Published online before print August 11, 2005, doi: 10.1161/01.RES.0000181160.31851.05
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© 2005 American Heart Association, Inc.
Cellular Biology |
Crosstalk of ß-Adrenergic Receptor Subtypes Through Gi Blunts ß-Adrenergic Stimulation of L-Type Ca2+ Channels in Canine Heart Failure
From the Departments of Medicine (R.C.B., T.J.K.), Physiology (J.-Q.H., T.J.K.), and Surgery (R.A.H.), University of Wisconsin–Madison.
Correspondence to Dr Timothy J. Kamp, Dept of Medicine, University of Wisconsin, H6/343 Clinical Science Center, Box 3248, 600 Highland Ave, Madison, WI 53792. E-mail tjk{at}medicine.wisc.edu
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Abstract |
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The mechanisms underlying the blunted contractile response to ß-adrenergic receptor (ß-AR) stimulation in heart failure (HF) are incompletely understood, especially with regard to ß-AR subtype–specific regulation of L-type Ca2+ channels. We evaluated the impact of HF induced by pacing tachycardia on ß-AR regulation of L-type Ca2+ channels in a canine model. To evaluate changes in the relative subcellular distribution of ß-AR subtypes, left ventricular membranes enriched in surface sarcolemma and T-tubular sarcolemma were prepared. Radioligand binding using [125I]cyanopindolol revealed that HF resulted in a comparable decrease in the density of ß1-ARs in both surface and T-tubule sarcolemma (55±4%, n=7, P<0.001; and 45±10%, n=7, P<0.01, respectively), but no significant change in ß2-AR density was observed. Whole-cell patch clamp studies demonstrated a markedly blunted increase in ICa,L in response to saturating concentrations of the nonselective ß-AR agonist isoproterenol (0.1 µmol/L) in failing myocytes compared with control (129±20%, n=11, versus 332±35%, n=7; P<0.001). Experiments testing ß1-AR– and ß2-AR–selective stimulation showed that the major component of the blunted response to nonselective ß-AR stimulation in HF was caused by ß2-AR activation, resulting in a pertussis toxin–sensitive, Gi-mediated inhibition of the ß1-AR–induced increase in ICa,L. In conclusion, canine HF results in the following: (1) a uniform reduction in ß1-AR density in surface and T-tubule membrane fractions without a change in ß2-AR density; and (2) the emergence of distinct Gi-coupling to ß2-ARs resulting in accentuated antagonism of ß1-AR–mediated stimulation of ICa,L. These results have implications for optimizing the use of ß-AR drugs in HF.
Key Words: heart failure • ventricular myocytes • ß-adrenergic receptor • calcium channels • electrophysiology
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Introduction |
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The importance of the ß-adrenergic receptor (ß-AR) system in the pathogenesis and treatment of heart failure (HF) is well accepted.1 For example, the extent of activation of the ß-ARs by elevated catecholamines is inversely correlated with survival in HF.2 Furthermore, ß-AR blockers represent the most powerful current pharmacological therapy for HF.1 The importance of the ß-AR signaling system is further emphasized by recent genetic studies that have identified polymorphisms in the ß2-AR that significantly impact prognosis in patients with HF.3 In addition, one of the hallmarks of the failing heart is a markedly blunted chronotropic and inotropic response to ß-AR stimulation.4,5 Therefore, understanding the molecular mechanisms underlying altered ß-AR regulation in HF remains a critical area of investigation.
Stimulation of ß-AR in the heart has classically been characterized as resulting in the Gs-mediated stimulation of adenylyl cyclase (AC), which leads to increased cellular cAMP activating protein kinase A (PKA). The catalytic subunit of PKA phosphorylates substrates including L-type Ca2+channels, ryanodine receptors, phospholamban, troponin I, and myosin-binding protein-C.1 This cascade is counterbalanced by phosphodiesterases and serine–threonine phosphatases. The situation is more complex because at least 3 different ß-AR receptor subtypes encoded by distinct genes have been identified in the heart.6 ß1-AR and ß2-AR have been subjected to the most study and dominate the known physiological responses. In the adult human and other large mammal hearts, the ß1-AR is the predominant ß-AR expressed (70% to 85%), and ß2-ARs make up most of the remaining 15% to 30% of ß-ARs.6 In addition, it has become clear that ß-AR signaling can regulate a number of other nonclassical signaling pathways in a receptor subtype–specific fashion.7
Prior investigations have identified multiple alterations in the classical ß-AR signaling pathway that contribute to the impaired ß-AR stimulation of the failing heart. Downregulation, specifically of ß1-ARs without changes in ß2-ARs, have commonly been described in HF.1,4 Additional studies have suggested that the remaining ß1-ARs in the failing heart are largely desensitized or uncoupled from Gs, in part, because of increased activity of G-protein–coupled receptor kinases 2 and/or 5.1,8 An increased abundance of G
i has also been found in HF, which could oppose Gs stimulation of AC.9–11 The combination of changes in the ß-AR/cAMP/PKA cascade ultimately leads to altered regulation of contraction in the failing heart; however, the impact of these changes on the target proteins of PKA regulation may be quite distinct and are incompletely understood. In the case of the L-type Ca2+ channel, blunted ß-AR stimulation of ICa,L in HF has been observed,12,13 but relatively little is known about the underlying mechanisms.
