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Am J Physiol Heart Circ Physiol 294: H2587-H2595, 2008. First published April 11, 2008; doi:10.1152/ajpheart.00985.2007
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Chronic β2-adrenoceptor stimulation impairs cardiac relaxation via reduced SR Ca2+-ATPase protein and activity

James G. Ryall,1 Jonathan D. Schertzer,1 Kate T. Murphy,1 Andrew M. Allen,2 and Gordon S. Lynch1

1Basic and Clinical Myology Laboratory and 2Central Cardiovascular Regulation Group, Department of Physiology, University of Melbourne, Victoria, Australia

Submitted 26 August 2007 ; accepted in final form 7 April 2008


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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We determined the cardiovascular effects of chronic β2-adrenoceptor (β2-AR) stimulation in vivo and examined the mechanism for the previously observed prolonged diastolic relaxation. Rats (3 mo old; n = 6), instrumented with implantable radiotelemeters, received the selective β2-AR agonist formoterol (25 µg·kg–1·day–1 ip) for 4 wk, with selected cardiovascular parameters measured daily throughout this period, and for a further 7 days after cessation of treatment. Chronic β2-AR stimulation was associated with an increase in heart rate (HR) of 17% (days 1–14) and 5% (days 15–28); a 11% (days 1–14) and 6% (days 15–28) decrease in mean arterial blood pressure; and a 24% (days 1–14) increase in the rate of cardiac relaxation (–dP/dt) compared with initial values (P < 0.05). Cessation of β2-AR stimulation resulted in an 8% decrease in HR and a 7% decrease in –dP/dt, compared with initial values (P < 0.05). The prolonged cardiac relaxation with chronic β2-AR stimulation was associated with a 30% decrease in the maximal rate (Vmax) of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity, likely attributed to a 50% decrease in SERCA2a protein (P < 0.05). glycogen synthase kinase-3β (GSK-3β) has been implicated as a negative regulator of SERCA2 gene transcription, and we observed a ~60% decrease (P < 0.05) in phosphorylated GSK-3β protein after chronic β2-AR stimulation. Finally, we found a 40% decrease (P < 0.05) in the mRNA expression of the novel A kinase anchoring protein AKAP18, also implicated in β2-AR-mediated cardiac relaxation. These findings highlight some detrimental cardiovascular effects of chronic β2-AR agonist administration and identify concerns for their current and future use for treating asthma or for conditions where muscle wasting and weakness are indicated.

β-agonist; glycogen synthase kinase-3β; sarco(endo)plasmic reticulum Ca2+-ATPase; A kinase anchoring protein

THE ACUTE EFFECTS OF β2-ADRENOCEPTOR (β2-AR) stimulation on cardiovascular function measured in vitro have shown a number of important changes in cardiovascular performance (17, 21–23, 29, 36, 51). These changes include the following: an increased force of contraction of the heart (positive inotropic response; Ref. 21); an increased rate of cardiac relaxation (positive lusitropic response; Refs. 23, 29, 49); a decrease in blood pressure (BP; Refs. 4, 48); and an increase in HR (chronotropic response; Ref. 7). Although these effects have been well described, our understanding of the underlying cellular mechanisms responsible for these responses remains incomplete.

In contrast to the well-documented acute cardiovascular effects of β2-AR stimulation (4, 22, 23, 28, 32, 36, 45), the chronic effects are less well understood. β2-AR agonists are currently prescribed for the treatment of asthma (8, 27, 30), and due to their well-described anabolic actions, they have also been trialed in conditions where muscle wasting and weakness are indicated (15, 24, 25). For these applications, a greater understanding of the cardiovascular effects of chronic β2-AR stimulation is essential.

We have demonstrated previously that chronic β2-AR stimulation (with fenoterol) elicits significant cardiac hypertrophy and impaired cardiac function in rats (16). Of particular importance was the finding that β2-AR stimulation prolonged diastolic relaxation in the hearts of both adult and old rats (16). A prolongation of diastolic relaxation could severely impair cardiac filling during times of stress, since an increase in HR is associated with a significantly reduced duration of diastole, the time when most ventricular filling occurs (14).

