Desensitization Of Beta-adrenergic Receptors Research Articles

Sympathetic activity markers in patients with resistant hypertension with renal dysfunction

Aim. To study the informativeness of sympathetic activity markers and perirenal adipose tissue (PRAT) in assessing renal function decline in patients with resistant hypertension (RH).Material and methods. The study included 63 patients with RH of both sexes aged 40-80 years. The main group included 19 patients with chronic kidney disease (CKD). The control group consisted of 44 patients with RH without CKD. All patients were assessed for uric acid, blood lipid profile, beta-adrenergic reactivity of erythrocyte mem­branes, free metanephrine and normetanephrine in plasma and 24-hour urine. The renal function was assessed by the glomerular filtra­tion rate calculated using the CKD-EPI equation. Twenty-four hour blood pressure monitoring was performed on the brachial artery of one arm at intervals of 15 minutes during the day and 30 minutes at night. Kidney size and PRAT were calculated based on the magnetic reso­nance imaging.Results. In both groups of patients, a significant increase in blood normetanephrine and beta-adrenergic reactivity of erythrocyte mem­branes relative to the reference values was shown. Blood and 24-hour urine metanephrine were within the reference intervals. No differences were found between the sympathetic activity indices in the main and control groups. In the main group patients, the PRAT thickness was greater (p=0,013), and the kidney diameter was smaller (p=0,046) than in the control group patients.Conclusion. CKD in RH patients is not accompanied by additional significant changes in sympathetic regulation, assessed by the blood and 24-hour urine catecholamine levels, the degree of beta-adrenergic receptor desensitization and variability of blood pressure. PRAT increase according to magnetic resonance imaging in patients with RH is associated with a decrease in renal filtration function.

Computational study of paroxetine-like inhibitors reveals new molecular insight to inhibit GRK2 with selectivity over ROCK1

The G-protein coupled receptor kinase 2 (GRK2) regulates the desensitization of beta-adrenergic receptors (β-AR), and its overexpression has been implicated in heart failure. Hence, the inhibition of GRK2 is considered to be an important drug target for the treatment of heart failure. Due to the high sequence similarity of GRK2 with the A, G, and C family (AGC family) of kinases, the inhibition of GRK2 also leads to the inhibition of AGC kinases such as Rho-associated coiled-coil kinase 1 (ROCK1). Therefore, unraveling the mechanisms to selectively inhibit GRK2 poses an important challenge. We have performed molecular docking, three dimensional quantitative structure activity relationship (3D-QSAR), molecular dynamics (MD) simulation, and free energy calculations techniques on a series of 53 paroxetine-like compounds to understand the structural properties desirable for enhancing the inhibitory activity for GRK2 with selectivity over ROCK1. The formation of stable hydrogen bond interactions with the residues Phe202 and Lys220 of GRK2 seems to be important for selective inhibition of GRK2. Electropositive substituents at the piperidine ring and electronegative substituents near the amide linker between the benzene ring and pyrazole ring showed a higher inhibitory preference for GRK2 over ROCK1. This study may be used in designing more potent and selective GRK2 inhibitors for therapeutic intervention of heart failure.

G Protein-coupled receptor kinase 2 impairs fatty acid metabolism in the failing heart through novel mechanisms

Compromised contractility and energetics are hallmarks of heart failure. Increased G protein-coupled receptor kinase (GRK)2 is central to HF pathogenesis, via desensitization of beta-adrenergic receptors and, therefore, loss of contractile reserve. We, and others, have reported that GRK2 also has non-canonical activity that contributes to its pathological functionality in HF. For example, GRK2 can compromise fatty acid (FA) metabolism in cardiomyocytes, the mechanism of which remains unknown. Our aim is to investigate the role of GRK2 in FA metabolism and bioenergetics in the heart. To do this, we measured FA uptake, cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination, and FA-driven bioenergetics in mice with cardiac-specific overexpression of GRK2 (TgGRK2) or a GRK2 inhibitory peptide (TgβARKct) or global heterozygous GRK2 knockout (GRK2+/-) mice. Additionally, we determined CD36 expression and phosphorylation following transverse aortic constriction (TAC). The results show a significant reduction in FA uptake rates (0.3-fold) and CD36 protein (0.8-fold), which is associated with an increase in its phospho-serine/threonine (0.7-fold increase) and ubiquitin-lysine (0.7-fold) content in TgGRK2 mice. These parameters were unchanged in TgβARKct or GRK2+/- mice. A reduction in CD36 mRNA (0.7-fold) and protein levels (0.2-fold) were detected in post-TAC, failing hearts, which was associated with an increase is its phosphorylation (0.5-fold). Notably, normalization of CD36 protein levels is observed in GRK2+/- mice post-TAC. Maximal respiratory capacity was also enhanced in TgβARKct (0.6-fold) and GRK2+/- cardiomyocytes (0.8-fold). Together, our results show that up-regulation of GRK2 induces CD36 phosphorylation, ubiquitination, and downregulation, which is associated with reduced FA uptake. Thus, we propose that the increase in GRK2 observed during HF is, at least partly, responsible for reduced FA uptake and oxidation and may be an important nodal switch in substrate utilization during HF.

