Published online before print May 8, 2008, doi: 10.1161/CIRCRESAHA.107.169011
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© 2008 American Heart Association, Inc.
Cellular Biology |
Arrhythmogenic Effects of β2-Adrenergic Stimulation in the Failing Heart Are Attributable to Enhanced Sarcoplasmic Reticulum Ca Load
From the Department of Pharmacology, University of California-Davis (J.D., D.M.B.), Department of Medicine, University of Illinois at Chicago (G.A., M.I.), Department of Physiology, Ohio State University (M.T.Z.), and Department of Medicine, Division of Cardiovascular Disease, University of Alabama at Birmingham (X.A., S.M.P.).
Correspondence to Steven M. Pogwizd, MD, Cardiac Rhythm Management Laboratory, University of Alabama at Birmingham, 1530 3rd Avenue South, Volker Hall-B140, Birmingham, AL 35294. E-mail spogwizd{at}uab.edu
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Abstract |
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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 β2-adrenergic receptor (β2-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies. Intravenous zinterol (2.5 µg/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 β2-AR antagonist ICI-118,551 (0.2 mg/kg). In field-stimulated myocytes (0.5 to 4 Hz, 37°C), β2-AR stimulation (1 µmol/L zinterol+300 nmol/L β1-AR antagonist CGP-29712A) induced aftercontractions and Ca aftertransients in 88% of HF versus 0% of control myocytes (P<0.01). β2-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 β-AR expression was reduced by 47% in HF rabbit left ventricle, β2-AR number was unchanged, indicating more potent β2-AR–dependent SR Ca uptake and arrhythmogenesis in HF. Human HF myocytes showed similar β2-AR–induced aftercontractions, aftertransients, and enhanced Ca transient amplitude, SR Ca load and twitch [Ca]i decline rate. Thus, β2-AR stimulation is arrhythmogenic in HF, mediated by SR Ca overload-induced spontaneous SR Ca release and aftercontractions.
Key Words: heart failure • β2-adrenergic stimulation • arrhythmia • sarcoplasmic reticulum • zinterol
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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Heart failure (HF) is associated with significant mortality, with nearly one-half of deaths occurring suddenly, primarily from ventricular tachycardia (VT) to ventricular fibrillation.1,2 In 3D cardiac mapping studies, both in an arrhythmogenic rabbit model of nonischemic HF3 and in the failing human heart,4 we have shown that VT initiates by a nonreentrant mechanism that may represent delayed afterdepolarizations (DADs), attributable to a transient inward current (Iti), or early afterdepolarizations (EADs).5
It is well known that arrhythmogenesis in the failing heart is enhanced by stimulation of β-adrenergic receptors (β-ARs).1,5 We recently showed that β-adrenergic stimulation with isoproterenol induced ventricular arrhythmias in vivo in HF but not control rabbits.5 Moreover, isoproterenol induced DADs and aftercontractions (ACs) by increasing sarcoplasmic reticulum (SR) calcium load, leading to spontaneous SR Ca release and activation of Iti (that underlies DADs and nonreentrant VT). We found that preserved β-AR responsiveness, as may occur in HF that is not end-stage,6 is an important contributor to arrhythmogenicity in HF myocytes.5
Isoproterenol stimulates β2- as well as β1-ARs; so, in HF, where there is β1-AR downregulation but relatively preserved β2-ARs,6 some of the arrhythmogenic effects of isoproterenol could arise from β2-AR stimulation. In fact, there is evidence of enhanced β2-AR responsiveness in HF. For instance, Altschuld et al7 have shown that β2-AR stimulation with zinterol can enhance Ca transients in HF myocytes to a greater degree than in controls (consistent with enhanced arrhythmogenesis). Although the second messenger pathways of β2-AR stimulation appear to differ from that of β1-AR stimulation,8 there is evidence in failing human myocardium that β2-AR stimulation can elicit localized increases in cAMP and phosphorylation of phospholamban (PLB) that may contribute to SR Ca overload.9
To determine whether stimulation of β2-AR per se may be arrhythmogenic in HF, we assessed the response of β2-AR stimulation on arrhythmogenesis in vivo and on intracellular Ca transients, SR Ca content, and Ca current (ICa) in ventricular myocytes from control rabbits and from an arrhythmogenic rabbit model of HF induced by combined aortic insufficiency and aortic constriction. These studies were complemented by molecular and biochemical studies of β-ARs and phosphorylation of PLB and were extended to human HF by selected experiments performed with isolated myocytes from human hearts. We found that in HF, β2-AR stimulation is arrhythmogenic by enhancing spontaneous SR Ca release and ACs and likely attributable to enhanced SR Ca load secondary to PLB phosphorylation.