Changes in the properties of L-type Ca2+ channels have also been seen in the canine tachycardia–induced cardiomyopathy model and human HF, with a decreased density of channels being detected and apparent compensatory changes in channel gating, resulting in unchanged macroscopic current density.12,14,15 The alterations in L-type Ca2+ channels, ß-AR, and other proteins in HF are associated with substantial subcellular remodeling, such as a decrease in density of T-tubules.14,16,17 Because localized regulation of L-type Ca2+ channels by ß-AR signaling may be critical,18,19 we hypothesized that alterations in the subcellular distribution of ß-AR subtype relative to L-type Ca2+ channels may contribute to impaired coupling. We secondly hypothesized that a part of the blunted ß-AR regulation of ICa,L is attributable to the altered regulation of the channel by ß2-ARs with increased coupling to Gi. Therefore, the purposes of the present work were (1) to evaluate changes in the relative distribution of ß1-ARs and ß2-ARs in surface and T-tubular sarcolemma; (2) to determine the functional regulation of ICa,L by ß-AR subtype–specific stimulation; and (3) to evaluate for crosstalk between ß1-AR and ß2-AR in the regulation of L-type Ca2+ channels.
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Materials and Methods |
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Materials
All reagents were purchased from Sigma Chemical (St Louis, Mo) unless otherwise stated. (+/–)isoproterenol (ISO) and [-]-norepinephrine (NE) were freshly made in H2O before each experiment. ICI118,551 (ICI) and carbachol (CCh) stock solutions were prepared in H2O. Prazosin (Praz) stock solution was dissolved in methanol. Atenolol and salbutamol (Salb) stock solutions were dissolved in DMSO. All stock solutions were stored at –20°C. The stock solutions were freshly diluted in bath solution immediately before experimental recording. Ascorbic acid (30 µmol/L) was added together with all other drugs in the bath to prevent oxidation of drugs tested. The final concentrations of methanol (0.01%) and DMSO (<0.1%) have no effect on ICa,L in control experiments (data not shown).
Pacing-Induced HF and Isolation of Canine Ventricular Myocytes
HF was induced with rapid ventricular pacing at 220 to 250 bpm for 4 to 5 weeks in adult mongrel dogs as previously described.20 The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). Dogs were obtained from Twin-Valley Kennel, Spring Green, Wis. Myocytes were isolated from 17 control dogs and 15 failing dogs using enzymatic digestion with collagenase (1 mg/mL, Worthington Type II) plus hyaluronidase (0.5 mg/mL, Sigma Type I-S), as described elsewhere.21
ß-AR Radioligand Binding
Membrane fractions highly enriched in sarcolemma were prepared from canine ventricular muscle as previously described17 and were then used for radioligand binding using [125I]iodocyanopindolol ([125I]CYP; New England Nuclear, Boston, MA), a nonselective high-affinity ß-AR antagonist using vacuum filtration method with a 24-well harvester (Brandel, Gaithersburg, Md), as described previously.22 One micromole per liter (-)propranolol was used to determine total and nonspecific binding. Saturation binding data were fit to a single site-binding model, and competition-binding curves were fit to single- or 2-site competition models using nonlinear regression analysis.
