Nuclear alpha1-adrenergic receptors signal activated ERK localization to caveolae in adult cardiac myocytes

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.     

Subtype specific roles of beta-adrenergic receptors in apoptosis of adult rat ventricular myocytes

We have previously reported that beta-adrenergic receptor (beta-AR) stimulation promotes apoptosis in adult ventricular myocytes through PKCepsilon-mediated suppression of ERK. In this study, we investigated differential effects of beta-AR subtypes on this signal pathway. The apoptosis induced by the non-specific beta-AR agonist isoproterenol was largely blocked by the beta(1)-selective antagonist CGP 20712A, but not by the beta(2)-selective antagonist ICI 118551. A pro-apoptotic effect of beta(1)-AR was also blocked by the PKA inhibitor H89, while the protein kinase A (PKA) activators forskolin and dibutyryl-cAMP both induced apoptosis. These results indicate that beta(1)-AR-mediated PKA activation is largely responsible for the apoptosis induced by beta-AR in adult rat cardiac myocytes. This conclusion was also supported by the finding that PKA was preferentially activated by beta(1)-AR over beta(2)-AR. beta(2)-AR selectively induced anti-apoptotic ERK activation in the presence of PKCepsilon suppression, and this ERK activation was sensitive to pertussis toxin. PKCepsilon itself as well as Akt, the other anti-apoptotic factor were activated by both beta-AR subtypes. Thus, beta(1)-AR induces pro-apoptotic signals mainly through PKA activation. In contrast, beta(2)-AR is linked to Gi-mediated ERK activation, which is involved in the anti-apoptotic pathway, and is regulated by PKCepsilon. Therefore, our findings suggest a rather complex role for beta-AR subtypes in the regulation of apoptosis in adult ventricular myocytes.     

Modulation of the pacemaker current If by beta-adrenoceptor subtypes in ventricular myocytes isolated from hypertensive and normotensive rats.

OBJECTIVE: Both beta 1- and beta 2-adrenoceptors (beta 1-AR and beta 2-AR) are functionally present in human and rat ventricular myocytes. The two receptor subtypes are differently regulated during the development of myocardial hypertrophy and failure. I(f) is expressed in human and rat ventricular myocytes. In hypertrophied myocytes isolated from old spontaneously hypertensive rats (SHR) the density is much larger than in age-matched normotensive Wistar Kyoto (WKY). Due to the possible relevance of I(f) as an arrhythmogenic mechanism in the rat and human ventricle, we studied and compared the effects of beta 1-AR and beta 2-AR stimulation on I(f) in both hypertrophied and normal left ventricular myocytes of 18-month old SHR and WKY. METHODS: The whole-cell configuration of the patch-clamp technique was employed. Noradrenaline (NA, 1 microM) was used to stimulate beta 1-AR and isoprenaline (ISO, 1 microM) in the presence of the beta 1-AR antagonist CGP 20712A (0.1 microM) to stimulate beta 2-AR. RESULTS: In SHR, NA increased I(f) by causing a 10.8 +/- 0.9 mV (n = 10) positive shift in the voltage of maximal activation (V1/2); this effect was completely reversed by CGP 20712A. beta 2-AR stimulation was effective in seven out of 13 cells tested, where it caused a small positive shift in V1/2 (4.0 +/- 1.7 mV). Cyclopentyladenosine (CPA), a selective A1-receptor agonist, reversed the effect of NA; the antiadrenergic action of CPA was abolished in cells pre-incubated with pertussis toxin (PTX) to block inhibitory G proteins (Gi). In PTX-treated cells the shift in V1/2 caused by both beta 2-AR (9.6 +/- 1.7 mV, n = 6, p < 0.05) and beta 1-AR (17.6 +/- 1.9 mV, n =7, p < 0.05) was significantly greater than in control cells. Both beta-AR subtypes modulated I(f) activation also in WKY: beta 1-AR shifted V1/2 by 16.0 +/- 1.4 mV (n = 15) and beta 2-AR by 4.2 +/- 1.1 mV (n = 7). However, in PTX-treated WKY cells only the beta 2-AR effect was potentiated (shift in V1/2: 11.4 +/- 1.4 mV, n = 9, p < 0.01), while the beta 1-AR response was unchanged (18.9 +/- 4.2 mV, n = 5, n.s.). CONCLUSIONS: I(f) expressed in SHR hypertrophied ventricular myocytes is modulated by catecholamines mainly through the stimulation of the beta 1-AR subtype. The beta 1-AR response is, however, significantly lower than that observed in myocytes from normotensive rats, probably as a consequence of the presence of an increased inhibitory activity of Gi proteins. This post-receptorial control may be seen as a mechanism to limit the arrhythmogenicity of beta-AR stimulation in myocardial hypertrophy and failure.     