Cardiac relaxation is controlled by numerous processes, including the rate of removal of Ca2+ from the sarcoplasm, the rate of release of Ca2+ from the contractile apparatus, and the dynamics between bound Ca2+ and the myofilaments. The removal of Ca2+ from the sarcoplasm occurs via the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA), the Na+/Ca2+ exchanger, sarcolemmal Ca2+-ATPases, and mitochondrial Ca2+ uniporters (18). The major determinant of Ca2+ reuptake into the sarcoplasmic reticulum (SR) is SERCA (5). The SERCA proteins belong to the highly conserved type E1-E2 ATPases family and comprise SERCA1, SERCA2, and SERCA3 isoforms (53). SERCA1 is expressed exclusively in fast-twitch skeletal muscle (26); SERCA3 is predominantly expressed in epithelial and hematopoietic cells (10), while SERCA2 is predominantly expressed in cardiac and slow-twitch muscle (54). SERCA2a is the predominant cardiac isoform and its expression is regulated via a number of transcriptional regulators, including specificity protein 1, thyroid hormone, protein phosphatases, and glycogen synthase kinase-3β [GSK-3β; among others (35, 53)].

Cardiac SERCA2 activity depends on numerous factors, including the cytoplasmic/SR Ca2+ gradient, the protein concentration of SERCA, the phosphorylation status of the inhibitory protein phospholamban (PLN; Ref. 9), and the presence of the A kinase anchoring protein AKAP18 (33). AKAP18 has been found to act as a scaffold protein for the cAMP-dependent protein kinase (PKA), SERCA2, and PLN and coordinates PKA-mediated phosphorylation of PLN (33). This novel protein may therefore play a previously unidentified role in the chronic β2-AR stimulation mediated prolongation of relaxation.

The aims of the present study were to characterize the effects of chronic β2-AR stimulation (with daily formoterol administration) on cardiovascular performance and to examine the role of SERCA2a in impaired Ca2+ handling. Specifically, we tested the hypothesis that chronic β2-AR stimulation would alter cardiovascular performance mediated, in part, by alterations in SERCA function.


   METHODS
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 METHODS
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The investigation conformed with 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). All experiments were approved by the Animal Experimentation Ethics Committee of The University of Melbourne.

Animals for radiotelemetry. Male 3-mo-old Fischer 344 rats (F344; n = 6; body mass = ~270 g) obtained from the Animal Resource Centre (Canning Vale, Western Australia) were instrumented with a surgically inserted radiotelemeter (TA11PA-C40, Data Sciences International, St. Paul, MN) as follows. Before surgery, each rat received a subcutaneous injection of the analgesic meloxicam (Metacam, 0.2 ml/kg, Boehringer Ingelheim, Germany). Briefly, rats were anesthetized with pentobarbital sodium [Nembutal, Rhone Merieux, Pinkenba, QLD, Australia (60 mg/kg ip)], with supplemental doses administered to maintain surgical anesthesia, defined by loss of the pedal withdrawal reflex. The descending aorta was exposed by a midline abdominal incision and cannulated, rostral to the femoral bifurcation, with the arterial pressure cannula of a radiotelemeter. The telemeter body was then placed in the abdominal cavity and secured to the abdominal musculature. The rats were allowed to recover from the surgical procedure for 14 days before any readings were taken.

All rats received a once daily injection of saline (0.5 ml) at 10:00 AM for a period of 7 days (control period), followed by 28 days of chronic β2-AR stimulation with formoterol [25 µg·kg–1·day–1 (AstraZeneca, Molndal, Sweden) in 0.5 ml saline (formoterol-treated period)]. The β2-AR stimulation period was split into periods of formoterol treatment, days 1–14 and days 15–28, to examine the early and late phases of prolonged β2-AR stimulation. Cardiovascular parameters were measured for a further 7 days after cessation of β2-AR stimulation to determine the physiologic effect of chronic β2-AR stimulation without the potentially confounding effects of increased PLN phosphorylation. During this period, the rats again received a once daily saline injection (0.5 ml, days 29–35).