Electrophysiological Recordings
Isolated ventricular myocytes were placed in the experimental chamber mounted on the stage of an inverted microscope and studied using the ruptured whole-cell configuration of the patch clamp technique at 22 to 23°C. In a subset of experiments, myocytes were treated for 3 hours with 2 µg/mL pertussis toxin (PTX) at 37°C to inactivate Gi.10,23,24 Patch pipette solution consisted of (in mmol/L) 90 Cs-glutamate, 20 CsCl, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate, 5 MgATP, and 10 HEPES (pH 7.2). The bath solution to measure L-type Ca2+ channel currents consisted of (in mmol/L) 140 tetraethylammonium-Cl, 1 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.4). Myocytes were held at a membrane potential of –80 mV, and 200-ms depolarizations to +20 mV at 10-mV step were applied at 20-s intervals to evaluate the effect of pharmacological interventions on ICa,L using whole-cell patch clamp techniques, as described previously.14
Statistics
All values are presented as mean±SEM. Statistical significance was evaluated by the paired or unpaired Student’s t test (2 tail) or ANOVA followed by Student–Newman–Keuls test when appropriate. Values of P<0.05 were considered statistically significant. Microsoft Excel 2000 (Redmond, Wash), WaveMetrics Igor 4.0 (Lake Oswego, Ore), and Microcal Origin 6.0 (Northampton, Mass) were used for data analysis and figure plotting.
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Results |
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Blunted ß-Adrenergic Stimulation of ICa,L With ISO in HF
We first used the ruptured whole-cell patch clamp technique to assess the effect of nonselective ß-AR stimulation (ISO) on ICa,L in control and tachycardia-induced failing canine ventricular myocytes. Representative current traces as well as the peak ICa,L are plotted in response to test pulses to +20 mV as a function of time in Figure 1A and 1B, and the effect of serial exposure to increasing concentrations of ISO, was assessed. In control myocytes, ICa,L is dramatically increased by 332±35% on superfusion of 0.1 µmol/L ISO (n=11, P<0.001). In contrast, ICa,L recorded from myocytes of failing dogs showed only a 129±20% (n=7) increase in response to 0.1 µmol/L ISO. Testing higher concentrations of ISO (1.0 and 10 µmol/L) showed no additional increase in ICa,L (Figure 1C). These results demonstrate that the maximal stimulation of ICa,L by the nonselective ß-AR agonist ISO is markedly reduced in HF. This blunted response of ICa,L to ISO is similar to that observed in patients with HF and earlier studies in animal models.12,13,15
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Experiments testing ß2-AR activation alone using the ß2-AR agonist Salb (10 µmol/L) in the presence of the ß1-AR antagonist atenolol (1.0 µmol/L) revealed no effect on ICa,L in either control (ICa,L at 20 mV, basal 5.3±0.6 pA/pF versus Salb+atenolol 5.1±0.6 pA/pF, n=3; P=not significant) or failing cells (ICa,L at 20 mV, basal 4.8±0.5 pA/pF versus Salb+atenolol, 4.6±0.4 pA/pF, n=4; P=not significant). A lack of effect of ß2-AR regulation of basal ICa,L in normal canine ventricular myocytes has been observed by some investigators,25 but others have suggested that ß2-AR activation can increase ICa,L in normal canine myocytes.26 Nevertheless, under our conditions, the present results indicate that ß1-AR signaling is primarily responsible for the increase in ICa,L observed in response to nonselective ß-AR stimulation with ISO in both control and failing myocytes.
Downregulation of Surface and T-Tubular Sarcolemma ß1-AR in HF
Previous studies have determined that ß1-ARs are downregulated in HF, but it is unknown whether changes in the subcellular distribution of ß-ARs further contribute to the impaired regulation of ICa,L in HF. We examined the abundance of ß1-AR and ß2-AR in control and failing dogs in membranes enriched in surface sarcolemma (FI) and T-tubular sarcolemma (FII). Equilibrium binding studies revealed that nonselective ß-AR antagonist [125I]CYP bound with comparable affinities in control and failing hearts with KD values of 0.058±0.01 and 0.056±0.02 nmol/L, respectively, for FI membranes, and comparable results were found with FII membranes (see Table I in the online data supplement available at http://circres.ahajournals.org). We determined the relative proportion of ß1-AR and ß2-AR by performing competition assays with the ß1-AR antagonist atenolol and the ß2-AR antagonist ICI (Figure 2 and online Table II). Atenolol, over the concentration range tested, displaced [125I]CYP bound to ß1-AR and was fit with a single-site displacement curve (Figure 2A and 2D and online Table II). In contrast, ICI displaced both a high-affinity component and a low-affinity component correlating with ß2-AR and ß1-AR, respectively, and the data were fit to a 2-site displacement curve (Figure 2B and 2E and online Table II). Both competition strategies revealed a significant decrease in the density of ß1-AR with HF and no significant change in the density of ß2-AR. Average data for ICI competition are displayed in Figure 2C and 2F and show that ß1-AR receptors are decreased in both the surface sarcolemma-enriched FI and the T-tubular–enriched FII by 55±4% (n=7, P<0.001) and 45±10% (n=7, P<0.01), respectively. Furthermore, the results suggest that the ß1-AR and ß2-AR have distinct subcellular distributions, with ß1-AR showing the greatest density in FI (surface sarcolemma enriched) and less binding in FII (T-tubular sarcolemma enriched) in contrast to the ß2-AR, which shows comparable binding in FI and FII. These results confirm previous studies showing a selective downregulation of ß1-AR in HF, and the present study extends these results to show that the decrease in ß1-AR density is present in both surface and T-tubular sarcolemma membrane fractions.
