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Background: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. Methods: To address this, we performed FoxO1 chromatin immunoprecipitation–deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg −1 ·mg −1 ), transverse aortic constriction, or vehicle injection/sham surgery. Results: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation–deep sequencing results were aligned with those of pol II chromatin immunoprecipitation–deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction–operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1– and, in vivo, pressure overload–induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. Conclusions: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II–regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

Aberrant changes in gene expression underlie the pathogenesis and progression of pressure-overload heart failure, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. Signaling through the G protein Gq triggers maladaptation and heart failure, in part through the activation of G protein-coupled receptor kinase-5 (GRK5). Hypertrophic stimuli induce the accumulation of GRK5 in the nuclei of cardiomyocytes, where it regulates pathological gene expression through multiple transcription factors including NFAT. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that binds to calmodulin (CaM). Here, we sought to prevent GRK5-mediated pathology in pressure-overload maladaptation and heart failure by expressing in cardiomyocytes a peptide encoding the GRK5 NT (GRK5nt) that encompasses the CaM binding domain. In cultured cardiomyocytes, GRK5nt expression abrogated Gq-coupled receptor-mediated hypertrophy, including attenuation of pathological gene expression and the transcriptional activity of NFAT and NF-κB. We confirmed that GRK5nt bound to and blocked Ca(2+)-CaM from associating with endogenous GRK5, thereby preventing GRK5 nuclear accumulation after pressure overload. We generated mice that expressed GRKnt in a cardiac-specific fashion (TgGRK5nt mice), which exhibited reduced cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis after chronic transverse aortic constriction. Together, our data support a role for GRK5nt as an inhibitor of pathological GRK5 signaling that prevents heart failure.

Significance Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. G protein-coupled receptor (GPCR) kinase 5 (GRK5) has been shown to cause deleterious effects on the cardiomyocyte during HF; however, its effects in cardiac fibroblasts, the crucial cell type responsible for maintaining the structural integrity of the heart, is not understood. Here, we use in vitro and in vivo methods to demonstrate that inhibition of GRK5 inhibits fibroblast activation and attenuates the fibrotic response in the heart., Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

Introduction: The induction of heart failure (HF) after aortic banding (TAC) in mice encompasses aberrations in gene regulation, leading to maladaptive cardiac hypertrophy and contractile dysfunction. Previously, our lab has shown that the nuclear targeting of GRK5, which is required for maladaptive hypertrophy, is controlled by its N-terminal (NT) calmodulin-binding domain. Hypothesis: We hypothesize that expression of a peptide encoding the NT of GRK5 will act as a GRK5-calmodulin inhibitor preventing the nuclear accumulation of GRK5 and inhibit its pathological activities. Methods: Neonatal rat ventricular cardiomyocytes (NRCM) overexpressing GRK5-NT or control virus were subjected to α-adrenergic stress with 50μM phenylephrine (PE) or vehicle for 30 minutes. Subcellular fractionation and western blotting were used to detect nuclear GRK5. Novel cardiac-specific transgenic mice were generated expressing HA-tagged GRK5-NT and these mice along with their control mice were characterized 8 weeks after TAC or sham surgery. Echocardiography, fibrosis, and lung and heart weight to tibia length metrics were used to evaluate hypertrophy and heart failure response post-TAC. Other cohorts were subjected to TAC for 1 week followed by Western blotting to assess acute signaling and nuclear accumulation of GRK5. Results: In vitro, expression of the GRK5-NT peptide reduced PE-induced nuclear accumulation of GRK5. In both NRCM and tissue, the GRK5-NT peptide was found to bind to calmodulin, which disrupts the endogenous calmodulin-GRK5 interaction. GRK5-NT expression abrogates nuclear GRK5 accumulation following hypertrophic stress in vitro and in vivo. Following 8 weeks of TAC, cardiac function, hypertrophy, and pulmonary congestion were significantly attenuated in GRK5-NT mice compared to NLC mice. Conclusion: We show that the GRK5-NT peptide binds to calmodulin and prevents nuclear accumulation of GRK5 following hypertrophic stress. Our in vivo data show that cardiac-restricted expression of GRK5-NT prevents HF by partially restoring cardiac dysfunction, hypertrophy, and pulmonary congestion following 8 weeks of TAC. Further in vitro work will reveal the calmodulin-dependence of GRK5-NT and the peptide’s effects on G-protein signaling.

