Anthony Yiu-Ho Woo

Activation of CaMKIIδC Is a Common Intermediate of Diverse Death Stimuli-induced Heart Muscle Cell Apoptosis

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is expressed in many mammalian cells, with the δ isoform predominantly expressed in cardiomyocytes. Previous studies have shown that inhibition of CaMKII protects cardiomyocytes against β1-adrenergic receptor-mediated apoptosis. However, it is unclear whether activation of CaMKII is sufficient to cause cardiomyocyte apoptosis and whether CaMKII signaling is important in heart muscle cell apoptosis mediated by other stimuli. Here, we specifically enhanced or suppressed CaMKII activity using adenoviral gene transfer of constitutively active (CA-CaMKIIδC) or dominant negative (DN-CaMKIIδC) mutants of CaMKIIδC in cultured adult rat cardiomyocytes. Expression of CA-CaMKIIδC promoted cardiomyocyte apoptosis that was associated with increased mitochondrial cytochrome c release and attenuated by co-expression of Bcl-XL. Importantly, isoform-specific suppression of CaMKIIδC with the DN-CaMKIIδC mutant similar to nonselective CaMKII inhibition by the pharmacological inhibitors (KN-93 or AIP) not only prevented CA-CaMKIIδC-mediated apoptosis but also protected cells from multiple death-inducing stimuli. Thus, activation of CaMKIIδC constitutes a common intermediate by which various death-inducing stimuli trigger cardiomyocyte apoptosis via the primary mitochondrial death pathway.

Stereochemistry of an Agonist Determines Coupling Preference of β2-Adrenoceptor to Different G Proteins in Cardiomyocytes

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most β2-adrenoceptor agonists activate both Gs and Gi proteins, fenoterol, a full agonist of β2-adrenoceptor, selectively activates Gs protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate Gs signaling, their S,R isomers were able to activate both Gs and Gi proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on Gs,Gi2, and Gi3 proteins. The inefficient Gi signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the β2-adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.

β-Adrenergic receptor subtype signaling in heart: From bench to bedside

β-adrenergic receptor (βAR) stimulation by the sympathetic nervous system or circulating catecholamines is broadly involved in peripheral blood circulation, metabolic regulation, muscle contraction, and central neural activities. In the heart, acute βAR stimulation serves as the most powerful means to regulate cardiac output in response to a fight-or-flight situation, whereas chronic βAR stimulation plays an important role in physiological and pathological cardiac remodeling.There are three βAR subtypes, β1AR, β2AR and β3AR, in cardiac myocytes. Over the past two decades, we systematically investigated the molecular and cellular mechanisms underlying the different even opposite functional roles of β1AR and β2AR subtypes in regulating cardiac structure and function, with keen interest in the development of novel therapies based on our discoveries. We have made three major discoveries, including (1) dual coupling of β2AR to Gs and Gi proteins in cardiomyocytes, (2) cardioprotection by β2AR signaling in improving cardiac function and myocyte viability, and (3) PKA-independent, CaMKII-mediated β1AR apoptotic and maladaptive remodeling signaling in the heart. Based on these discoveries and salutary effects of β1AR blockade on patients with heart failure, we envision that activation of β2AR in combination with clinically used β1AR blockade should provide a safer and more effective therapy for the treatment of heart failure.

Cardioprotective and Survival Benefits of Long-Term Combined Therapy with β2 Adrenoreceptor (AR) Agonist and β1 AR Blocker in Dilated Cardiomyopathy Postmyocardial Infarction