Abstract 109: G Protein-coupled Receptor Kinase 2 Impairs Fatty Acid Metabolism in the Failing Heart Through Novel Mechanisms

Compromised contractility and energetics are hallmarks of the failing heart. Increased G protein-coupled receptor kinase (GRK)2 is central to the pathogenesis of heart failure (HF), via desensitization of beta-adrenergic receptors and, therefore, loss of contractile reserve. Additionally, we, and others, have reported that GRK2 has non-canonical activity that contributes to its pathological functionality in HF. For example, GRK2 can compromise fatty acid (FA) metabolism in cardiomyocytes, the mechanism of which remains unknown. Thus, our aim is to investigate the role of GRK2 in FA metabolism and bioenergetics in the heart. To do this, we measured FA uptake, cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination, and FA-driven bioenergetics in mice with cardiac-specific overexpression of GRK2, to levels observed in the human failing myocardium, or a GRK2 inhibitory peptide (TgGRK2 and TgβARKct, respectively) or in global heterozygous GRK2 knockout (GRK2 +/- ) mice. Additionally, we determined CD36 expression and phosphorylation following cardiac dysfunction induced by transverse aortic constriction (TAC). The results show a significant reduction in FA uptake rates (0.3-fold) and CD36 protein (0.8-fold), which was associated with an increase in its phospho-serine/threonine (0.7-fold increase) and ubiquitin-lysine (0.7-fold) content in the TgGRK2 mice. In contrast, these parameters were unchanged in the TgβARKct or GRK2 +/- mice. A reduction in CD36 mRNA (0.7-fold) and protein levels (0.2-fold) were detected in post-TAC, failing hearts, which was associated with an increase is its phosphorylation (0.5-fold). Notably, normalization of CD36 protein levels is observed in GRK2 +/- mice post-TAC. Maximal respiratory capacity was also enhanced in TgβARKct and GRK2 +/- cardiomyocytes (0.6- and 0.8-fold, respectively). Together, our results show that up-regulation of GRK2 induces CD36 phosphorylation, ubiquitination, and downregulation, which is associated with reduced FA uptake. Thus, we propose that the increase in GRK2 observed during HF is, at least partly, responsible for reduced FA uptake and oxidation and may be an important nodal switch in substrate utilization during HF pathogenesis.

Effect of beta-agonists on LAM progression and treatment

Lymphangioleiomyomatosis (LAM), a rare disease of women, is associated with cystic lung destruction resulting from the proliferation of abnormal smooth muscle-like LAM cells with mutations in the tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2 The mutant genes and encoded proteins are responsible for activation of the mechanistic target of rapamycin (mTOR), which is inhibited by sirolimus (rapamycin), a drug used to treat LAM. Patients who have LAM may also be treated with bronchodilators for asthma-like symptoms due to LAM. We observed stabilization of forced expiratory volume in 1 s over time in patients receiving sirolimus and long-acting beta-agonists with short-acting rescue inhalers compared with patients receiving only sirolimus. Because beta-agonists increase cAMP and PKA activity, we investigated effects of PKA activation on the mTOR pathway. Human skin TSC2+/- fibroblasts or LAM lung cells incubated short-term with isoproterenol (beta-agonist) showed a sirolimus-independent increase in phosphorylation of S6, a downstream effector of the mTOR pathway, and increased cell growth. Cells incubated long-term with isoproterenol, which may lead to beta-adrenergic receptor desensitization, did not show increased S6 phosphorylation. Inhibition of PKA blocked the isoproterenol effect on S6 phosphorylation. Thus, activation of PKA by beta-agonists increased phospho-S6 independent of mTOR, an effect abrogated by beta-agonist-driven receptor desensitization. In agreement, retrospective clinical data from patients with LAM suggested that a combination of bronchodilators in conjunction with sirolimus may be preferable to sirolimus alone for stabilization of pulmonary function.