A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes

Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-AR regulation of I(Ca,L), whereas only PDE4 controlled Glu-R regulation of I(Ca,L) thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.     

Beta1-adrenergic receptors promote focal adhesion signaling downregulation and myocyte apoptosis in acute volume overload

Numerous studies demonstrated increased expression of extracellular matrix (ECM) proteins and activation of focal adhesion (FA) signaling pathways in models of pressure overload-induced cardiac hypertrophy. However, little is known about FA signaling in response to volume overload where cardiac hypertrophy is associated with ECM loss. This study examines the role of beta1-adrenergic receptors (β(1)-ARs) in FA signaling changes and myocyte apoptosis induced during acute hemodynamic stress of volume overload. Rats with eccentric cardiac hypertrophy induced after aorto-caval fistula (ACF) develop reduced interstitial collagen content and decreased tyrosine phosphorylation of key FA signaling molecules FAK, Pyk(2) and paxillin along with an increase in cardiac myocyte apoptosis. ACF also increased activation of PTEN, a dual lipid and protein phosphatase, and its interaction with FA proteins. β(1)-AR blockade (extended-release of metoprolol succinate, 100mg QD) markedly attenuated PTEN activation, restored FA signaling and reduced myocyte apoptosis induced by ACF at 2days, but failed to reduce interstitial collagen loss and left ventricular dilatation. Treating cultured myocytes with β(1)-AR agonists or adenoviral expression of β(1)-ARs caused PTEN activation and interaction with FA proteins, thus leading to FA signaling downregulation and myocyte apoptosis. Adenoviral-mediated expression of a catalytically inactive PTEN mutant or wild-type FAK restored FA signaling downregulation and attenuated myocyte apoptosis induced by β(1)-ARs. Collectively, these data show that β(1)-AR stimulation in response to ACF induces FA signaling downregulation through an ECM-independent mechanism. This effect involves PTEN activation and may contribute to adverse cardiac remodeling and function in the course of volume overload.     

The beta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G(i)-dependent coupling to phosphatidylinositol 3'-kinase

Recent studies have shown that chronic beta-adrenergic receptor (beta-AR) stimulation alters cardiac myocyte survival in a receptor subtype-specific manner. We examined the effect of selective beta(1)- and beta(2)-AR subtype stimulation on apoptosis induced by hypoxia or H(2)O(2) in rat neonatal cardiac myocytes. Although neither beta(1)- nor beta(2)-AR stimulation had any significant effect on the basal level of apoptosis, selective beta(2)-AR stimulation protected myocytes from apoptosis. beta(2)-AR stimulation markedly increased mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) activation as well as phosphatidylinositol-3'-kinase (PI-3K) activity and Akt/protein kinase B phosphorylation. beta(1)-AR stimulation also markedly increased MAPK/ERK activation but only minimally activated PI-3K and Akt. Pretreatment with pertussis toxin blocked beta(2)-AR-mediated protection from apoptosis as well as the beta(2)-AR-stimulated changes in MAPK/ERK, PI-3K, and Akt/protein kinase B. The selective PI-3K inhibitor, LY 294002, also blocked beta(2)-AR-mediated protection, whereas inhibition of MAPK/ERK activation at an inhibitor concentration that blocked agonist-induced activation but not the basal level of activation had no effect on beta(2)-AR-mediated protection. These findings demonstrate that beta(2)-ARs activate a PI-3K-dependent, pertussis toxin-sensitive signaling pathway in cardiac myocytes that is required for protection from apoptosis-inducing stimuli often associated with ischemic stress.     

心力衰竭患者抗β3 肾上腺素能受体自身抗体的分布及特性

目的探讨心力衰竭(心衰)患者血清中的抗β3肾上腺素能受体(β3-AR)自身抗体的生物学效应,为临床治疗心衰提供新的思路和线索.方法 (1)以人β3-AR细胞外第二环的合成肽段作为抗原,采用ELISA筛选正常人和心衰患者血清中抗β3-AR自身抗体.(2)提纯该抗体阳性的患者血清中的IgG.(3)设立各组对照观察该抗体对成年大鼠心肌细胞收缩效应的影响.(4)设立各组对照观察该抗体对乳鼠心肌细胞跳动频率的影响.结果 (1)正常人抗β3-AR自身抗体的阳性率为11.0%,平均滴度为1∶ 14.59±1.61;心衰患者抗β3-AR自身抗体的阳性率为26.7%,平均滴度为1∶ 43.27±2.71;与正常人相比,P＜0.05.(2)与空白对照组相比,该抗体可降低成年大鼠心肌细胞收缩幅度/初长度(3.84%±0.33%)、收缩速率(-0.47 μm/s±0.07 μm/s)和舒张速率(0.17 μm/s±0.02 μm/s),P＜0.05.该作用不能被纳多洛尔(nadolol,β1-AR和β2-AR受体拮抗剂)阻断,但可被布拉洛尔(bupranolol,非特异性β受体拮抗剂)或β3抗原阻断.(3)与空白对照组(94.3次/min±10.7次/min)相比,该抗体可降低乳鼠心肌细胞跳动频率(47.1次/min±8.11次/min),P＜0.05.同样该作用不能被纳多洛尔阻断,但可被布拉洛尔或β3抗原阻断.此外,该抗体的负性变时效应在观察时间内(6 h)无衰减.结论心衰患者血清中含有较高滴度的抗β3-AR自身抗体并具有负性变力和变时效应,提示该抗体可能参与心衰的病理生理机制.     