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Materials and Methods |
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An expanded Methods section is available in the online data supplement at http://circres.ahajournals.org.
Rabbit Heart Failure Model and Myocyte Isolation
In New Zealand white rabbits of either sex, HF was induced by aortic insufficiency and 2 to 4 weeks later by abdominal aortic constriction (both during isoflurane anesthesia) as previously described.3 Progression of HF was assessed by 2D echocardiography.3,5,10 HF rabbits were studied when left ventricular (LV) end-systolic dimension exceeded 1.40 cm.3,5,10 At that stage, IV bolus administration of zinterol (1.0 or 2.5 µg/kg over 5 seconds) with or without the β2-AR blocker ICI-118,551 (0.2 mg/kg) was performed in conscious control and HF rabbits with monitoring of the surface ECG for at least 3 minutes. Protocols were approved by the University of Illinois at Chicago Animal Studies Committee. Rabbit LV myocytes were isolated as described,5,10 with back flow across the incompetent aortic valve in HF rabbits blocked by a balloon-tipped catheter inflated in the LV outflow tract.
Contraction, [Ca]i, and Patch Clamp
Ventricular myocytes were stored at 22°C and plated on laminin-pretreated glass-bottomed chambers. Cells were loaded with fluo-3-acetoxymethyl ester, and [Ca]i was measured as previously described.11 SR Ca load was determined by rapid caffeine (10 mmol/L) application after 20 stimulated pulses.5 Myocyte shortening was measured by video edge detection. The normal Tyrode (NT) solution contained (in mmol/L): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 5 HEPES, pH 7.4. Myocytes were field-stimulated (0.5 to 4 Hz, 20 beats, 37°C), followed by 10 seconds of observation for the presence of ACs in the absence or presence of the β2-AR agonist zinterol (300 nmol/L or 1 µmol/L; generous gift from Bristol-Myers Squibb) with or without the β1-AR antagonist CGP 20712A (300 nmol/L; Sigma) or ICI-118,551 (100 nmol/L; TOCRIS).
In other studies, ruptured patch voltage clamp was done to measure L-type ICa with or without similar doses of zinterol and CGP with pipettes containing (mmol/L) CsOH 110, CsCl 20, EGTA 5, MgCl2 2, aspartic acid 100, HEPES 5, Mg-ATP 2, Na3GTP 0.1, pH 7.2, 25°C. Membrane capacitance was measured from responses to 5-mV hyperpolarizing and depolarizing pulses.12
Phosphorylation of PLB
Freshly isolated control and HF rabbit myocytes were incubated for 10 minutes with zinterol (1 µmol/L), zinterol plus CGP 20712A (300 nmol/L) or zinterol plus CGP plus ICI-118,551 (100 nmol/L). Cell pellets were then spun down and homogenized for protein quantification and Western blotting.
β-AR Assays and Competitive Binding Studies
Saturation binding studies were carried out on LV homogenates by incubation with various concentrations (0 to 500 pmol/L) of the β-AR antagonist [125I]-(-)iodocyanopindolol as previously described.13 The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were determined by Scatchard analysis using GraphPad Prism. For competitive binding studies, homogenates were incubated with 500 pmol/L 125I-CYP plus increasing dilutions of 1 µmol/L ICI-118,551, a selective β2-AR antagonist, or 100 nmol/L CGP 20712A, a selective β1-AR antagonist. Results were adjusted to femtomoles per milligram protein to calculate for the specific binding, and determine the percentage of β1 and β2 receptors.