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, n=7) and failing (
, n=7) canine left ventricles. C and F, Average data for calculated density of ß1-AR and ß2-AR in FI (C) and FII (F) membranes from control and failing hearts. **P<0.01, ***P<0.001 compared with control.
ß2-AR Antagonist Unmasks Stimulation of ICa,L With ISO in HF
Although a decrease in ß1-AR density could explain the reduced ability of ISO to stimulate ICa,L in HF, a variety of confounding factors, such as spare receptors and alterations in coupling of the ß-ARs to downstream signaling molecules, raise the possibility of more complex alterations. Because ISO is a nonselective ß-AR agonist, we used additional pharmacological probes to understand receptor subtype–specific roles. The effect of the ß2-AR–selective antagonist ICI on ISO stimulation of ICa,L was tested in both control and failing myocytes (Figure 3). ICI alone has no significant effect on ICa,L in control and failing myocytes, suggesting that there was no significant agonist-free activation of the receptor. In control myocytes, application of 1 µmol/L ISO in the continued presence of ICI increased in ICa,L (352±26%, n=14) to a comparable degree compared with ISO effect in the absence of ICI (332±33%, n=8). However, in failing cells, ICI unmasked a significantly larger stimulatory effect by 1 µmol/L ISO on ICa,L (325±36%) than without ICI pretreatment (179±22%, n=7, P<0.01). These results suggest that specifically in failing, but not control ventricular myocytes, ß2-AR activation blunts the ß1-AR stimulation of ICa,L.
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Activation of ß2-AR Inhibits ICa,L Stimulated by ß1-AR Agonist in HF
We performed a complementary experiment examining the regulation of ICa,L by specific ß1-AR stimulation using NE with Praz to inhibit 1-AR. Then we superfused the ß2-AR agonist Salb in the continued presence of ß1-AR stimulation, as shown in Figure 4. In control canine myocytes, NE (1 µmol/L)+Praz (1 µmol/L) increased ICa,L 325±23% (n=7) comparable to the effect of ISO on control myocytes (332±35%, n=11). In failing myocytes, NE+Praz increased ICa,L to a similar extent as observed in control myocytes (365±51%, n=6). Superfusion of 10 µmol/L Salb in the continued presence of NE+Praz had no effect in control myocytes, but Salb resulted in an inhibition of NE+Praz–stimulated ICa,L in failing myocytes (183±25% increase relative to basal, n=6, P<0.01). These results show that the response of ICa,L to specific ß1-AR stimulation is not significantly impaired in this model of HF, but the opposing action of ß2-AR stimulation blunts the ß1-AR effect when the receptors are simultaneously activated.
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Inhibitory Action of ß2-AR in HF Requires Gi
Because an increased abundance of Gi protein has been identified in various models of HF,9,27 and because ß2-AR are known to be capable of coupling with Gi,19,28 we tested the impact of PTX inactivation of Gi on ß-AR regulation of ICa,L (Figure 5). First, we demonstrated that PTX pretreatment is effective by testing the ability of CCh, a muscarinic receptor agonist acting via Gi, to inhibit ISO-stimulated ICa,L. Figure 5A and 5B shows that 200 µmol/L CCh inhibited ISO-stimulated ICa,L in both control (n=3) and failing cells (n=6), but these effects were abolished in PTX-treated control (n=8) and failing (n=7) cells (Figure 5C and 5D). Thus, PTX treatment functionally inactivates Gi-mediated regulation of ICa,L in both control and failing myocytes. Inactivation of Gi by PTX pretreatment in control myocytes did not alter the effect of 1 µmol/L ISO on ICa,L (compared in Figure 5E). However, the response of failing cells to 1 µmol/L ISO was significantly greater with PTX treatment than without (463±36%, n=7 and 129±49%, n=6; P<0.05). These results demonstrate that, specifically in failing myocytes, Gi-activated pathways serve to blunt the response of ICa,L to nonspecific ß-AR stimulation with ISO, and this suggests that ß2-AR–mediated inhibition of ß1-AR stimulation of ICa,L in failing cells occurs via Gi.