Cardiovascular parameters measured included the following: mean BP [systolic BP, (SBP) and diastolic BP (DBP)], mean arterial pressure, pulse pressure, and heart rate (HR) and were obtained using six receiver pads (Data Sciences International) connected to a receiver multiplexer (RMX10, Data Sciences International) and one channel on the consolidation matrix (BCM100, Data Sciences International). Data were obtained and analyzed using the Dataquest A.R.T. program (v2.2, Data Sciences International). BP and HR were recorded at a sampling rate of 64 Hz for a period of 20 s every 5 min for a total duration of 70 min. Individual recordings included a preinjection (t = 0) period and a postinjection period. Peak cardiovascular changes were determined each day as the difference between the initial preinjection period and the maximum (or minimum) value each day. Mean cardiovascular parameters (Table 1) were obtained from the last 30 min of recording so as to exclude any response from handling the rats. Mean relaxation parameters including the time to one-half relaxation (1/2RT) and the rate of relaxation (–dP/dt) were determined from no less than five consecutive heart beats obtained at the end of the recording period.


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  		Table 1. Mean cardiovascular parameters of rats throughout the four phases of treatment

 
Animals for biochemical analyses. As SERCA is primarily responsible for restoring cytosolic Ca2+ concentration during diastole we determined the basal and maximal rate (Vmax) of cardiac SERCA activity. A second group of F344 rats (n = 12; body mass = ~270 g) also obtained from the Animal Resource Centre were allocated into either control or chronic β2-AR-stimulated groups. Treated rats were administered 25 µg/kg formoterol in isotonic saline (~0.5 ml volume), delivered by intraperitoneal injection once daily for 28 consecutive days. Control rats received an equivolume injection of isotonic saline. Twenty-four hours after the final formoterol injection rats were anesthetized with pentobarbital sodium (60 mg/kg ip, Nembutal) and then killed by rapid surgical excision of the heart. The heart was trimmed of atria, and all connective tissue was blotted once on filter paper, weighed, and frozen in thawing isopentane for biochemical analyses.

Vmax of SERCA activity. Crude ventricular homogenates and enriched SR vesicles were prepared simultaneously based on previous techniques (44). The left and right ventricles were diluted 1:5 (wt/vol) in ice cold homogenizing buffer (containing in mM: 250 sucrose, 5 HEPES, 0.2 PMSF, and 0.2% sodium azide, pH 7.5). The ventricular tissue was homogenized at 16,500 rpm for 3 x 15-s bursts, separated by 45-s breaks. SR isolation was carried out on the same day as cardiac homogenization and accomplished by sucrose gradient and differential centrifugation using a Beckman ultracentrifuge with a 70.1 Ti fixed-angle rotor to remove unwanted cellular organelles, fat, and debris. During the entire homogenization and SR vesicle isolation procedure, the samples were immersed in ice. It is critical to keep the samples on ice to avoid temperature-dependent reductions in SERCA activity (43). Protein determination of homogenates and SR vesicles was made by the method of Bradford and analyzed in triplicate. The Vmax of Ca2+-induced SERCA activity in enriched cardiac SR membranes was analyzed according to methods described previously (31). The reaction buffer contained (in mM) 100 KCl, 20 HEPES, 10 MgCl2, 10 NaN3, 10 phosphoenolpyruvate, 5 ATP, and 1 EGTA. The pH of the reaction buffer was adjusted to 7.0 at 37°C. Immediately before the reaction was started, 18 U/ml lactate dehydrogenase, 18 U/ml pyruvate kinase, 0.3 mM NADH, 1 µM Ca2+ ionophore A-23187 (Sigma-Aldrich, Castle Hill, NSW, Australia), and 170 µl of enriched SR membrane were added to 1 ml of reaction buffer. Assays were performed at 37°C and 340 nm (Multiskan Spectrum, Thermo Electron, Waltham, MA). In all cases, the Vmax of SERCA activity was established by adding between 9–11 µl free Ca2+ ([Ca2+]f; 100 mM as CaCl2) until a plateau and subsequent decline in SERCA activity occurred. In-house tests showed that cardiac Vmax for all samples occurred between these levels of [Ca2+]f (data not shown). Basal (or Mg2+-ATPase) activity was determined by the addition of cyclopiazonic acid, a specific inhibitor of the SERCA enzyme, to a final concentration of 40 µM.