Human HF LV Myocytes
Human HF LV myocytes were isolated from LV tissue obtained at the time of clinically indicated cardiac transplantation in patients with end-stage idiopathic dilated cardiomyopathy (n=3) or ischemic cardiomyopathy (n=3) performed at Loyola University Hospital and the University of Illinois at Chicago Hospital. This protocol has been approved by the Human Studies Committees of Loyola and University of Illinois at Chicago.
Data Analysis
Data are shown as means±SEM, and statistical significance was based on P<0.05 (Student t test, ANOVA and 
2).
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Results |
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HF Rabbit Preparation
HF was induced in 27 rabbits. After an average of 8.0±1.4 months following combined aortic regurgitation and aortic constriction, rabbits developed progressive cardiac enlargement and decreased systolic function. LV end-diastolic dimension increased by 41% (1.51±0.03 to 2.13±0.03 cm, P<0.001) and LV end-systolic dimension increased by 60% (0.98±0.02 to 1.57±0.03 cm, P<0.001), whereas fractional shortening decreased by 25% (34.9±1.1 to 26.1±0.95%, P<0.001).
In Vivo Arrhythmogenic Effects of β2-AR Agonist Zinterol
To determine whether stimulation of β2-ARs may contribute to the arrhythmogenic effects of isoproterenol, we performed in vivo drug infusion studies in 5 control and 6 HF rabbits. Zinterol (2.5 µg/kg IV bolus over 5 seconds) did not significantly alter heart rate or mean arterial blood pressure in either control or HF rabbits. Figure 1 shows that zinterol at this dose led to ventricular arrhythmias including premature ventricular complexes and runs of VT (up to 13 beats long) in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect that was blocked by the β2-AR antagonist ICI-118,551 (0.2 mg/kg). Zinterol at a lower dose (1 µg/kg IV, n=4) did not induce ventricular arrhythmias in HF rabbits.
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In Vitro Arrhythmogenic Effects of Zinterol
To determine the underlying cellular mechanism of ventricular arrhythmias induced by zinterol, we measured contractions in isolated LV myocytes from control and HF rabbits. Field stimulation (0.5 to 4 Hz) of fluo-3–loaded myocytes (37°C) demonstrated that zinterol (1 µmol/L) induced ACs in 7 of 7 HF myocytes, compared to 0 of 6 controls (P<0.01), an effect that was blocked by ICI-118,551 (100 nmol/L, Figure 1, bottom). These ACs were associated with spontaneous SR Ca release or aftertransients. To verify that the effects of zinterol were attributable to stimulation of β2-ARs, additional β2-AR stimulation studies were performed with 1 µmol/L zinterol plus 300 nmol/L of the β1-AR blocker CGP-20712A (CGP). Zinterol plus CGP induced ACs (associated with aftertransients, Figure 2) in 7 of 8 HF myocytes, compared to 0 of 7 controls (P<0.01), an effect that was blocked by 100 nmol/L ICI-118,551. Similar results were found in HF and control myocytes on glass slides without laminin, ruling out potential effects of laminin on β2-AR signaling.14 Lower concentrations of zinterol (300 nmol/L) with CGP failed to induce aftertransients in 4 HF myocytes, consistent with a dose-dependent specific effect.
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Effects of β2-AR Stimulation on Cell Shortening, Ca Transients, and SR Ca Load
Zinterol plus CGP had no effects on cell shortening in control myocytes (at 1 or 3 Hz; Figure 3C). However, this β2-AR stimulation significantly increased cell shortening in HF myocytes (42% and 41% increase at 1 and 3 Hz, respectively, n=7, P<0.05; Figure 4C).