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca 2+ ·CaM–GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca 2+ ·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca 2+ ·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca 2+ ·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca 2+ ·CaM-dependent hypertrophy.

Heart failure (HF) is a global epidemic with the highest degree of mortality and morbidity of any disease presently studied. G protein-coupled receptors (GPCRs) are prominent regulators of cardiovascular function. Activated GPCRs are “turned off” by GPCR kinases (GRKs) in a process known as “desensitization”. GRKs 2 and 5 are highly expressed in the heart, and known to be upregulated in HF. Over the last 20 years, both GRK2 and GRK5 have been demonstrated to be critical mediators of the molecular alterations that occur in the failing heart. In the present review, we will highlight recent findings that further characterize “non-canonical” GRK signaling observed in HF. Further, we will also present potential therapeutic strategies (i.e. small molecule inhibition, microRNAs, gene therapy) that may have potential in combating the deleterious effects of GRKs in HF.

Background— Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased β-adrenergic receptor (βAR) responsiveness. This led us to hypothesize that V1AR signaling regulates βAR responsiveness and in doing so contributes to development of heart failure. Methods and Results— Transaortic constriction resulted in decreased cardiac function and βAR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased βAR ligand affinity, as well as βAR-induced Ca 2+ mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of βAR responsiveness was demonstrated to occur in a previously unrecognized Gq protein–independent/G protein receptor kinase–dependent manner. Conclusions— This newly discovered relationship between cardiac V1AR and βAR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.

Circulating levels of arginine vasopressin (AVP) are elevated during hypovolemia and during cardiac stress. AVP activates arginine vasopressin type 1A (V(1A))/Gα(q)-coupled receptors in the heart and vasculature and V(2)/Gα(s)-coupled receptors in the kidney. However, little is known regarding the signaling pathways that influence the effects of V(1A) receptor (V(1A)R) activation during cellular injury. Using hypoxia-reoxygenation (H/R) as a cell injury model, we evaluated cell survival and caspase 3/7 activity in H9c2 myoblasts after treatment with AVP. Pretreatment of H9c2 cells with AVP significantly reduced H/R-induced cell death and caspase 3/7 activity, effects that were blocked via both selective V(1A)R inhibition and mitogen-activated protein kinase (MEK1/2) inhibition. AVP increased extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation in a concentration-dependent manner that was sensitive to MEK1/2 inhibition and V(1A)R inhibition, but not V(1B)R or V(2)R inhibition. Discrete elements of the V(1A)/Gα(q)-protein kinase C (PKC) and V(1A)/G protein-coupled receptor kinase (GRK)/β-arrestin signaling cascades were inhibited to dissect the pathways responsible for the protective effects of V(1A)R signaling: Gα(q) (overexpression of Gq-I-ires-green fluorescent protein), PKC (administration of Ro 31-82425; 2-[8-(aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide, HCl, bisindolylmaleimide X, HCl), GRK2 [C-terminal GRK2 peptide overexpression and small interfering RNA (siRNA) knockdown], GRK5 (siRNA knockdown), and β-arrestin1 (siRNA knockdown). These studies demonstrated that both Gα(q)/PKC- and GRK2/β-arrestin1-dependent V(1A)R signaling were capable of inducing ERK1/2 phosphorylation in response to AVP stimulation. However, AVP-mediated protection against H/R was elicited only via GRK2- and β-arrestin1-dependent signaling. These results suggest that activation of the V(1A)R in H9c2 cells mediates protective signaling via a GRK2/β-arrestin1/ERK1/2-dependent mechanism that leads to decreased caspase 3/7 activity and enhanced survival under conditions of ischemic stress.