Abstract 469: Targeting Beta Adrenergic Receptor Resensitization Aids in Preservation of Receptor Function

Beta adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and is an integral contributor to heart failure. We have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on in silico predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also generated a small molecule from our in silico predictions that could target the interface of I2PP2A and PP2A binding to find that disruption of the PP2A/I2PP2A interaction underlies receptor function. We will use this small molecule to look at preservation of βAR function and amelioration of cardiac function. To test whether resensitization is altered in heart failure we used plasma membrane and endosomal fractions from non-failing and paired pre- and post-LVAD samples to show PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase activity as a measure of recovery in βAR function. Our studies showed that endosomal fractions from human heart failure samples had elevated PI3K activity associated with reduced PP2A activity supporting the idea that βAR resensitization is inhibited in human heart failure samples. Since human heart failure samples have inhibited resensitization we tested the underlying mechanisms regulating βAR resensitization. Thus ongoing studies suggest that targeting the resensitization of βAR could provide beneficial cardiac remodeling in conditions of chronic mechanical overload and will be further discussed.

Abstract 16596: Disruption of Gravin’s Scaffolding as a Potential Therapy for Heart Failure in Combination With Carvedilol

Beta-adrenergic receptor (β-AR) desensitization culminating in impaired β-AR signaling triggers the vicious cascade of HF. Gravin; an A-kinase anchoring protein (AKAP) is crucial for desensitization of the β-ARs upon agonist stimulation. Our previous studies have shown that disruption of gravin’s scaffolding imparts protection against isoproterenol induced HF in mice. The goal of this study was to further compare the protective effects of absence of gravin in HF to that of the standard β blocker HF therapy such as Carvedilol and Bisoprolol. After inducing HF with 2-weeks administration of isoproterenol (60mg/kg/day), wild-type (WT) and gravin-t/t mice (harboring non-functional truncated gravin) were started on blocker therapy. There was no significant reduction in the HW/BW ratio or the fibrotic lesions after Carvedilol and Bisoprolol treatment in the WT mice; whereas, gravin-t/t mice did not show any significant increase in either of the two after isoproterenol treatment. Gravin-t/t mice treated with either of these blockers showed enhanced hemodynamic function compared to the WT mice on the blocker therapy. Interestingly, the gravin-t/t mice after treatment with Carvedilol showed significant increase in the β2-AR expression (579.83 ± 0. 476 fmol/mg) compared to the WT mice (113.74 ± 0. 359 fmol/mg) treated with Carvedilol; whereas, no significant changes were observed in the β1-AR expression in WT (310.03 ± 0. 284 fmol/mg) or gravin-t/t mice (282.54 ± 0. 392 fmol/mg). Bisoprolol treatment on the other hand showed significant increase only in the β1-AR expression post treatment for both WT (405.62 ± 0. 264 fmol/mg) and gravin-t/t (421.01 ± 0. 382 fmol/mg) mice. Though Carvedilol is the gold standard for HF, and it increases the contractile function of the failing heart, it does not rescue the decline in the receptor density. Absence of gravin not only increases the β2-AR expression 2-fold at baseline but also increases it 5-fold after Carvedilol treatment. Disruption of gravin’s scaffolding is a potential drug target and can be further developed as a combination therapy with Carvedilol. Increase in β2-ARs expression can provide beneficial effects via activation of anti-apoptotic and survival pathways.

Abstract 207: Left Ventricular Assist Device (LVAD) Reverses Inhibition on Beta-adrenergic Receptor Resensitization

Beta-adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Agonist occupied βAR undergo desensitization through phosphorylation by G-protein coupled receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by protein phosphatase 2A (PP2A) in the endosomes and we have shown previously shown that PP2A activity is regulated by phosphoinositide3-kinaseγ (PI3Kγ). Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and an integral contributor to heart failure. To test whether resensitization mechanisms are altered in heart failure and could play a role in recovery of βAR function post-LVAD, we used paired pre- and post-LVAD human heart samples along with non-failing hearts. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from these human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of recovery in βAR function by G-protein coupling. Significant PI3Kγ activity was observed in the endosomes of pre-LVAD compared post-LVAD and non-failing human heart samples while plasma membrane PI3K activity remained similar across all samples. Similarly, AC activity was markedly reduced in plasma membranes and endosomes of pre-LVAD samples compared to post-LVAD and non-failing showing reduced ability of receptors to couple to G-proteins indicating reduced recovery of receptor function in post-LVAD heart samples. Consistent with the reduced recovery in βAR function in pre-LVAD samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of pre-LVAD compared to post-LVAD and non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in the pre-LVAD samples which was remarkably reversed in post-LVAD samples similar to the activity in non-failing. These studies suggest that resensitization is inhibited in end-stage human heart failure and may critically contribute to cardiac remodeling and hypertrophic response upon cardiac stress.