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In control rabbit myocytes, β2-AR stimulation (zinterol plus CGP) did not increase Ca transient amplitude or SR Ca load (assessed by caffeine contractures) at either 1 or 3 Hz stimulation frequency (Figure 3A and 3B). However, in HF myocytes, β2-AR stimulation significantly increased Ca transient amplitude and SR Ca load at 1 Hz (1.28±0.05 versus 0.99±0.06 and 1.82±0.06 versus 1.42±0.06 
F/Fo, n=12, P<0.05, Figure 4A and 4B). Similar effects were seen at 3 Hz (Figure 4A and 4B). All of these effects were blocked by the β2-AR blocker ICI-118,551 (Figure 4B). Thus, β2-AR stimulation enhances SR Ca content and Ca transients and contractions in HF, but not control myocytes, and this enhancement of SR Ca load and spontaneous SR Ca release may be arrhythmogenic in HF by enhancing ACs and afterdepolarizations.5
To assess the effects of β2-AR stimulation on SERCA and Na/Ca exchanger (NCX) function, we measured the rate of [Ca]i decline during twitch and caffeine-induced Ca transients, respectively (Figure 5). Zinterol plus CGP had no effect on 
of twitch [Ca]i decline in control myocytes but significantly accelerated twitch [Ca]i decline in HF myocytes (156±11 versus 199±12 ms at 1 Hz, 104±8 versus 125±8 ms at 3 Hz, n=12, n=12, P<0.05). Zinterol plus CGP had no effect on the of Ca decline of the caffeine transient in either HF or control myocytes, suggesting unaltered NCX (Figure 5).
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β2-AR Effects on Ca Current in HF
Figure 6A shows that in control myocytes, zinterol (1 µmol/L) increased ICa (eg, by 
40% for Em between –10 and +20 mV). This is much smaller than the 300% increase in ICa that we previously observed with 1 µmol/L isoproterenol (combined β1- and β2-AR activation)5 in this same HF myocyte preparation. However, when we repeated the ICa measurements in control myocytes on zinterol challenge in the presence of the β1-AR antagonist CGP, zinterol did not change ICa. This implies that the modest ICa stimulation by zinterol alone in control myocytes in Figure 6A is likely attributable to a minor crosstalk from the extremely potent β1-AR–mediated effect on ICa. In HF myocytes, ICa was unaltered by zinterol (Figure 6B). We conclude that β2-AR does not appreciably alter ICa in either control or HF rabbit myocytes.
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β2-AR Effects on PLB Phosphorylation
We assessed the effects of β2-AR stimulation on PLB phosphorylation at Ser16 (protein kinase A site) and Thr17 (Ca/calmodulin kinase [CaMKII] site). At baseline, HF myocytes showed decreased Ser16 and increased Thr17 phosphorylation, as we have previously demonstrated.15 Control and HF rabbit myocytes were exposed to 1 µmol/L zinterol plus 300 nmol/L CGP 20712A in the presence or absence of 100 nmol/L ICI-118,551. HF (but not control) myocytes exhibited a 78% increase in PLB phosphorylation at the Ser16 (protein kinase A [PKA]) site (P<0.05) but no significant change at the Thr17 (CaMKII) site, which was already elevated in HF (n=5, n=4, Figure 7A). The slight tendency for zinterol-induced increase in Thr17 phosphorylation in HF (not significant) could be secondary to the zinterol-induced enhancement of Ca transients, which was seen only in HF myocytes.
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β2-AR Density and Subtype Distribution in HF
Based on β-AR binding assays, HF rabbits LV exhibit a 47% downregulation of total β-ARs (P<0.05, Figure 7B), and our results here in LV homogenates are nearly identical to those reported using LV membrane preparations from a similar HF rabbit model,16 as well as in the failing human heart.6 In control rabbit LV, β2-ARs represent only 8% of total β-ARs (similar to Brodde et al,17). With HF, there is a 50% downregulation of β1-AR, but the amount of β2-ARs is unaltered in HF. This implies that the prominent β2-AR–mediated SR Ca uptake and arrhythmogenic effects are attributable to altered coupling of β2-AR to the PKA-dependent SR effects, rather than increased β2-AR number or effects on ICa.
Human HF LV Myocytes
Studies in rabbit myocytes were validated by experiments in human myocytes from 6 patients with end-stage HF (3 ischemic and 3 nonischemic) at the time of cardiac transplantation. Human HF hearts exhibited severely depressed LV systolic function with a mean LV ejection fraction of 18±5%. Four of the 6 HF patients had an implantable defibrillator for documented VT and 3 were known to be taking β-AR blockers. No apparent differences were seen among myocytes from the different HF hearts, so data from these 6 failing hearts were pooled.