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To test directly whether PTX pretreatment in failing myocytes altered the ß2-AR–mediated blunting of ß1-AR stimulation of ICa,L, we stimulated ICa,L with NE+Praz (ß1-AR stimulation) and then added the ß2-AR agonist Salb (Figure 6). In PTX-pretreated control myocytes, 1 µmol/L NE+1 µmol/L Praz strongly stimulated ICa,L (374±31%, n=7; Figure 6A and 6C), and no effect of Salb was observed. These results were similar to that seen in non–PTX-pretreated myocytes (compare to Figure 4). PTX effectively inactivated Gi in these experiments as CCh did not inhibit the NE+Praz–stimulated ICa,L. In failing myocytes pretreated with PTX, NE+Praz stimulated ICa,L to a similar extent as in control myocytes (383±62%, n=6); however, 10 µmol/L Salb failed to inhibit the effect of NE+Praz on ICa,L (Figure 6B). Thus, inactivation of Gi in the failing myocytes prevented ß2-AR–mediated inhibition of ß1-AR stimulation of ICa,L.
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Discussion |
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The ability of the nonselective ß-AR agonist ISO to stimulate ICa,L in canine HF is markedly reduced, and this is accompanied by a downregulation of specifically ß1-ARs in both surface and T-tubular membrane fractions. Using ß-AR subtype–specific agonists and antagonists, we demonstrated that the major part of the blunted response to ISO in failing cells is attributable to ß2-AR–induced inhibition of the ß1-AR stimulation of ICa,L. Studies using PTX have revealed that ß2-ARs act via Gi in failing cells to blunt the ß1-AR response. This form of crosstalk between ß1-AR and ß2-AR was not observed in control myocytes and suggests that, despite an unchanged density of ß2-AR in failing hearts, there are important alterations in the coupling to downstream signaling molecules. Furthermore, the present results suggest that previous studies of ß-AR desensitization in HF using nonselective ß-AR agonists (eg, ISO) could have been complicated by crosstalk between ß-AR subtypes.
Gi-Mediated Crosstalk Between ß2-AR and ß1-AR Receptors
The interaction of sympathetic and parasympathetic inputs to precisely regulate cardiac function is an important example of crosstalk between signaling pathways at the whole organ level. The concept of accentuated antagonism was first used by Levy to describe the phenomenon of vagal stimulation reducing heart rate to a greater extent in the presence of elevated sympathetic tone.29 Studies at the cardiomyocyte level measuring ICa,L have refined the idea of accentuated antagonism to describe the M2-muscarinic–mediated inhibition of ICa,L, which occurs to a greater extent or is only observed in the presence of ß-AR stimulation of ICa,L.30 The M2-muscarinic receptors are coupled via Gi or Go to exert this inhibitory effect.31,32 Initially, it was proposed that a simple inhibition of AC by Gi relative to the Gs-mediated AC stimulation explained the competing effects of muscarinic and ß-AR stimulation on ICa,L33; however, there are other possible mechanisms including the activation of phosphatases or NO-induced activation of cGMP-dependent phosphodiesterases.34,35
The possibility that ß2-AR stimulation can oppose the ß1-AR/Gs–mediated stimulation of AC has been anticipated since the original observations that the ß2-AR can variably couple to Gs and Gi.19,28 Opposing effects of ß1-AR and ß2-AR stimulation have been described in the regulation of cell survival and apoptosis in the heart.36,37 Opposing effects of Gs- and Gi-coupled ß2-AR in myocytes on contraction, Ca2+ transients, and ICa,L have been extensively investigated in rat ventricular myocytes, but the present work describes a new paradigm where ß2-AR coupled to Gi can inhibit ß1-AR stimulation of ICa,L, analogous to previously described examples of accentuated antagonism. This finding bares similarity to a recent study using rat ventricular myocytes that specifically, when overexpressing the Na/Ca exchanger, exhibited ß2-AR/Gi–mediated inhibition of ß1-AR stimulation of contraction.38
Altered coupling of ß2-AR with Gi in the setting of HF observed in this study is consistent with the increased abundance of Gi found in failing human hearts and in this animal model of HF.9,11 In addition, agonist-mediated conversion of ß2-AR from Gs coupling to Gi coupling may be particularly relevant in the failing heart, where persistent elevations in adrenergic signaling are present. The ability of PTX treatment to partially restore the blunted contractile response to nonspecific ß-AR stimulation has been observed previously in a rat myocardial infarction HF model and in myocytes from failing human heart.23,39 More recently, the role of Gi signaling on ß-AR subtype–specific regulation of contraction was examined in the end-stage SHR rat model of HF, and Xiao et al10 found evidence for increased Gi signaling by ß2-AR activation, negating the positive inotropic effect of ß2-AR stimulation in the failing hearts, but the ß2-AR-Gi pathway did not impact the ß1-AR–positive inotropic effect, in apparent contrast to our results. These apparently conflicting results could be attributable to differences in species studied, HF model, or parameters measured.