Effects of direct and indirect activation of G protein of adenylyl cyclase on the subsequent response to beta-adrenergic receptor agonists in human trachealis.

To examine the response to beta-adrenergic receptor agonists (beta-agonists) following prolonged activation of the stimulatory G protein of adenylyl cyclase (Gs), relaxation by isoproterenol (isoprenaline, CAS 949-36-0) and formoterol (CAS 73573-87-2), a long-acting beta-agonist, after exposure to formoterol was measured in human tracheal smooth muscle, using isometric tension records. Prior exposure to formoterol (0.3-30 nmol/l) for 45 min reduced the subsequent relaxation induced by this drug in a concentration-dependent manner, but only modestly reduced that induced by isoproterenol. Next, the effects of cholera toxin (CTX, CAS 9012-63-9) an irreversible direct activator of Gs and formoterol on the reduced responsiveness to isoproterenol after continuous and repeated exposure to isoprotenerol were examined. Preincubation with cholera toxin (0.02-2 micrograms/ml) caused concentration-dependent inhibition of the desensitization induced by isoproterenol, but preincubation with formoterol did not. These results indicate that prolonged activation of Gs via beta-adrenergic receptors does not cause cross-desensitization to short-acting beta-agonists. However, it also fails to inhibit the desensitization of beta-adrenergic receptors after excessive exposure to short-acting beta-agonists. Activation of Gs via a pathway that bypasses the receptors may be beneficial for the prevention of this phenomenon.

Desensitization of β‐adrenergic receptors in lung injury induced by 2‐chloroethyl ethyl sulfide, a mustard analog

2-Choloroethyl Ethyl Sulfide (CEES) exposure causes inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. This may be associated with oxidative stress, which has been implicated in the desensitization of beta-adrenergic receptors (beta-ARs). The objective of this study was to investigate whether lung injury induced by intratracheal CEES exposure (2 mg/kg body weight) causes desensitization of beta-ARs. The animals were sacrificed after 7 days and lungs were removed. Lung injury was established by measuring the leakage of iodinated-bovine serum albumin ([(125)I]-BSA) into lung tissue. Receptor-binding characteristics were determined by measuring the binding of [(3)H] dihydroalprenolol ([(3)H] DHA) (0.5-24 nM) to membrane fraction in the presence and absence of DLDL-propranolol (10 micro M). Both high- and low-affinity beta-ARs were identified in the lung. Binding capacity was significantly higher in low-affinity site in both control and experimental groups. Although CEES exposure did not change K(D) and B(max) at the high-affinity site, it significantly decreased both K(D) and B(max) at low affinity sites. A 20% decrease in beta(2)-AR mRNA level and a 60% decrease in membrane protein levels were observed in the experimental group. Furthermore, there was significantly less stimulation of adenylate cyclase activity by both cholera toxin and isoproterenol in the experimental group in comparison to the control group. Treatment of lungs with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase (PDE) could not abolish the difference between the control group and the experimental group on the stimulation of the adenylate cyclase activity. Thus, our study indicates that CEES-induced lung injury is associated with desensitization of beta(2)-AR.

Uncoupling of Myocardial β-Adrenergic Receptor Signaling During Coronary Artery Bypass Grafting: The Role of GRK2

Cardiopulmonary bypass (CPB) and cardioplegic arrest during cardiac surgery leads to desensitization of myocardial beta-adrenergic receptors (beta-ARs). Impaired signaling through this pathway can have a detrimental effect on ventricular function and increased need for inotropic support. The mechanism of myocardial beta-AR desensitization during cardiac surgery has not been defined. This study investigates the role of G protein-coupled receptor kinase-2 (GRK2), a serine-threonine kinase which phosphorylates and desensitizes agonist-occupied beta-ARs, as a primary mechanism of beta-AR uncoupling during coronary artery bypass grafting (CABG) with CPB and cardioplegic arrest. Forty-eight patients undergoing elective CABG were enrolled in this study. Myocardial beta-AR signaling was assessed by measuring total beta-AR density and adenylyl cyclase activity in right atrial biopsies obtained before CPB and just before weaning from CPB. Myocardial GRK2 expression and activity were also measured before CPB and just before weaning from CPB. Myocardial beta-AR signaling was significantly impaired after CPB and cardioplegic arrest during CABG. Cardiac GRK2 expression was not altered; however, there was a twofold increase in GRK2 activity during CABG. There was an even greater elevation in cardiac GRK2 activity in patients with severely depressed ventricular function. Increased myocardial GRK2 activity appears to be the primary mechanism of impaired beta-AR signaling during CABG with CPB and cardioplegic arrest. This may contribute to the greater need for inotropic support in patients with severe ventricular dysfunction. Strategies to inhibit activation of GRK2 during CABG may decrease morbidity in this patient population.