In HF human myocytes, zinterol (1 µmol/L) plus CGP (300 nmol/L; 1 Hz, 37°C) significantly increased cell shortening (10.7±3.0 versus 4.2±0.9, n=6, P<0.05) and induced ACs in 6 of 6 cells (eg, see Figure 8A, middle). In human HF myocytes loaded with fluo-3 (37°C; Figure 8), zinterol plus CGP significantly increased both Ca transient amplitude (3.34±0.72 versus 1.48±0.18 F/Fo, n=6, P<0.05, Figure 8B) and SR Ca load (assessed by caffeine Ca transient, 4.82±0.60 versus 3.94±0.44 F/Fo, n=5, P<0.05, Figure 8B). Moreover, zinterol plus CGP enhanced the rate of twitch [Ca]i decline ( of 306±26 versus 436±85 ms, n=6, P<0.05, Figure 8C), consistent with enhanced SERCA activity, whereas the rate of [Ca]i decline of the caffeine contracture was unchanged (indicating unaltered NCX function after β2-AR). All of these effects of zinterol plus CGP were reversed by the β2-AR antagonist ICI-118,551.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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We found that β2-AR stimulation is arrhythmogenic in the failing heart. In conscious HF rabbits (but not controls), infusion of the β2-AR agonist zinterol induces ventricular arrhythmias that are prevented by the β2-AR antagonist ICI-118,551. In HF (but not control) rabbit myocytes, zinterol plus the β1-AR antagonist CGP-29712A induces ACs, as well as increased Ca transient amplitude, SR Ca load, and PLB phosphorylation. This increase of SR Ca content in HF is not attributable to changes on ICa or NCX activity but increased SR Ca pump rate (indicated by acceleration of twitch Ca decline). Similar results were found in human failing myocytes. Thus, β2-AR stimulation enhances arrhythmogenesis in HF by increasing SR Ca load and consequent spontaneous SR Ca release (presumably via DADs).
In Vivo Arrhythmogenesis
We assessed the in vivo arrhythmogenic effects of β2-AR stimulation in our well-characterized arrhythmogenic rabbit HF model.3,5,10 This HF model of combined aortic insufficiency and aortic constriction is ideally suited for these studies for a number of reasons. We previously found that HF rabbits exhibit spontaneously occurring VT in vivo that initiates by a nonreentrant mechanism3 and that these arrhythmias are enhanced by β-AR stimulation with isoproterenol.5 Moreover, HF rabbits have an 10% incidence of sudden cardiac death.5,10
Zinterol infusion led to spontaneous ventricular arrhythmias and VT in HF but not control rabbits. Although zinterol has predominantly β2-AR agonist effects, at high doses it might stimulate β1-ARs. However, we think these effects are β2-AR–mediated because there were no changes in heart rate (as expected for β1-AR effects) and we were able to block the enhancement of VT by zinterol in the HF rabbits with the β2-AR antagonist ICI-118,551.
