Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure.

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of beta-adrenergic receptor (betaAR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting alpha2AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial alpha2AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein-coupled receptor kinase 2 (GRK2). Adrenal gland-specific GRK2 inhibition reversed alpha2AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac betaAR signaling and function, and increased sympatholytic efficacy of a alpha2AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target.

Receptor-independent sensitization of the adenylyl cylase after chronic treatment with cyclosporine A.

Chronic treatment with cyclosporine A (CyA) is often complicated by severe hypertension. If activation of the beta-adrenergic-receptor-linked adenylyl cyclase (AC) system contributes to hypertension is unresolved. Rats were treated with CyA (20 mg kg(-1) day(-1)) for 7 days. beta-adrenergic, muscarinic, and alpha-adrenergic receptors, G-proteins, and the activity of AC were determined in cardiac and pulmonary plasma membranes. The density of cardiac beta-adrenergic receptors, muscarinic receptors, alpha-adrenergic receptors, G(alphas) and, G(alphai) remained unchanged after treatment with CyA. However, CyA increased the responsiveness of AC to different stimulators. The responsiveness of AC was even more pronounced after solubilization and partial purification, suggesting a direct modulation of the enzyme. These data suggest that CyA modulates the activity of the sympathoadrenergic system by a direct, receptor-independent sensitization of AC, suggesting that this pathway contributes to hypertension in patients treated with CyA.

AAV6-ßARKct gene delivery mediated by molecular cardiac surgery with recirculating delivery (MCARD) in sheep results in robust gene expression and increased adrenergic reserve.

OBJECTIVE: Genetic modulation of heart function is a novel therapeutic strategy. We investigated the effect of molecular cardiac surgery with recirculating delivery (MCARD)-mediated carboxyl-terminus of the ß-adrenergic receptor kinase (ßARKct) gene transfer on cardiac mechanoenergetics and ß-adrenoreceptor (ßAR) signaling. METHODS: After baseline measurements, sheep underwent MCARD-mediated delivery of 10(14) genome copies of self-complimentary adeno-associated virus (scAAV6)-ßARKct. Four and 8 weeks after MCARD, mechanoenergetic studies using magnetic resonance imaging were performed. Tissues were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ßAR density, cyclic adenosine monophosphate levels, and physiologic parameters were evaluated. RESULTS: There was a significant increase in dP/dt(max) at 4 weeks: 1384 ± 76 versus 1772 ± 182 mm Hg/s; and the increase persisted at 8 weeks in response to isoproterenol (P < .05). Similarly, the magnitude of dP/dt(min) increased at both 4 weeks and 8 weeks with isoproterenol stimulation (P < .05). At 8 weeks, potential energy was conserved, whereas in controls there was a decrease in potential energy (P < .05) in response to isoproterenol. RT-qPCR confirmed robustness of ßARKct expression throughout the left ventricle and undetectable expression in extracardiac tissues. Quantitative Western blot data confirmed higher expression of ßARKct in the left ventricle: 0.46 ± 0.05 versus 0.00 in lung and liver (P < .05). Survival was 100% and laboratory parameters of major organ function were within normal limits. CONCLUSIONS: MCARD-mediated ßARKct delivery is safe, results in robust cardiac-specific gene expression, enhances cardiac contractility and lusitropy, increases adrenergic reserve, and improves energy utilization efficiency in a preclinical large animal model.

Role of 90-kDa heat shock protein (Hsp 90) and protein degradation in regulating neuronal levels of G protein-coupled receptor kinase 3.

Cellular levels of G protein-coupled receptor kinase (GRK)3 determine the sensitivity of the alpha(2A/B)-adrenoceptor (alpha(2)-AR) to agonist-induced down-regulation. Using human neuroblastoma BE(2)-C cells, this study examines how cellular GRK3 levels are affected by several mechanisms reported to influence stability and degradation of other GRKs. We first examined the interaction between the 90-kDa heat shock protein (Hsp90) and GRK3; Hsp90 reportedly affects the maturation and stability of GRK2. In unstimulated cells, GRK3 coimmunoprecipitates with Hsp90, suggesting a physical interaction. Moreover, when GRK3 protein expression was increased by 24-h epinephrine (EPI) treatment, Hsp90 protein expression increased with a similar but slightly delayed time course. To investigate the influence of Hsp90 on GRK3 protein stability, we determined the effect of the Hsp90 inhibitor geldanamycin (GA) on cellular GRK3 levels. GA eliminated the interaction between Hsp90 with GRK3 and produced a rapid, proteasome-mediated, 70% decrease in GRK3 levels within 24 h. To investigate the influence of Hsp90 on up-regulation of GRK3 expression, we examined the effect of GA on EPI-induced up-regulation. GA reduced the absolute increase in GRK3; however, the percentage of increase in GRK3 by EPI was not significantly different in the absence versus presence of GA (141 +/- 41 versus 94 +/- 12%). Finally, we examined the influence of Ca(2+)-activated proteases on cellular GRK3. Treatment with the calcium ionophore ionomycin produced a rapid decrease in GRK3 levels that was inhibited by the calpain inhibitor calpeptin. In conclusion, several mechanisms influence the degradation of GRK3 and therefore have the potential to affect GPCR signaling by regulating GRK3 levels in neurons.