SDS-PAGE for Western blotting. SDS-PAGE was performed to separate proteins according to molecular mass and isolate SERCA2a, total PLN and phospho-(Ser16) PLN, GSK-3β and phospho-(Ser9) GSK-3β, Akt, and phospho-(Ser473) Akt as described previously (44). The protein concentration of enriched SR vesicles or crude cardiac homogenates was equalized before gel electrophoresis. Samples were loaded into an 8% polyacrylamide gel, with all samples run in duplicate, at two different concentrations.

After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride (PVDF; Bio-Rad, NSW, Australia) membrane by placing the gel in transfer buffer and applying a low voltage (32 V) for 60 min (Novex Xcell II blot module, InVitrogen). Nonspecific binding sites were blocked with 10% nonfat skim milk powder in phosphate buffered saline (pH 7.5), applied for 60 min at room temperature. Immunoblotting was performed on enriched SR vesicles, or crude cardiac homogenates using primary antibodies specific for rat SERCA2a (95 kDa; 7E6, Affinity Bioreagents), PLN (25 kDa; #05-205, Upstate Cell Signaling Solutions), phospho-(Ser16) PLN (25 kDa; #07-052, Upstate Cell Signaling Solutions), GSK-3β (47 kDa; AB8687, Upstate Cell Signaling Solutions), phospho-(Ser9) GSK-3β (47 kDa; #07-835, Upstate Cell Signaling Solutions), Akt (60 kDa; #9272, Cell Signaling), or phospho-(Ser473) Akt (60 kDa; #9271, Cell Signaling) that were diluted 1:1,000 (SERCA2a, phospho-/GSK-3β, and phospho-/Akt), or 1:2,000 (phospho-/PLN) in 10% nonfat milk and incubated for 60 min at room temperature. In response to β-AR stimulation PLN has been shown to be phosphorylated at two adjacent amino acid residues, Ser16 and Thr17. In vitro studies (29) have shown that β-AR administration strongly phosphorylates the Ser16 residue in a PKA-dependent manner and to a lesser degree the Thr17 residue via activation of Ca2+-calmodulin-dependent protein kinase. Thus, only phosphorylation at the Ser16 residue was examined in the present study.

After application of an appropriate IgG secondary antibody that was diluted 1:2,500 in 10% nonfat milk and applied for 60 min at room temperature, protein quantification was performed using an enhanced chemiluminescence immunodetection procedure (ECL plus; Amersham Biosciences). After exposure to photographic film (Hyperfilm-ECL; Kodak), the blot was developed for 60 s in Kodak GBX developing solution, washed in tap water, and fixed in Kodak GBX fixer. Blots were digitized, and relative protein levels were determined by scanning densitometry. Values were normalized to control values.

After immunoblotting, PVDF membranes were stained with Ponceau stain to visualize protein levels to confirm equal protein loading (data not shown).

Real-time RT-PCR measurement of mAKAP and AKAP18 mRNA expression. Total RNA was extracted from 10–20 mg of ventricular tissue using a commercially available kit, according to the manufacturer's instructions (no. 74704, Qiagen, Valencia, CA). RNA concentration was determined spectrophotometrically at 260 {eta}m, and the samples were stored at –80°C. RNA was transcribed into cDNA using the Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (no. 18080–051, Invitrogen, Carlsbad, CA), and the resulting cDNA was stored at –20°C for subsequent analysis.