These findings are, to our knowledge, the first demonstration that β2-AR stimulation is arrhythmogenic in the failing heart, although β2-AR stimulation has been shown to be arrhythmogenic in other pathophysiologic setting (eg, dogs with increased sympathetic stimulation (from exercise) and acute coronary occlusion develop ventricular fibrillation that is prevented by the β2-AR antagonist ICI-118,551.18
Arrhythmogenicity In Vitro (ACs and After Transients)
We have previously shown in HF rabbit myocytes that isoproterenol (β1- and β2-AR activation) induced DADs, ACs, spontaneous SR Ca release and activation of Iti that is mediated by NCX (that is upregulated 2-fold).5 Here, we report that β2-AR stimulation in HF (but not control) myocytes induced ACs and spontaneous SR Ca release (aftertransients). This was associated with increased SR Ca load, but no change in NCX or ICa, and suggests that the arrhythmogenicity of β2-AR stimulation is mediated through the increased SR Ca load that likely activates SR Ca release and Iti. We previously showed how Iti induction depends critically on SR Ca content in this model.5
We found that β2-AR stimulation enhanced SR Ca uptake in both HF rabbit and HF human myocytes, based on an enhanced rate of twitch [Ca]i decline in the absence of altered NCX function. This strongly suggests enhanced SERCA function, which is further substantiated by our findings that β2-AR stimulation specifically enhanced PLB phosphorylation the Ser16 (PKA) site in HF but not control myocytes. This is in line with findings of Kaumann et al9 in failing human LV myocytes. With a trend for enhanced PLB phosphorylation at the Thr17 (CaMKII) site in our quiescent myocytes, we cannot rule out that activation of CaMKII may contribute to increasing SR Ca load. Our findings of decreased PLB phosphorylation at Ser16 and increased phosphorylation at Thr17 with HF are similar to what we have previously reported.15 Although β2-AR stimulation could potentially decrease the threshold level of SR Ca load at which spontaneous SR Ca release occurs in HF myocytes, we previously showed that isoproterenol did not alter this threshold level in HF rabbit myocytes,5 making the enhanced uptake rate and load by a PKA-mediated (and possibly CaMKII-mediated) phosphorylation of PLB a more likely mechanism. In these rabbit HF myocytes, ryanodine receptor (RyR) phosphorylation by CaMKII (but not PKA) might also increase the diastolic SR Ca leak, which may be arrhythmogenic15; so, some contribution of RyR phosphorylation to the observed β2-AR–induced arrhythmias cannot be excluded. However, because SR Ca content is elevated (versus lowered) in that setting, we infer that the SR Ca loading effect of β2-AR activation is the predominant arrhythmogenic basis here. That is, if RyR activation were predominant, β2-AR activation should lower SR Ca load.
Validation in Failing Human Myocytes
Focused parallel studies in human HF myocytes gave results comparable to those in HF rabbits, with ACs as well as enhanced Ca transient amplitude, SR Ca load, and rate of Ca decline in response to β2-AR stimulation (and block of all of these effects by ICI-118,551). Our findings, which are consistent with those of del Monte et al,19 validate our results in the HF rabbit model and further support its applicability to the electrophysiological substrate of the failing human heart. These findings also suggest that even at end-stage HF, when overall β-AR responsiveness is diminished,6 preserved β2-AR responsiveness can contribute to the altered electrophysiological substrate that underlies sudden death in patients with end-stage HF.1
Increased β2-AR Responsiveness in HF
Our findings of increased arrhythmogenicity (and contractile response) with β2-AR stimulation in HF further supports work by others7,9 that HF may be characterized by increased β2-AR responsiveness. For example, β2-AR stimulation with zinterol can enhance Ca transients in HF myocytes to a greater degree than in controls in a way that could potentially be arrhythmogenic.7 We began to explore the basis for this increased β2-AR responsiveness. As in failing human LV,6 HF rabbit LV exhibits a decrease in the total amount of expressed β-ARs, which is attributable to downregulation of β1-ARs but with β2-ARs being unaltered. The unaltered β2-AR expression here, but dramatic enhancement of β2-AR effects on SR Ca uptake and arrhythmogenic SR Ca release events, suggest altered coupling to G proteins (eg, preserved Gs
protein coupling in HF)9 and/or changes in other downstream signaling pathways.8,20 Recent studies have delineated some of the complexity of the β2-AR signal transduction pathway, including functional compartmentalization of signaling mediated by Gi and phosphatidylinositol 3-kinase.8,21 and alterations of β2-AR phosphorylation state.22 Membrane microdomains such as caveolae or lipid rafts may play an important role in localizing β2-AR response and downstream signaling, especially in the failing heart. A recent report that disrupting of caveolae led to an increase in responsiveness of β2-AR stimulation further supports this.23
Some aspects of signaling appear to be species-specific. For instance, β2-AR stimulation elicits positive inotropic effects in control rat and dog myocytes that are mediated by an increase in ICa (with action potential prolongation)24 but no lusitropic effect and no significant phosphorylation of PLB.25,26 However, in human HF the situation appears to be quite different, with β2-AR stimulation leading to both inotropic and lusitropic effects, including PLB phosphorylation.9 Our findings suggest that β2-AR pathways in rabbit heart (HF and control) may more closely resemble that of humans. The enhanced SR Ca uptake and inotropic state induced by β2-AR stimulation in HF would be potentially beneficial, but the increased arrhythmogenic propensity may limit this functional benefit. This raises the challenge of therapeutically enhancing SR Ca function without enhancing arrhythmogenesis.