Right ventricular beneficial effects of beta adrenergic receptor kinase inhibitor (βARKct) gene transfer in a rat model of severe pressure overload

Heart failure is associated with abnormalities in βAR cascade regulation, calcium cycling, expression of inflammatory mediators and apoptosis. Adenoviral mediated gene transfer of βARKct has beneficial indirect effects on these pathologic processes upon the left ventricular myocardium. The concomitant biochemical changes that occur in the right ventricle have not been well characterized. Sprague–Dawley rats underwent aortic banding and were followed by echocardiography. After a decrease in fractional shortening of 25% from baseline, intracoronary injection of adenoviral-βARKct (n=14) or adenoviral-β-galactosidase (control, n=13) was performed. Rats were randomly euthanized on post-operative day 7, 14 or 21. Protein analysis including RV myocardial levels of βARKct, βARK1, SERCA2a, inflammatory tissue mediators (IL-1, IL-6 and TNF-α), apoptotic markers (bax and bak), and MAP kinases (jnk, p38 and erk) was performed. ANOVA was employed for group comparison. Adenoviral-βARKct treated animals showed increased expression of βARKct and decreased levels of βARK1 compared with controls. This treatment group also demonstrated normalization of SERCA2a expression and decreased levels of the inflammatory markers IL-1, IL-6 and TNF-α. The pro-apoptotic markers bax and bak were similarly improved. Ventricular levels of the MAP kinase jnk were increased. Differences were most significant 7 days after gene transfer, but the majority of these changes persisted at 21 days. These results suggest that attenuation of the pathologic mechanisms of beta adrenergic receptor desensitization, SERCA2a expression, inflammation and apoptosis, not only occur in the left ventricle but also in the right ventricular myocardium after intracoronary gene transfer of βARKct during heart failure.

Acute β-blockade prevents myocardial β-adrenergic receptor desensitization and preserves early ventricular function after brain death

Beta-adrenergic receptor desensitization through activation of the G protein-coupled receptor kinase 2 is an important mechanism of early cardiac dysfunction after brain death. We hypothesized that acute beta-blockade can prevent myocardial beta-adrenergic receptor desensitization after brain death through attenuation of G protein-coupled receptor kinase 2 activity, resulting in improved cardiac function. Adult pigs underwent either sham operation, induction of brain death, or treatment with esmolol (beta-blockade) for 30 minutes before and 45 minutes after brain death (n = 8 per group). Cardiac function was assessed at baseline and for 6 hours after the operation. Myocardial beta-adrenergic receptor signaling was assessed 6 hours after operation by measuring sarcolemmal membrane adenylate cyclase activity, beta-adrenergic receptor density, and G protein-coupled receptor kinase 2 expression and activity. Baseline left ventricular preload recruitable stroke work was similar among sham, brain death, and beta-blockade groups. Preload recruitable stroke work was significantly decreased 6 hours after brain death versus sham, and beta-blockade resulted in maintenance of baseline preload recruitable stroke work relative to brain death and not different from sham. Basal and isoproterenol-stimulated adenylate cyclase activities were preserved in the beta-blockade group relative to the brain death group and were not different from the sham group. Left ventricular G protein-coupled receptor kinase 2 expression and activity in the beta-blockade group were markedly decreased relative to the brain death group and similar to the sham group. Beta-adrenergic receptor density was not different among groups. Acute beta-blockade before brain death attenuates beta-adrenergic receptor desensitization mediated by G protein-coupled receptor kinase 2 and preserves early cardiac function after brain death. These data support the hypothesis that acute beta-adrenergic receptor desensitization is an important mechanism in early ventricular dysfunction after brain death. Future studies with beta-blocker therapy immediately after brain death appear warranted.