We have reported therapeutic effectiveness of pharmacological stimulation of β2 adrenoreceptors (ARs) to attenuate the cardiac remodeling and myocardial infarction (MI) expansion in a rat model of dilated cardiomyopathy (DCM) post-MI. Furthermore, the combination of β2 AR stimulation with β1 AR blockade exceeded the therapeutic effectiveness of β1 AR blockade. However, these studies were relatively short (6 weeks). In this study, in the same experimental model, we compared different effects, including survival benefit, of combined therapy with the β1 AR blocker, metoprolol, plus the β2 AR agonist, fenoterol (β1–β2+), and either therapy alone (β1– or β2+) during the 1-year study. Therapy was started 2 weeks after permanent ligation of the left coronary artery. Cardiac remodeling, MI expansion, and left ventricular function were assessed by serial echocardiography and compared with untreated animals (nT). Sixty-seven percent mortality in nT was reduced to 33% in the β1–β2+ (p < 0.01). Progressive cardiac remodeling observed in nT and β1– was significantly attenuated in β1–β2+ during the first 6 months of treatment. In β1–β2+, MI expansion was completely prevented, and functional decline was significantly attenuated during the entire year. Myocardial apoptosis was significantly reduced in both β1–β2+ and β1–. A reduction of cardiac β1 AR density and decreases in chronotropic and contractile responses to β2 AR-specific stimulation in the absence of a reduction of β2 AR density in nT were precluded in rats receiving combined therapy. The results demonstrate the cardioprotective and survival benefit of long-term combination therapy of β2 AR agonists and β1 AR blockers in a model of DCM.

Comparative Molecular Field Analysis of Fenoterol Derivatives: A Platform Towards Highly Selective and Effective β2 Adrenergic Receptor Agonists

PURPOSE To use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the beta(2)-adrenergic receptor. RESULTS Out of 21 computationally designed structures 6 compounds were synthesized and characterized for beta(2)-AR binding affinities, subtype selectivities and functional activities. CONCLUSION the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with K(i)beta(2)-AR=0.28microm, K(i)beta(1)-AR/K(i)beta(2)-AR=573, EC(50cAMP)=3.9nm, EC(50cardio)=16nm. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure.

RGS2 is a primary terminator of β2-adrenergic receptor-mediated Gi signaling

Two major β-adrenergic receptor (βAR) subtypes, β(1)AR and β(2)AR, are expressed in mammalian heart with β(1)AR coupling to G(s) and β(2)AR dually coupling to G(s) and G(i) proteins. In many types of chronic heart failure, myocardial contractile response to both β(1)AR and β(2)AR stimulation is severely impaired. The dysfunction of βAR signaling in failing hearts is largely attributable to an increase in G(i) signaling, because disruption of the G(i) signaling restores myocardial contractile response to β(1)AR as well as β(2)AR stimulation. However, the mechanism terminating the β(2)AR-G(i) signaling remains elusive, while it has been shown activation of the G(i) signaling is dependent on agonist stimulation and subsequent PKA-mediated phosphorylation of the receptor. Here we demonstrate that regulator of G protein signaling 2 (RGS2) is a primary terminator of the β(2)AR-G(i) signaling. Specifically, prolonged absence of agonist stimulation for 24h impairs the β(2)AR-G(i) signaling, resulting in enhanced β(2)AR- but not β(1)AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased β(2)AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or G(s), G(i) or βAR subtypes. Administration of a βAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the β(2)AR-G(i) signaling in cultured cells. Furthermore, RGS2 ablation, similar to βAR agonist stimulation, sustains the β(2)AR-G(i) signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated β(2)AR-G(i) signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the β(2)AR-G(i) signaling but also provide a potential novel target for the treatment of chronic heart failure.

Biased β2‐adrenoceptor signalling in heart failure: pathophysiology and drug discovery