Real-time RT-PCR was performed using the Bio-Rad iCycler Thermal Cycler (Bio-Rad, Hercules, CA) and was run for one cycle (95°C for 3 min) and 40 cycles (95°C for 20 s, 55°C for 30 s, and 72°C for 30 s). PCR was conducted in triplicate with reactions containing SYBR Green (iQ SYBR Green Supermix, Bio-Rad), forward and reverse primers, and cDNA template. Measurements included a no-template control as well as an RT negative control. The forward and reverse primers used were; mAKAP (also referred to as AKAP100), 5'-CGACTGAAAAAGCCACACAA-3' and 5'-TGTCCACTGCGATTTCTCTG-3'; AKAP18 (also referred to as AKAP7 or AKAP15), 5'-CTGGCTGGAGAAAGCAGAAC-3' and 5'-CCACCAGCCTCTTACTGAGC-3'; and 18S 5'-ACGGAAGGGCACCACCAGGA-3' and 5'-CACCACCACCCACGGAATCG-3', respectively. 18S was used as a control to account for any variations in the amount of RNA input and the efficiency of reverse transcription. Formoterol treatment had no effect on 18S mRNA expression, when expressed in the linear form (2–CT) (control: 1.23 x 10–3 ± 0.15 x 10–3; formoterol: 1.20 x 10–3 ± 0.14 x 10–3, NS). Gene expression was quantified using a cycle threshold (CT) method, whereby a {Delta}CT was calculated by subtracting the 18S CT from the gene CT. The relative gene expression was then calculated using the expression 2–{Delta}CT.

Statistical analyses. All values in the text and table are means ± SE. Mean data were compared between treatment groups using a ANOVA for influences of treatment and withdrawal, using Fisher's least significant differences post hoc multiple comparison procedure to identify differences between specific groups. Western blotting and SERCA activity were analyzed using a nonpaired two-tailed t-test. In all cases, differences between groups were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Body mass of rats instrumented with radiotelemeters was not altered by formoterol treatment. However, formoterol-treated rats exhibited a 17% increase in absolute heart mass compared with saline-treated control rats (control: 743 ± 31 mg vs. formoterol: 868 ± 50 mg, P < 0.05).

Radiotelemetry measurement of cardiovascular parameters. Immediately after a single injection of 0.5 ml of saline (control period), HR increased by 23% above basal (t = 0, the preinjection baseline level) and remained elevated for ~35 min (P < 0.05; Fig. 1) and was an unavoidable and expected response to handling of the rats.


Figure 1
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  		Fig. 1. Increased heart rate (HR) in response to saline or formoterol injection or saline injection after cessation of chronic β2-adrenoreceptor (AR) stimulation with formoterol. A: timeline of HR changes after injection with saline or formoterol (indicated by arrow). Dashed lines under the data points indicate significantly increased (P < 0.05) HR compared with basal (t = 0) throughout each phase of treatment (n = 6/group). B: peak change in HR elicited by formoterol or saline. *P < 0.05 vs. control; #P < 0.05 vs. formoterol days 1–14; {dagger}P < 0.05 vs. formoterol days 15–28.

 
Throughout days 1–14 of β2-AR stimulation, formoterol injection increased HR by a maximum of 32% above basal and remained elevated throughout the 70-min recording period (P < 0.05). By days 15–28 of β2-AR stimulation, basal HR was decreased by 11% compared with the basal level during the control period. Formoterol administration during days 15–28 was associated with a 39% increase in HR above basal (P < 0.05).

After cessation of β2-AR stimulation with formoterol (days 29–35), basal HR was decreased by 8% compared with basal levels during the control period (P < 0.05). A single saline injection during this period resulted in a 13% increase in HR compared with basal values (P < 0.05), which remained elevated for 30 min.

When HR values were averaged over the last 30 min of recording (to remove the influence of the handling response), a 17 and 5% increase in HR was observed throughout days 1–14 and 15–28 of formoterol treatment, respectively (P < 0.05; Table 1), compared with control. During days 29–35, after cessation of formoterol treatment, HR was decreased by 11% compared with control (P < 0.05).

Changes in SBP and DBP associated with treatment are presented in Fig. 2. After a single saline injection, SBP and DBP increased by a maximum of 14 and 13%, respectively, and remained elevated above initial values for 35 min (P < 0.05; Fig. 2), indicative of a response to animal handling. Formoterol treatment during days 1–14 was associated with a 12 and 16% decrease in SBP and DBP, compared with basal, respectively (P < 0.05). The decrease in SBP was first evident 20 min after the formoterol injection during days 1–14, while the decrease in DBP was evident immediately after formoterol administration (Fig. 2, A and C).