Inhibition of protein kinase Cα improves myocardial β-adrenergic receptor signaling and ventricular function in a model of myocardial preservation

The specific effect of protein kinase C alpha, the primary ventricular calcium-dependent protein kinase C isoform, on myocardial protection is unclear. The objective of this study was to determine the role of protein kinase C alpha in myocardial protection and recovery of function after cardioplegic arrest, cold preservation, and normothermic reperfusion, as relevant to cardiac transplantation. We used an ex vivo murine model, and hearts were arrested with cold crystalloid cardioplegia or saline as a control and maintained at 4 degrees C for 4 hours. This was followed by normothermic reperfusion for 90 minutes. Transgenic hearts with cardiac-specific activation or inhibition of protein kinase C alpha were then studied to specifically examine the effects of protein kinase C alpha on myocardial preservation in this model. Cardioplegic arrest with University of Wisconsin solution led to significantly improved postreperfusion hemodynamics and inhibition of myocardial protein kinase C alpha activity relative to that seen in saline-treated control hearts. Beta-adrenergic receptor signaling was also preserved with University of Wisconsin solution. Transgenic hearts with enhanced protein kinase C alpha activity had poor postreperfusion hemodynamics, impaired beta-adrenergic receptor signaling, and increased G protein-coupled receptor kinase 2 activity compared with those seen in nontransgenic control hearts. In contrast, transgenic hearts with inhibited protein kinase C alpha activity had even better myocardial protection relative to control hearts and preserved beta-adrenergic receptor signaling. Current techniques of myocardial preservation are associated with inhibition of protein kinase C alpha activity and maintenance of intact beta-adrenergic receptor signaling. Activation of protein kinase C alpha leads to enhanced beta-adrenergic receptor desensitization and impaired signaling and ventricular function as a result of increased G protein-coupled receptor kinase 2 activity. This is a novel in vivo mechanism of G protein-coupled receptor kinase 2 activation. Strategies to specifically inhibit these kinases might improve long-term myocardial protection.

Role of PI3 Kinase Gamma in Excitation-Contraction Coupling and Heart Disease

Class I phosphoinositide 3-kinases (PI3Ks) are enzymes with both protein and lipid kinase activities that have ubiquitous cellular functions. In the heart, subclass IA PI3Ks, PI3K-alpha and beta, regulate cell growth, apoptosis, cell division and cell size, whereas PI3Kgamma, the only member of subclass IB, has been shown to regulate myocardial contractility. Loss of p110gamma, the catalytic subunit of PI3Kgamma, enhances cardiac excitation-contraction coupling by modulating cyclic adenosine monophosphate (cAMP) levels in subcellular domains containing the sarcoplasmic reticulum (SR) leading to increased cAMP-mediated phosphorylation of phospholamban. The ability of p110gamma to modulate cAMP is likely mediated by the protein-protein interactions with the cAMP-degrading enzymes, phosphodiesterases, independent of its lipid kinase activity. PI3Kgamma also plays a key role in modulating the cAMP response and desensitization of beta-adrenergic receptors. Loss of p110gamma gamma leads to acute decompensation and rapid progression into heart failure in response to pathological biomechanical stress while lipid kinase-dead mutants were relatively resistant suggesting that elevated intracellular cAMP (and its secondary effects) is an important predisposing factor for heart failure. The commercial availability of specific PI3Kgamma inhibitors may be used as therapeutic agents in inflammatory and cardiovascular diseases. In this review article, we discuss the key role of PI3Kgamma gamma in regulating cAMP, Ca(2+) cycling, beta-adrenergic signaling and myocardial structure and function in heart disease.

Beta-adrenergic Receptor Antagonism Preserves Myocardial Function After Brain Death in a Porcine Model