Extracellular signal-regulated kinase 1/2-mediated transcriptional regulation of G-protein-coupled receptor kinase 3 expression in neuronal cells.

Relatively small changes in G-protein-coupled receptor kinase (GRK) 3 expression (approximately 2-fold) profoundly affect alpha2-adrenergic receptor (AR) function and preferentially regulate neuronal alpha2A- and alpha2B-AR signaling. In the present study, we provide evidence that epinephrine (EPI)-induced up-regulation of GRK3 protein expression in two neuronal cell lines, BE(2)-C cells (endogenously express alpha2A- and beta2AR) and BN17 cells [endogenously express alpha2B (NG108) and transfected to express beta2-AR] is due in part to increased GRK3 gene expression. In both cell lines, the increase in GRK3 transcription occurred via an extracellular signal-regulated kinase (ERK) 1/2-dependent mechanism because the increase in GRK3 mRNA is eliminated in the presence of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2-amino phenylthiobutadiene)]. EPI-induced GRK3 mRNA up-regulation also is prevented in the presence of propranolol or phentolamine. Moreover, GRK3 mRNA did not increase in response to EPI treatment in NG108 cells (endogenously express alpha2B-AR with no beta2-AR). Both these results suggest that simultaneous activation of alpha2- and beta2-AR by EPI is required for the ERK1/2-dependent increase in GRK3 mRNA. The EPI-induced increase in GRK3 mRNA was unaffected in the presence of the protein kinase C inhibitor, chelerythrine chloride. Finally, EPI treatment resulted in increased nuclear translocation and accumulation of the transcription factors, Sp-1 and Ap-2, in BE(2)-C cells. Taken together, our results demonstrate the involvement of the ERK1/2 pathway in selective up-regulation of GRK3 mRNA expression, possibly via activation of Sp-1 and Ap-2 transcription factors in neuronal cells.

Lymphocyte G-protein receptor kinase (GRK)3 mRNA levels in bipolar disorder.

Linkage studies in bipolar disorder were positive for markers in the region of chromosome 22q12.1 including the gene coding for G-protein receptor kinase (GRK)3. Two of six variants of the GRK3 5'-UTR/promoter were reported to be associated with bipolar disorder. GRK3 protein levels in lymphoblastoid cell lines derived from bipolar patients originating from families with linkage to chromosome 22q11 were reported to be decreased compared to those of control subjects and correlated with disease severity. We compared GRK3 mRNA levels in fresh lymphocytes from 31 bipolar patients vs. 26 control subjects, using real-time RT-PCR. No overall difference was found between patients and controls. However, GRK3 mRNA levels were markedly and significantly reduced in the subgroup of patients with no family history of a major psychiatric disorder compared with patients with family history.

Down regulation of the L-type Ca2+ channel, GRK2, and phosphorylated phospholamban: protective mechanisms for the denervated failing heart.

We previously found that a canine model of selective surgical ventricular denervation (VD), which does not permit increased sympathetic tone during the pathogenesis of heart failure (HF), tolerated the development of HF better than controls. To investigate the cellular mechanisms, we examined cellular contraction and L-type Ca(2+) channel currents (I(Ca)) and their responses to beta-adrenergic receptor (beta-AR) stimulation in left ventricular myocytes from 1) control, 2) VD, 3) HF induced by rapid pacing, and 4) HF induced in VD (VD-HF) dogs. The magnitude of myocyte contraction and rate of relaxation in VD were similar to control but were depressed in both HF and VD-HF. These changes were associated with reduced protein expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and protein kinase A phosphorylated phospholamban (PLB), which was reduced in HF, but essentially abolished in VD-HF. beta-AR kinase (GRK2) was increased in HF but reduced in VD-HF. Basal I(Ca) density did not differ among control, VD, and HF groups, but VD-HF myocytes showed a markedly reduced I(Ca) density (approximately 40%). Compared to controls, the sensitivity of I(Ca) to isoproterenol (ISO), was significantly higher in VD, but reduced in HF. While I(Ca) responses to ISO in VD-HF were maintained at control levels, the amplitude of the ISO-stimulated I(Ca) was significantly smaller (approximately 50%) compared with HF myocytes. The relative decrease in Ca(2+) influx due to downregulation of I(Ca) density may contribute to the cardioprotective effects in VD-HF hearts by preventing Ca(2+) overload during the development of HF. These findings, in combination with the virtual abolition of phosphorylated PLB in VD-HF and the decrease in GRK2, may explain, in part, why VD dogs tolerate the development of HF better than control dogs.

