pathological cardiac hypertrophy involves the activation of the nuclear factor of activated T cells (NFAT) because GRK5 causes enhancement of NFAT-mediated hypertrophic gene transcription. Transgenic mice with cardiomyocyte-specific GRK5 overexpression activate an NFAT-reporter in mice basally and after hypertrophic stimulation, including transverse aortic constriction and phenylephrine treatment. Complimentary to this, GRK5 null mice exhibit less NFAT transcriptional activity after transverse aortic constriction. Furthermore, the loss of NFATc3 expression in the heart protected GRK5 overexpressing transgenic mice from the exaggerated hypertrophy and early progression to heart failure seen after transverse aortic constriction. Molecular studies suggest that GRK5 acts in concert with NFAT to increase hypertrophic gene transcription in the nucleus via GRK5's ability to bind DNA directly without a phosphorylation event.GRK5, acting in a kinase independent manner, is a facilitator of NFAT activity and part of a DNA-binding complex responsible for pathological hypertrophic gene transcription.
Project description:The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3?), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase ? (PI3K?). Moreover, inhibition of PI3K? as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3K? mediated Akt phosphorylation and subsequent inactivation of GSK3?, resulting in enhanced NFAT activity.
Project description:Heart failure (HF) is often the end phase of maladaptive cardiac hypertrophy. A contributing factor is activation of a hypertrophic gene expression program controlled by decreased class II histone deacetylase (HDAC) transcriptional repression via HDAC phosphorylation. Cardiac-specific overexpression of G proteinen-coupled receptor kinase-5 (GRK5) has previously been shown to possess nuclear activity as a HDAC5 kinase, promoting an intolerance to in vivo ventricular pressure overload; however, its endogenous requirement in adaptive and maladaptive hypertrophy remains unknown.We used mouse models with global or cardiomyocyte-specific GRK5 gene deletion to determine the absolute requirement of endogenous GRK5 for cardiac hypertrophy and HF development after chronic hypertrophic stimuli.Mice with global deletion of GRK5 were subjected to transverse aortic constriction. At 12 weeks, these mice showed attenuated hypertrophy, remodeling, and hypertrophic gene transcription along with preserved cardiac function. Global GRK5 deletion also diminished hypertrophy and related gene expression due to chronic phenylephrine infusion. We then generated mice with conditional, cardiac-specific deletion of GRK5 that also demonstrated similar protection from pathological cardiac hypertrophy and HF after transverse aortic constriction.These results define myocyte GRK5 as a critical regulator of pathological cardiac growth after ventricular pressure overload, supporting its role as an endogenous (patho)-physiological HDAC kinase. Further, these results define GRK5 as a potential therapeutic target to limit HF development after hypertrophic stress.
Project description:Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels.
Project description:G protein-coupled receptor (GPCR) kinases (GRKs) are critical regulators of cellular signaling and function. In cardiomyocytes, GRK2 and GRK5 are two GRKs important for myocardial regulation, and both have been shown to be up-regulated in the dysfunctional heart. We report that increased levels and activity of GRK5 in failing myocardium may have unique significance due to its nuclear localization, a property not shared by GRK2. We find that transgenic mice with elevated cardiac GRK5 levels have exaggerated hypertrophy and early heart failure compared with control mice after pressure overload. This pathology is not present in cardiac GRK2-overexpressing mice or in mice with overexpression of a mutant GRK5 that is excluded from the nucleus. Nuclear accumulation of GRK5 is enhanced in myocytes after aortic banding in vivo and in vitro in myocytes after increased G alpha q activity, the trigger for pressure-overload hypertrophy. GRK5 enhances activation of MEF2 in concert with Gq signals, demonstrating that nuclear localized GRK5 regulates gene transcription via a pathway critically linked to myocardial hypertrophy. Mechanistically, we show that this is due to GRK5 acting, in a non-GPCR manner, as a class II histone deacetylase (HDAC) kinase because it can associate with and phosphorylate the myocyte enhancer factor-2 repressor, HDAC5. Moreover, significant HDAC activity can be found with GRK5 in the heart. Our data show that GRK5 is a nuclear HDAC kinase that plays a key role in maladaptive cardiac hypertrophy apparently independent of any action directly on GPCRs.