Cardiac dysfunction after brain death decreases the already limited number of potential donors for cardiac transplantation. Acute beta-adrenergic receptor (betaAR) desensitization after the brain death-associated catecholamine surge is an important mechanism. We hypothesized that acute betaAR antagonism could improve myocardial function after brain death by preserving betaAR signaling. Pigs were randomly assigned to three study groups (n = 5): sham; brain death; and brain death with betaAR antagonist (200 microg/kg/min esmolol), 30 minutes before brain death until 45 minutes after brain death. Functional data were collected for 6 hours after brain death and tissues procured. Compared with baseline, pre-load recruitable stroke work (PRSW), a pre-load-independent measure of systolic function (21.4 +/- 7.5 vs 43.3 +/- 6.8, slope of regression line during vena caval occlusion, p < 0.001), diastolic function (Tau, 101 +/- 54.7 vs 36.4 +/- 5.4 ms, p = 0.03) and systemic oxygen delivery (151 +/- 79.7 vs 298 +/- 78.7 ml/min, p < 0.001) deteriorated in untreated animals at 6 hours after brain death. In contrast, betaAR antagonist maintained baseline systolic function (PRSW, 37.8 +/- 5.6 vs 38.2 +/- 4.7, slope of regression line during vena caval occlusion, p = 0.92), diastolic function (Tau, 32.6 +/- 5.1 vs 48.5 +/- 28.3 ms, p = 0.57) and oxygen delivery (427 +/- 116 vs 397 +/- 98.8 ml/min, p = 0.36) at 6 hours after brain death. betaAR antagonist preserved betaAR signaling, as demonstrated by similar left ventricular (LV) basal (55.4 +/- 32.8 vs 58.8 +/- 10.9 pmol/mg/min, p = 0.40) and isoproterenol-stimulated (125 +/- 70.5 vs 124 +/- 52.0 pmol/mg/min, p = 0.49) adenylate cyclase activity at 6 hours after brain death, upon comparing betaAR antagonist and sham treatment groups. Both LV basal and isoproterenol-stimulated adenyl cyclase activity were higher with betaAR antagonist (25.9 +/- 4.8 pmol/mg/min, p = 0.03) than with untreated brain death (55.6 +/- 17.3 pmol/mg/min, p = 0.02). Beta-adrenergic receptor antagonism before brain death preserves cardiac function by preventing betaAR desensitization. This therapy in potential donors might increase the number of organs available for transplantation.

Suppression of Autonomic‐Mediated Triggered Firing in Pulmonary Vein Preparations, 24 Hours Postcoronary Artery Ligation in Dogs

Rapid arrhythmia originating within pulmonary veins (PVs) precipitates atrial fibrillation (AF) in man. To determine a possible basis for an increased incidence of AF observed peri-myocardial infarction in man, we compared AF induction in vivo and triggered arrhythmia formation within isolated PVs in vitro in normal dogs and dogs studied 24 hours postcoronary artery ligation. The incidence of AF initiated by atrial premature stimuli was increased in dogs postcoronary artery ligation (6/9) versus normal (2/12) (P = 0.03). In isolated PVs from normal hearts, pause-dependent early afterdepolarizations (EADs) were enhanced by catecholamines. Rapid arrhythmia (1,182 +/- 213 beats/min) was triggered during isoproterenol/norepinephrine (32 nM) + acetylcholine (100 nM) (N = 16/23) using the same pacing pauses eliciting EADs. Rapid arrhythmia (802 +/- 161 beats/min) was also triggered by local autonomic nerve stimulation (ANS; N = 18/23). Despite equivalent pause-dependent afterdepolarization formation in PVs from infarcted hearts, a rightward 45-fold and 28-fold shift in the dose-response curve for afterdepolarization enhancement was observed for isoproterenol and norepinephrine, respectively (P < 0.02). ANS (N = 1/19) and isoproterenol/norepinephrine (32 nM) + acetylcholine (100 nM) (N = 0/9 and 0/12, respectively) (P = 0.0001) failed to elicit arrhythmia formation. Beta-adrenergic receptor desensitization was associated with a 2.5-fold increase in PV beta-adrenergic receptor kinase (ARK). The data demonstrate decreased susceptibility of isolated canine PVs for arrhythmia triggered by local ANS, or pacing pauses in the presence of a catecholamine + acetylcholine, postmyocardial infarction, despite a greater susceptibility of the intact heart to AF. The decreased arrhythmia susceptibility was observed coincident with an increase in beta-ARK and a decreased responsiveness to beta-adrenergic receptor agonists.

Noninvasive Imaging of Heart Failure: Neuronal Dysfunction and Risk Stratification

Congestive heart failure (CHF) is a clinical syndrome of impaired cardiac contractile function resulting from structural damage, and is characterized by a vicious cycle in which reduced cardiac output results in neurohumoral activation. The plasma norepinephrine level, which acts as a major neurotransmitter of the sympathetic nervous system, has been shown to increase in heart failure patients in response to decreased systemic pressure [1]. This hyperadrenergic state in turn causes desensitization and downregulation of cardiac beta-adrenergic receptors and alterations in postsynaptic signal transduction, which causes further impairment of myocardial performance [2]. The close relationship between these alterations of the cardiac autonomic nervous system in heart failure and disease progression, and prognosis, including pump failure and sudden death, has been discussed intensively [3]. Nuclear medicine techniques, such as single photon emission tomography (SPECT) and positron emission tomography (PET), are unique methods of evaluating global and regional myocardial autonomic innervation [4]. These techniques have facilitated and refined substantially the study of the nervous system in both healthy and diseased hearts and have contributed significantly to an improved understanding of heart failure pathophysiology. Radiolabeled catecholamine and catecholamine analogs are available and their tracer physiologic properties are well under-