Protein kinase A-mediated phosphorylation contributes to enhanced contraction observed in mice that overexpress beta-adrenergic receptor kinase-1.

Transgenic mice with cardiac specific overexpression of beta-adrenergic receptor kinase-1 (betaARK-1) exhibit reduced contractility in the presence of adrenergic stimulation. However, whether contractility is altered in the absence of exogenous agonist is not clear. Effects of betaARK-1 overexpression on contraction were examined in mouse ventricular myocytes, studied at 37 degrees C, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microelectrodes (18-26 MOmega; 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in betaARK-1 cells than in wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1-3 MOmega; 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl(2), 2.5 mM KH(2)PO(4), 0.12 mM CaCl(2), 0.5 mM EGTA, and 10 mM HEPES), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. Furthermore, when cells were dialyzed with solutions that contained phosphodiesterase-sensitive sodium-cAMP (50 microM), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. However, when patch solutions were supplemented with phosphodiesterase-resistant 8-bromo-cAMP (50 muM), contractions were larger in betaARK-1 than wild-type cells. This difference was eliminated by the protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). Interestingly, Ca(2+) current amplitudes and inactivation rates were similar in betaARK-1 and wild-type cells in all experiments. These results suggest components of the adenylyl cyclase-protein kinase A pathway are sensitized by chronically increased betaARK-1 activity, which may augment contractile function in the absence of exogenous agonist. Thus, changes in contractile function in myocytes from failing hearts may reflect, in part, effects of chronic up-regulation of betaARK-1 on the cAMP-protein kinase A pathway.

Activation of beta2-adrenergic receptor plays a pivotal role in generating the protective effect of ischemic preconditioning in rat hearts.

BACKGROUND: Ischemic preconditioning (IPC) protects hearts against ischemia by reducing infarct size. However, IPC does not preserve cardiac function, such as left ventricular peak developed pressure (LVPDP). Moreover, IPC fails to protect the post-myocardial infarct (MI) heart. DESIGN: Rat hearts were transfected with beta2-adrenergic receptor (B2AR) cDNA by the hemagglutinating virus of Japan-liposome method. After the gene transfer, the hearts were perfused in a Langendorff mode and preconditioned with two cycles of 5 min of ischemia and reperfusion. After 20 min of global ischemia, the hearts were reperfused under aerobic conditions for 90 min. LVPDP was measured as an indicator of the cardiac function. RESULTS: LVPDP of ischemic hearts was well preserved by the combination treatment of IPC and gene transfer of B2AR, but not IPC or gene transfer of B2AR alone. Moreover, the treatment was beneficial to even the post-MI heart. On the contrary, gene transfer of beta-adrenergic receptor kinase 1 (BARK1) reduced the protective effect of IPC. We also found that the mRNA ratio of B2AR and BARK1 was well correlated with the preservation of the LVPDP. CONCLUSIONS: The combination treatment of IPC and gene transfer of B2AR protects cardiac function against ischemia and it shows the beneficial effect also in post-MI hearts.

Reduction of vascular intimal-medial hyperplasia in polytetrafluoroethylene arteriovenous grafts via expression of an inhibitor of G protein signaling.

Polytetrafluoroethylene (PTFE) arteriovenous (AV) grafts are performed routinely for vascular access. The limited life span of PTFE grafts is a major cause of morbidity. Graft failure is attributed to venous outflow tract vascular smooth muscle (VSM) hyperplasia, which is linked to heterotrimeric G protein signaling. We proposed that expression of a peptide inhibitor of G(betagamma) signaling (betaARKct) in the venous outflow of PTFE grafts would reduce hyperplasia and prolong graft patency. Left carotid to right external jugular vein PTFE AV grafts were placed in swine. The isolated external jugular vein was treated with an adenovirus encoding betaARKct, empty adenovirus, or phosphate-buffered saline for approximately 25 min. After 7 or 28 days, flow probe analysis was performed and the vein was harvested and analyzed for cross-sectional area comparison. After both 7 and 28 days, when compared to controls, treated animals demonstrated a statistically significant reduction in VSM hyperplasia with a reduction in cross-sectional intimal and medial areas of >40% (p < 0.05). Flow was maintained in treated grafts, while control groups demonstrated a >50% reduction (p < 0.05) at 7 days. Further, treated grafts demonstrated significant improvement in graft patency at 28 days (100% vs. 12% for treated and untreated grafts, respectively). The inhibition of G(betagamma) signaling reduces intimal-medial hyperplasia and prolongs graft patency in PTFE AV grafts. This represents a novel molecular therapeutic strategy for improving the patency of vascular access grafts.