Subcellular Localization and Compartmentalization of ß-AR Signaling
The impact of HF on ß-AR regulation of ICa,L may also be affected by changes in subcellular localization of signaling molecules and channels. At the level of the surface sarcolemma and T-tubule sarcolemma, we found that the percentage decrease in ß1-ARs was comparable in both surface and T-tubular sarcolemma fractions; however, this does not exclude alterations in distribution on a smaller scale. For example, ß2-ARs are preferentially localized to caveolae in heart cells according to some investigations, and this membrane pool is not resolved by the present membrane fractionation studies.40 ß2-AR regulation of AC and downstream L-type Ca2+ channels is highly compartmentalized to the sarcolemma relative to the more diffuse ß1-AR regulation.19 Scaffold proteins play a critical role in enabling efficient and localized signaling by bringing the needed molecules in the signaling cascade together. In addition, in rat brain, the ß2-AR was found to be associated directly with Cav1.2 in a macromolecular signaling complex.18 Therefore, the precise localization and composition of macromolecular signaling complexes could easily be altered in the failing heart. In particular, it is striking that in HF, the typically localized ß2-AR signaling impacts what has previously been defined as a more diffuse ß1-AR regulation of ICa,L.
Clinical Implications and Future Directions
There are potential clinical implications of the present study for human HF, where ß-AR agonists and antagonists play a prominent role in therapy. For example, short-term inotropic support of failing hearts typically uses relatively selective ß1-AR agonists such as dobutamine and dopamine rather than the nonselective ß-AR agonist ISO. This clinical preference has developed based on many factors, but it is possible that a greater efficacy of ß1-AR stimulation in the absence of ß2-AR stimulation is partly responsible. The present study does not address the effects of long-term ß-AR–receptor stimulation or blockade in HF. For example, recent studies have pointed to the potentially beneficial effect of long-term ß2-AR–specific stimulation in the failing heart in reducing apoptosis and cardiac remodeling.37,41 Such long-term effects improving overall contractility with ß2-AR agonists may seem in conflict with the short-term inhibition ß1-AR–mediated stimulation of ICa,L by ß2-AR signaling in failing myocytes, but a reduction of Ca2+ influx may be beneficial in the long-term for cell survival and, thus, overall cardiac function. Furthermore, alterations in a wide variety of signaling molecules in HF add significant complexity, which makes simple extrapolation of the present results to clinical HF difficult.
The downstream signaling pathways from Gi responsible for the ß1-AR and ß2-AR crosstalk are unknown. Does Gi act to directly blunt AC activation or are alternative pathways active, such as the phosphatidylinositol 3-kinase pathway leading to activation of NO synthase-3, important, as suggested by some studies in other cardiac preparations?24,42 The role of NO is of particular interest, given accumulating evidence that NO is critically involved in the reduced effect of ß-AR stimulation in the failing heart.43 There are many steps in the ß1-AR regulation of ICa,L that could potentially be modulated by NO, but future studies will be necessary to evaluate HF-induced alterations in this signaling. Could changes in the colocalization of the critical downstream molecules and ß-AR be involved? Ultimately, unraveling the complexities of ß-AR signaling in the failing heart will provide new opportunities to refine therapy.
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Acknowledgments |
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This work was supported by NIH/NHLBI grants R01 HL61537 (to T.J.K. and R.A.H.) and RO1 HL61534 (to R.A.H. and T.J.K.). The technical support in preparation of the canine model and myocyte isolation by Larry F. Whitesell, Jennifer Buck, and Kathy Potter is gratefully acknowledged. Excellent assistance with manuscript preparation by Thankful Sanftleben is acknowledged.
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Footnotes |
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Original received December 29, 2004; revision received February 25, 2005; resubmission received July 7, 2005; revised resubmission received July 28, 2005; accepted August 1, 2005.
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