The body is constantly faced with a dynamic requirement for blood flow. The heart is able to respond to these changing needs by adjusting cardiac output based on cues emitted by circulating catecholamine levels. Cardiac β‐adrenoceptors transduce the signal produced by catecholamine stimulation via Gs proteins to their downstream effectors to increase heart contractility. During heart failure, cardiac output is insufficient to meet the needs of the body; catecholamine levels are high and β‐adrenoceptors become hyperstimulated. The hyperstimulated β1‐adrenoceptors induce a cardiotoxic effect, which could be counteracted by the cardioprotective effect of β2‐adrenoceptor‐mediated Gi signalling. However, β2‐adrenoceptor‐Gi signalling negates the stimulatory effect of the Gs signalling on cardiomyocyte contraction and further exacerbates cardiodepression. Here, further to the localization of β1‐ and β2‐adrenoceptors and β2‐adrenoceptor‐mediated β‐arrestin signalling in cardiomyocytes, we discuss features of the dysregulation of β‐adrenoceptor subtype signalling in the failing heart, and conclude that Gi‐biased β2‐adrenoceptor signalling is a pathogenic pathway in heart failure that plays a crucial role in cardiac remodelling. In contrast, β2‐adrenoceptor‐Gs signalling increases cardiomyocyte contractility without causing cardiotoxicity. Finally, we discuss a novel therapeutic approach for heart failure using a Gs‐biased β2‐adrenoceptor agonist and a β1‐adrenoceptor antagonist in combination. This combination treatment normalizes the β‐adrenoceptor subtype signalling in the failing heart and produces therapeutic effects that outperform traditional heart failure therapies in animal models. The present review illustrates how the concept of biased signalling can be applied to increase our understanding of the pathophysiology of diseases and in the development of novel therapies.

Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Background: Ligand-specific receptor signaling is often referred to as functional selectivity or biased agonism. Results: Single amino acid substitution on β2-adrenoreceptor (Y308F) converts a ligand-specific signaling from Gs-biased to promiscuous Gs and Gi dual signaling. Conclusion: Specific ligand-receptor interaction results in receptor conformation(s) sufficient to convey biased signaling. Significance: Our work reveals a molecular mechanism for biased agonism. Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R′)-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R′)-4′-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R′)-4′-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

Interaction of β1-adrenoceptor with RAGE mediates cardiomyopathy via CaMKII signaling

Stimulation of β1-adrenergic receptor (β1AR), a GPCR, and the receptor for advanced glycation end-products (RAGE), a pattern recognition receptor (PRR), have been independently implicated in the pathogenesis of cardiomyopathy caused by various etiologies, including myocardial infarction, ischemia/reperfusion injury, and metabolic stress. Here, we show that the two distinctly different receptors, β1AR and RAGE, are mutually dependent in mediating myocardial injury and the sequelae of cardiomyopathy. Deficiency or inhibition of RAGE blocks β1AR- and RAGE-mediated myocardial cell death and maladaptive remodeling. Ablation or blockade of β1AR fully abolishes RAGE-induced detrimental effects. Mechanistically, RAGE and β1AR form a complex, which in turn activates Ca2+/calmodulin-dependent kinase II (CaMKII), resulting in loss of cardiomyocytes and myocardial remodeling. These results indicate that RAGE and β1AR not only physically crosstalk at the receptor level, but also functionally converge at the common mediator, CaMKII, highlighting a combined inhibition of RAGE and β1AR as a more effective therapy to treat diverse cardiovascular diseases, such as myocardial infarction, ischemia/reperfusion injury, and diabetic cardiovascular complications.

β-adrenergic receptor subtype signaling in the heart: from bench to the bedside.

Publisher Summary This chapter discusses the β-adrenergic receptor (βAR) subtype signaling in the heart. The stimulation of βAR, a prototypical member of G protein-coupled receptor (GPCR) superfamily, is broadly involved in metabolic regulation, growth control, muscle contraction, cell survival, and cell death. The major βAR subtypes—β 1 AR and β 2 AR—couple to distinct G proteins and differentially regulate cardiac function and remodeling. Three major discoveries have marked the recent research line with respect to βAR subtype-specific signal transduction. Heart failure (HF) is a complex clinical syndrome featured by extensive abnormalities in the βAR system, including elevated circulating catecholamine levels, selective down regulation and desensitization of β 1 AR, and increased β 2 AR-coupled G i signaling. In particular, the enhanced G i signaling negates β 1 AR- as well as β 2 AR-mediated contractile response, thus contributing to the pathogenesis of HF. Recent translational studies support the concept that inhibition of the G i signaling or selective β 2 AR-G s stimulation with fenoterol markedly improves cardiac remodeling and the function of the failing heart.