Figure 2
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  		Fig. 2. Response of systolic blood pressure (SBP) and diastolic blood pressure (DBP) to saline or formoterol injection or saline injection after cessation of chronic β2-AR stimulation with formoterol. A: timeline of SBP changes after injection (indicated by arrow). Dashed lines under the data points indicate a significant change (P < 0.05) in SBP compared with basal (t = 0) throughout each phase of treatment (n = 6/group). B: peak change in SBP elicited by formoterol or saline. C: timeline of DBP changes after injection (indicated by arrow). Dashed lines under the data points indicate a significant change (P < 0.05) in DBP compared with basal (t = 0) throughout each phase of treatment (n = 6/group). D: peak change in DBP elicited by formoterol or saline. *P < 0.05 vs. control; #P < 0.05 vs. formoterol days 1–14; {dagger}P < 0.05 vs. formoterol days 15–28.

 
During days 15–28 of β2-AR stimulation, basal SBP was 5% higher than basal control levels (P < 0.05), while formoterol administration during this period was associated with a 7 and 9% decrease in SBP and DBP, compared with basal, respectively (P < 0.05; Fig. 2). The decrease in SBP was evident 30 min after formoterol administration, while the decrease in DBP occurred 20 min after formoterol injection. Both SBP and DBP remained below control saline values throughout the 70-min recording period.

After cessation of β2-AR stimulation with formoterol, basal SBP was 6% higher than basal control levels (P < 0.05), while saline injection during this period increased both SBP and DBP by 9% above basal values (P < 0.05), SBP remained elevated for 35 min, and DBP was increased above basal for 25 min (P < 0.05; Fig. 2, A and C).

Cardiac relaxation. Representative BP traces from rats during the control saline period, the first day of the formoterol treatment period, and the first day after cessation of formoterol treatment are shown in Fig. 3A. 1/2RT and –dP/dt were determined from no less than five heartbeats·rat–1·day–1 exactly 65 min after injection with either saline or formoterol and are presented in Fig. 3, B and C. Days 1–14 of formoterol treatment were associated with a 15% decrease in 1/2RT and a concomitant 24% increase in –dP/dt compared with control. In contrast, compared with days 1–14, days 15–28 of formoterol treatment were associated with an increase in 1/2RT and a decrease in –dP/dt (P < 0.05), such that they were restored to control values. After cessation of formoterol treatment (days 29–35), 1/2RT was increased by 18%, which was associated with a 7% decrease in –dP/dt compared with control (P < 0.05).


Figure 3
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  		Fig. 3. Prolonged cardiac relaxation after chronic β-agonist treatment. A: representative BP traces (corrected to control) during the control period (solid line), after a single injection of formoterol (dotted line), and immediately after cessation of formoterol treatment (dashed line). B: rate of relaxation (–dP/dt) during the control period (white bars), during formoterol treatment (black bars, days 1–14 and 15–28), and after a period of formoterol withdrawal (horizontal lines). C: one-half relaxation time (1/2RT) during the control period (white bars), during formoterol treatment (black bars, days 1–14 and 15–28), and after cessation of formoterol treatment (horizontal lines). *P < 0.05 vs. control; #P < 0.05 vs. formoterol days 1–14; {dagger}P < 0.05 vs. formoterol days 15–28.

 
SERCA activity, protein levels, and regulatory proteins. Crude homogenates and enriched SR vesicles were prepared from a separate group of rats treated with formoterol or saline (control) for 28 days. The Vmax of Ca2+-ATPase activity in enriched cardiac SR vesicles was decreased by 31% in rats treated with formoterol for 4 wk (P < 0.05; Fig. 4A). Basal or Mg2+-ATPase activity was not altered by formoterol treatment (data not shown).


Figure 4
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  		Fig. 4. Impaired ATP hydrolysis by sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) was observed in hearts from rats treated chronically with formoterol. A: mean data for maximal sarcoplasmic reticulum Ca2+-ATPase activity (Vmax) of enriched SR vesicles. B: representative Western blots and relative concentration of SERCA2a in ventricles from control (Con) and formoterol-treated (Form) rats. C: representative Western blots and relative concentration of total and phospho-(Ser16) phospholamban (PLN) in control (Con) and formoterol-treated (Form) rats. D: representative Western blots and relative concentration of total and phospho-(Ser9) GSK-3β. E: representative Western blots and relative concentration of total and phospho-(Ser473) Akt. *P < 0.05 vs. control. OD, optical density.