Project description:AlphaB-crystallin (CryAB) is the most abundant small heat shock protein (HSP) constitutively expressed in cardiomyocytes. Gain- and loss-of-function studies demonstrated that CryAB can protect against myocardial ischemia/reperfusion injury. However, the role of CryAB or any HSPs in cardiac responses to mechanical overload is unknown. This study addresses this issue. Nontransgenic mice and mice with cardiomyocyte-restricted transgenic overexpression of CryAB or with germ-line ablation of the CryAB/HSPB2 genes were subjected to transverse aortic constriction or sham surgery. Two weeks later, cardiac responses were analyzed by fetal gene expression profiling, cardiac function analyses, and morphometry. Comparison among the 3 sham surgery groups reveals that CryAB overexpression is benign, whereas the knockout is detrimental to the heart as reflected by cardiac hypertrophy and malfunction at 10 weeks of age. Compared to nontransgenic mice, transgenic mouse hearts showed significantly reduced NFAT transactivation and attenuated cardiac hypertrophic responses to transverse aortic constriction but unchanged cardiac function, whereas NFAT transactivation was significantly increased in cardiac and skeletal muscle of the knockout mice at baseline, and they developed cardiac insufficiency at 2 weeks after transverse aortic constriction. CryAB overexpression in cultured neonatal rat cardiomyocytes significantly attenuated adrenergic stimulation-induced NFAT transactivation and hypertrophic growth. We conclude that CryAB suppresses cardiac hypertrophic responses likely through attenuating NFAT signaling and that CryAB and/or HSPB2 are essential for normal cardiac function.
Project description:G protein-coupled receptor kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is upregulated in heart failure. Although GRK5 is a critical regulator of cardiac G protein-coupled receptor signaling, recent data has uncovered noncanonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early heart failure after transverse aortic constriction (TAC) because of GRK5 nuclear accumulation.In this study, we investigated the role of GRK5 in physiological, swimming-induced hypertrophy (SIH).Cardiac-specific GRK5 transgenic mice and nontransgenic littermate control mice were subjected to a 21-day high-intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed, including nuclear localization of GRK5, and compared with TAC. Unlike after TAC, swim-trained transgenic GRK5 and nontransgenic littermate control mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC transgenic GRK5 mice was able to preserve cardiac function.These data suggest that although nuclear-localized GRK5 is a pathological mediator after stress, this noncanonical nuclear activity of GRK5 is not induced during physiological hypertrophy.
Project description:Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca(2+) cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca(2+) signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKC? and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca(2+) current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKC?, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKC?-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.
Project description:G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5's newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca(2+)/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.
Project description:Combined ablation of all 3 cardiac-expressed GRKs in the developing mouse heart impaired Hedgehog/Smoothened and GATA signaling, recapitulating heart abnormalities of human Complete Atrioventricular Septal Defect. Overall design: Cardiac transcriptomes from mouse E13.5 hearts (8 wt, 8 haploinsufficient GRK2+/-GRK5-/-GRK6-/- and 7 GRK-triple knockout GRK2-/-GRK5-/-GRK6-/-) were generated via Illumina RiboZeroGold rRNA depletion and subsequent single-end 50 nt sequencing on Illumina HiSeq 2500
Project description:BACKGROUND:Although the complex roles of macrophages in myocardial injury are widely appreciated, the function of neutrophils in nonischemic cardiac pathology has received relatively little attention. METHODS:To examine the regulation and function of neutrophils in pressure overload-induced cardiac hypertrophy, mice underwent treatment with Ly6G antibody to deplete neutrophils and then were subjected to transverse aortic constriction. RESULTS:Neutrophil depletion diminished transverse aortic constriction-induced hypertrophy and inflammation and preserved cardiac function. Myeloid deficiency of Wnt5a, a noncanonical Wnt, suppressed neutrophil infiltration to the hearts of transverse aortic constriction-treated mice and produced a phenotype that was similar to the neutropenic conditions. Conversely, mice overexpressing Wnt5a in myeloid cells displayed greater hypertrophic growth, inflammation, and cardiac dysfunction. Neutrophil depletion reversed the Wnt5a overexpression-induced cardiac pathology and eliminated differences in cardiac parameters between wild-type and myeloid-specific Wnt5a transgenic mice. CONCLUSIONS:These findings reveal that Wnt5a-regulated neutrophil infiltration has a critical role in pressure overload-induced heart failure.