Implications of Arrhythmogenic Effects of β2-AR Stimulation
The β2-AR is being targeted as a potential therapeutic target for the treatment of HF, and approaches to increase β2-AR number by transgenic and adenoviral gene transfer27,28 have yielded enhancement of cardiac function that is likely attributable to enhancement of SR Ca load.
There may be a therapeutic window in which β2-AR stimulation would lead to increased contractility without much additional arrhythmogenic risk. However, the results of the present study suggest that this approach in failing myocardium will need to be balanced by the potential for enhanced arrhythmogenesis from SR Ca overload in an environment that makes myocytes more prone to developing triggered arrhythmias (eg, upregulated NCX which can mediate the arrhythmogenic Iti, enhanced β2-AR–induced SR Ca loading, and downregulated inward rectifying current IK1 which could destabilize resting potential and enhance depolarizing effects of Iti).5 It will, thus, be important to examine the effects of these approaches on the electrophysiological substrate of the failing heart.
Although "cardioselective" β1-AR blockers such as metoprolol have been used to treat HF patients and decrease mortality and sudden cardiac death,29 there is evidence that nonselective (β1- and β2-AR) blockers such as carvedilol may have greater efficacy in preventing sudden death.30 Our results suggest that "nonselective" β-AR blockade could offer therapeutic advantages by limiting catecholamine-mediated increases in SR Ca load mediated by stimulation of the β2-AR and, thereby, further stabilize the arrhythmogenic substrate of the failing heart. Although there is a potential concern that β2-AR could have negative inotropic effects, these have not been borne out in clinical trials.30
Limitations
Zinterol has been used for many β2-AR studies,7,21,24–26 but at high doses (eg, 10–5 mol/L), zinterol may also stimulate β1-AR.31 We therefore performed most of these studies with zinterol in the presence of the β1-AR antagonist CGP 20712A (this was more difficult for in vivo studies, where we found β1-AR blockade was not always well tolerated hemodynamically in rabbits with HF). In some in vitro studies, we also used zinterol alone and found results comparable to zinterol plus CGP. This suggests that at the doses used, the effects of zinterol were predominantly attributable to stimulation of β2-ARs21,24–26 (with the exception that zinterol could slightly increase ICa via β1-AR). Overall, the ability to reverse the effects of zinterol by the specific β2-AR blocker ICI-118,551 supports our conclusions about β2-AR–induced effects. Wang et al14 reported that myocytes on laminin-coated cover slips exhibited greater β2-AR responsiveness to zinterol. Our findings of enhanced arrhythmogenicity with β2-AR stimulation in vivo as well as in vitro (whether on laminin-coated cover slips or on glass cover slips without laminin) suggest that the laminin was not a confounding issue. Moreover, laminin may help simulate the extracellular protein environment in intact tissue that involves interactions with laminin and integrins.14
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Conclusion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionReferences |
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We found that β2-AR stimulation is arrhythmogenic in the failing rabbit and human heart by enhancing spontaneous SR Ca release and ACs (secondary to enhanced SR Ca uptake and load). Notably, β2-AR activation is more arrhythmogenic in the failing versus nonfailing heart. Thus, β2-AR blockade may offer therapeutic advantages to stabilize the altered electrophysiological substrate of failing myocardium and help decrease the incidence of sudden death in HF patients.
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Acknowledgments |
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We thank Jodi Jeanes for technical assistance.
Sources of Funding
This work was supported by NIH grants HL46929 and HL73966 (to S.M.P.) and NIH grants HL30077 and HL64724 (to D.M.B.).
Disclosures
None.
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Footnotes |
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Original received October 22, 2004; resubmission received December 3, 2007; revised resubmission received April 10, 2008; accepted April 28, 2008.
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References |
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