 
Representative immunoblots for SERCA2a, PLN and phospho-(Ser16) PLN, GSK-3β and phospho-(Ser9) GSK-3β, Akt, and phospho-(Ser473) Akt are presented in Fig. 4, B-E. Crude homogenates prepared from ventricles isolated from formoterol-treated rats exhibited a 52% decrease in SERCA2a protein levels compared with control rats (P < 0.05; Fig. 4B). Formoterol treatment did not alter total PLN protein levels but increased phosphorylation of the Ser16 site of PLN by approximately twofold (P < 0.05; Fig. 4C). Similarly, formoterol treatment did not alter total GSK-3β protein levels but decreased the phosphorylation of GSK-3β at Ser9 by ~60% (P < 0.05; Fig. 4D). Formoterol treatment did not alter total Akt or phospho-Akt levels (Fig. 4E).

AKAP18 and mAKAP mRNA expression. Ventricles from formoterol-treated rats demonstrated a tendency for lower mAKAP mRNA expression; however, this did not reach statistical significant (Fig. 5A). In comparison, chronic formoterol treatment reduced cardiac AKAP18 mRNA expression by 40% (P < 0.05; Fig. 5B).


Figure 5
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  		Fig. 5. Expression levels of mAKAP (A) and AKAP18 (B) mRNA in ventricles from control or formoterol-treated rats. Formoterol treatment significantly reduced the expression of AKAP18, while the decrease in mAKAP mRNA did not reach statistical significance. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The most important finding of this study was that chronic β2-AR stimulation (with formoterol) resulted in a significant prolongation of cardiac relaxation, which was associated with a ~30% decrease in SERCA activity and a ~50% decrease in SERCA2a protein levels. SERCA2a is the major regulator of diastolic Ca2+ concentration (5), and as such, these changes would likely result in an increased time for cardiac relaxation and impair diastolic filling, as observed after cessation of formoterol treatment. The results provide important information regarding the cardiovascular safety of chronic β2-AR stimulation with formoterol.

Our finding that chronic β2-AR stimulation is detrimental to cardiac function is in contrast to previous studies in animal models of heart failure, where chronic β2-AR stimulation improved cardiac function in dilated cardiomyopathy (Refs. 1–3). Furthermore, we have reported previously that chronic β2-AR stimulation does not have the same detrimental effects on cardiac function in old rats as in young healthy rats (16). These results suggest that chronic β2-AR stimulation can have either beneficial or detrimental effects on cardiac function depending on the pretreatment condition of the heart. Seminal work by Xiao and colleagues (50, 55) showed that the β2-AR (via a G{alpha}i-dependent mechanism) signals to promote cell survival and therefore may attenuate cell death and prevent the progression of a number of cardiac pathologies. However, under control conditions the activation of the β2-AR-G{alpha}i signaling pathway can reduce the beneficial effects of β2-AR-G{alpha}s signaling to inhibit the positive chronotropic, inotropic, and lusitropic effects of β2-AR stimulation (50), as observed in the present study.

The increased rate of cardiac relaxation observed throughout the first 14 days (and to a lesser extent, days 15–28) of formoterol administration was likely due to the β-adrenergic-mediated dysinhibition of SERCA by PLN phosphorylation (29, 45). In the unphosphorylated state, PLN inhibits SERCA activity by reducing the affinity of SERCA for Ca2+; phosphorylation of PLN removes this inhibition of SERCA activity (29, 32, 45). While phosphorylation of PLN is traditionally believed to be the primary mechanism for βAR-mediated changes in Ca2+ transport and cardiac relaxation, our in vivo radiotelemetry results demonstrate that 24 h after the final formoterol injection, despite clear phosphorylation of PLN, cardiac relaxation is impaired.