A new look at phospholamban in pressure-overload hypertrophy

THE QUESTION OF THE MECHANISMS of pathological cardiac hypertrophy and the relationship of changes in cellular function to a myriad of clinical symptoms has long been an important and intriguing puzzle to researchers. How is it that exercise induces signaling changes that are beneficial to heart function but pathological conditions such as hypertension or valvular disease are associated with a loss of hemodynamic function and neurohumoral response, although both produce muscular hypertrophy? What are the interactions between hypertrophic and contractile signaling pathways? One point of interest is the increase in sympathetic drive observed in the pressure-overloaded (PO) hypertrophic heart that is accompanied by decreased -adrenergic sensitivity and diminished contractile reserve. This condition is significant to patients in that it causes a loss of ability to tolerate exercise and can lead to myocyte death and heart failure, but the cellular changes underlying the loss of contractile reserve are not entirely clear. A number of researchers have identified mechanisms for this loss of contractile reserve, but this picture is not complete. One prominent mechanism involved in the loss of adrenergic response is a downregulation of -adrenergic receptor (-AR) density, which is improved by administration of -blockers (4). Another is upregulation of -receptor kinase 1 (or G proteincoupled receptor kinase 2, GRK2; see Ref. 1), which inactivates -ARs and is reversible by exercise training (2). In the current issue of the American Journal of PhysiologyHeart and Circulatory Physiology, Mills et al. (3) have addressed the question of what downstream mechanisms may be involved in the diminution of adrenergic contractile reserve using a feline model of PO-induced left ventricular hypertrophy. They propose that altered abundance or basal phosphorylation state of target regulatory proteins, specifically phospholamban (PLB), is a central cause of the loss of inotropic responsiveness in PO animals. They have presented compelling in vivo evidence that aortic banded animals have a blunted systolic function that is not restored with dobutamine, concurrent with reduced total PLB and a significant increase in total PLB phosphorylation that is mainly attributed to the calmodulin kinase II site, Thr 17 . They also report that the less the response to dobutamine, the greater the level of Thr 17 phos

Role of the β-adrenergic receptor kinase in myocardial dysfunction after brain death

Significant cardiac dysfunction after brain death leading to exclusion from procurement for cardiac transplantation is seen in up to 25% of potential organ donors in the absence of structural heart disease. The cause includes uncoupling of the myocardial beta-adrenergic receptor signaling system. The mechanism, however, has not yet been described. This study investigates our hypothesis that brain death causes acute activation of the betaAR kinase and leads to desensitization of myocardial beta-adrenergic receptors and impaired ventricular function.Adult pigs underwent a sham operation or induction of brain death by means of subdural balloon inflation (n = 8 in each group). Cardiac function was assessed by using sonomicrometry at baseline and for 6 hours after the operation. beta-Adrenergic receptor signaling was assessed at 6 hours after the operation by measuring myocardial sarcolemmal membrane adenylate cyclase activity, beta-adrenergic receptor density, beta-adrenergic receptor kinase expression, and activity.Induction of brain death led to significantly decreased left ventricular systolic and diastolic function. Basal and isoproterenol-stimulated adenylate cyclase activity was blunted in the brain dead group compared with the sham-operated group (28.3 +/- 4.3 vs 48.3 +/- 7.6 pmol of cyclic adenosine monophosphate.mg(-1) x min(-1) [P = .01] and 54.8 +/- 9.6 vs 114.5 +/- 18 pmol of cyclic adenosine monophosphate x mg(-1) x min(-1) [P < .02]). There was no difference in beta-adrenergic receptor density between the brain dead and sham-operated groups. Myocardial beta-adrenergic receptor kinase expression was 3-fold greater in the brain dead versus sham-operated groups, and membrane beta-adrenergic receptor kinase activity was 2.5-fold greater in the brain dead group compared with that seen in the sham-operated group.Induction of brain death leads to significant left ventricular dysfunction in this porcine model. Cardiac beta-adrenergic receptors are clearly uncoupled after brain death, and our data suggest that the mechanism is acute increase of myocardial beta-adrenergic receptor kinase activity, leading to beta-adrenergic receptor desensitization and ventricular dysfunction.