Phosphorylation of eukaryotic translation initiation factor 2Bepsilon by glycogen synthase kinase-3beta regulates beta-adrenergic cardiac myocyte hypertrophy

Glycogen synthase kinase 3beta (GSK-3beta) negatively regulates cardiac hypertrophy. A potential target mediating the antihypertrophic effect of GSK-3beta is eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon). Overexpression of GSK-3beta increased the cellular kinase activity toward GST-eIF2Bepsilon in neonatal rat cardiac myocytes, whereas LiCl (10 mmol/L) or isoproterenol (ISO) (10 micromol/L), a treatment known to inhibit GSK-3beta, decreased it. Immunoblot analyses using anti-S535 phosphospecific eIF2Bepsilon antibody showed that S535 phosphorylation of endogenous eIF2Bepsilon was decreased by LiCl or ISO, suggesting that GSK-3beta is the predominant kinase regulating phosphorylation of eIF2Bepsilon-S535 in cardiac myocytes. Decreases in eIF2Bepsilon-S535 phosphorylation were also observed in a rat model of cardiac hypertrophy in vivo. Overexpression of wild-type eIF2Bepsilon alone moderately increased cell size (+31+/-11%; P<0.05 versus control), whereas treatment of eIF2Bepsilon-transduced myocytes with LiCl (+73+/-22% versus eIF2Bepsilon only; P<0.05) or ISO (+84+/-33% versus eIF2Bepsilon only; P<0.05) enhanced the effect of eIF2Bepsilon. Overexpression of eIF2Bepsilon-S535A, which is not phosphorylated by GSK-3beta, increased cell size (+107+/-35%) as strongly as ISO (+95+/-25%), and abolished antihypertrophic effects of GSK-3beta, indicating that S535 phosphorylation of eIF2Bepsilon critically mediates antihypertrophic effects of GSK-3beta. Furthermore, expression of eIF2Bepsilon-F259L, a dominant-negative mutant, inhibited ISO-induced hypertrophy, indicating that eIF2Bepsilon is required for beta-adrenergic hypertrophy. Interestingly, expression of eIF2Bepsilon-S535A partially increased cytoskeletal reorganization, whereas it did not increase expression of atrial natriuretic factor gene. These results suggest that GSK-3beta is the predominant kinase mediating phosphorylation of eIF2Bepsilon-S535 in cardiac myocytes, which in turn plays an important role in regulating cardiac hypertrophy primarily through protein synthesis.     

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.     

Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure

J. D. Port and M. R. Bristow. Altered Beta-adrenergic Receptor Gene Regulation and Signaling in Chronic Heart Failure. Journal of Molecular and Cellular Cardiology (2001) 33, 887-905. Beta adrenergic receptors (beta -ARs) are critical regulators of cardiac function in both normal and pathophysiological states. Under normal conditions, beta -ARs and their signaling pathways modulate both the rate and force of myocardial contraction and relaxation, allowing individuals to respond appropriately to physiological stress or exercise. However, in chronic heart failure, sustained activation of the beta -AR signaling pathways can have overtly negative biological consequences. This notion is reinforced by the positive outcomes of a number of clinical trials demonstrating the usefulness of beta-blocker therapy in chronic congestive heart failure. During the last few years, significant progress has been made in understanding the molecular biological basis of beta -AR function, both at the biochemical and genetic levels. In this review, the biological basis of adrenergic signaling and how this changes in heart failure is discussed. Aspects of adrenergic receptor pharmacology relevant to heart failure are reviewed, including the recently emerging differences described for beta(1)- v beta(2)-AR signaling pathways. Highlighting these differences is recent evidence that over-stimulation of the beta(1)-AR pathway in cardiac myocytes appears to be pro-apoptotic, whereas stimulation of the beta(2)-AR pathway may be anti-apoptotic. Overview of beta -AR gene regulation, transgenic models of beta -AR overexpression, and beta -AR polymorphisms as they relate to heart failure progression are also discussed.     

Arrhythmogenic effects of beta2-adrenergic stimulation in the failing heart are attributable to enhanced sarcoplasmic reticulum Ca load

Ventricular tachycardia in heart failure (HF) can initiate by nonreentrant mechanisms such as delayed afterdepolarizations. In an arrhythmogenic rabbit model of HF, we have shown that isoproterenol induces ventricular tachycardia in vivo and aftercontractions and transient inward currents in HF myocytes. To determine whether beta(2)-adrenergic receptor (beta(2)-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies. Intravenous zinterol (2.5 microg/kg) led to ventricular arrhythmias, including ventricular tachycardia up to 13 beats long in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect blocked by beta(2)-AR antagonist ICI-118,551 (0.2 mg/kg). In field-stimulated myocytes (0.5 to 4 Hz, 37 degrees C), beta(2)-AR stimulation (1 micromol/L zinterol+300 nmol/L beta(1)-AR antagonist CGP-29712A) induced aftercontractions and Ca aftertransients in 88% of HF versus 0% of control myocytes (P<0.01). beta(2)-AR stimulation in HF (but not control) myocytes increased Ca transient amplitude (by 29%), sarcoplasmic reticulum (SR) Ca load (by 28%), the rate of [Ca](i) decline (by 28%; n=12, all P<0.05), and phospholamban phosphorylation at Ser16, but Ca current was unchanged. All of these effects in HF myocytes were blocked by ICI-118,551 (100 nmol/L). Although total beta-AR expression was reduced by 47% in HF rabbit left ventricle, beta(2)-AR number was unchanged, indicating more potent beta(2)-AR-dependent SR Ca uptake and arrhythmogenesis in HF. Human HF myocytes showed similar beta(2)-AR-induced aftercontractions, aftertransients, and enhanced Ca transient amplitude, SR Ca load and twitch [Ca](i) decline rate. Thus, beta(2)-AR stimulation is arrhythmogenic in HF, mediated by SR Ca overload-induced spontaneous SR Ca release and aftercontractions.     

Cardiac alpha 1-adrenergic drive in pathological remodelling

The heart is richly innervated by sympathetic nerves, and both acute and chronic regulation of cardiac function via sympathetically released catecholamines acting on cardiomyocyte adrenergic receptors (ARs), is critical for circulatory homeostasis. Cardiomyocytes express alpha 1A- and alpha 1B-, and beta 1- and beta 2-AR subtypes, which are all members of the G-protein-coupled receptor superfamily that signal via interaction with heterotrimeric G-proteins. Cardiac function - both inotropy and chronotropy - is regulated predominantly by beta 1-AR. Activation of alpha 1-ARs also results in increased contractility, as well as changes in the electrophysiological properties and metabolic responses of the heart. Nonetheless, there is little evidence that cardiac alpha 1-ARs play a major functional role under normal physiological conditions. In pathological settings, alpha 1-ARs may function in a compensatory fashion to maintain cardiac inotropy when the beta-AR system is downregulated and uncoupled from G-proteins and effectors. In addition, as we consider here, recent evidence from clinical studies and from genetically engineered animal models indicates that alpha 1-ARs are importantly involved in both developmental cardiomyocyte growth, as well as pathological hypertrophy. In the presence of pressure overload or with myocardial infarction, activation of alpha 1-ARs, particularly the alpha 1A-subtype, also appears to produce important pro-survival effects at the level of the cardiomyocyte, and to protect against maladaptive cardiac remodelling and decompensation to heart failure.     

Direct analysis of beta-adrenergic receptor subtypes on intact adult ventricular myocytes of the rat

beta 1- and beta 2-Adrenergic receptors co-exist in the adult rat ventricle. We have employed radioligand binding and cell purification techniques to determine the cellular origin of these receptors. The beta-adrenergic antagonist ligand (+/-)-[125I] iodocyanopindolol binds to 2 X 10(5) receptors per purified adult rat cardiomyocyte, with a dissociation constant of 70 pM. The subtype-selective antagonists betaxolol (beta 1), practolol (beta 1), and zinterol (beta 2) compete for [125I]iodocyanopindolol-binding sites on intact myocytes in monophasic manners with dissociation constants of 46, 845, and 923 nM, respectively. [125I]iodocyanopindolol binding to membranes prepared from nonmyocyte elements of rat ventricle occurs with a dissociation constant of 43 pM and a capacity of 88 fmol/mg membrane protein. Computer analysis of competition of [125I]iodocyanopindolol binding by betaxolol, practolol, and zinterol in nonmyocyte membranes demonstrates biphasic curves that comprise binding to both beta 1- and beta 2-receptors. These data demonstrate that purified adult ventricular myocytes possess only beta 1-receptors, and that the beta 2-receptors found in rat ventricle are located on nonmyocyte cell types.