Interestingly, AKAP18, which acts as a scaffold protein for the PKA, SERCA2, and PLN, is required for phosphorylation of PLN. Lygren et al. (22) found that inhibiting PLN binding to AKAP18{delta} (a large splice variant of the AKAP18 gene) resulted in a significant decrease in PLN phosphorylation, which was associated with a dramatic decline in SR Ca2+ reuptake (33). The 40% reduction in AKAP18 mRNA observed in the present study indicates that in addition to a reduction in maximal SERCA activity, chronic β2-AR stimulation is likely associated with a decline in the amount of PLN available for phosphorylation. This hypothesis is supported by the finding that 1/2RT was not reduced and the increase in –dP/dt was blunted during days 14–28 of formoterol treatment, suggesting a diminishing lusitropic response to β2-AR stimulation.

Boluyt et al. (6) demonstrated that chronic βAR stimulation with the non-selective βAR agonist isoproterenol (2.4 mg·kg–1·day–1) in rats, resulted in a ~50% downregulation of SERCA mRNA after 3 days in the heart. We have found a similar decrease in SERCA2a protein levels, with a concomitant decrease in SERCA activity, after 4 wk of treatment with the selective β2-AR agonist formoterol.

The reduction in SERCA2a protein levels observed in the present study is likely due to decreased transcription of the gene, possibly via a GSK-3β-dependent mechanism. GSK-3β is highly active in unstimulated cells and generally acts to inhibit gene expression; however, when phosphorylated at serine 9, GSK-3β inhibition is removed and gene transcription progresses (47). In a study by Michael et al. (35), SERCA2a protein levels were reduced by ~40% in transgenic mice overexpressing GSK-3β. These authors suggested that SERCA2a downregulation occurred as a result of a direct interaction of GSK-3β with the promoter region of the SERCA2a gene (35).

While acute activation of the β2-AR has been linked to phosphoinositol 3-kinase-Akt dependent phosphorylation of GSK-3β (52), the findings from the present study demonstrate that chronic β2-AR stimulation results in a reduction in GSK-3β phosphorylation, likely increasing the activity of this kinase and resulting in a decrease in SERCA2a gene transcription. Interestingly, the reduced GSK-3β phosphorylation was not associated with changes in Akt phosphorylation, indicating that another mechanism is responsible for the decrease in GSK-3β phosphorylation. Fang et al. (12) showed that synthetic analogs of cAMP can phosphorylate and inactivate cardiac GSK-3β in a PKA-dependent manner, while a more recent study (19) found epinephrine phosphorylated GSK-3β in soleus skeletal muscle independent of Akt signaling. These results indicate that changes in cAMP-PKA signaling, such as that associated with chronic β2-AR stimulation (11, 16, 20), could have dramatic effects on GSK-3β phosphorylation. Further research into PKA-mediated phosphorylation of GSK-3β is warranted.

While the present study focused predominantly on β2-AR-induced changes to SERCA2 and cardiac relaxation, we also examined mRNA expression levels of the most well-described cardiac AKAP mAKAP (13, 34). mAKAP has been found to act as a link between PKA and the ryanodine receptor and therefore likely plays a role in the βAR-mediated chronotropic and inotropic response (34). While no significant difference was observed in the mRNA levels of mAKAP after chronic β2-AR stimulation, the results suggest a trend towards a decrease in mAKAP expression, which would likely impair the PKA-mediated increased rate and force of contraction. The role of AKAPs in the regulation of PKA signaling is an emerging field, and further research is required to examine the role of these scaffold proteins in the response to chronic βAR stimulation and under pathological conditions where βAR signaling is impaired.

While the focus of the present study was to examine the role of SERCA and SERCA-related proteins in the prolonged relaxation associated with chronic β2-AR stimulation, it is important to recognize that there are many proteins and signaling pathways that regulate the rate of cardiac relaxation (5, 45). It has been shown previously that chronic β2-AR stimulation with the β2-AR agonist clenbuterol resulted in a 6.5-fold increase in the level of the slow β-myosin heavy chain in the left ventricles of mice (39). Therefore, our treatment protocol with formoterol would also likely produce a similar shift in cardiac myosin heavy chains, which could result in a decreased rate of myocyte relengthening and a prologation of relaxation. Formoterol is currently prescribed for the treatment of asthma at a similar absolute dose to that used in the present study. Previous clinical studies examining the safety of formoterol treatment in asthma patients have focused on the immediate effects of formoterol on HR, BP, and ECG (27, 37), whereas longer term studies have reported (patient described) adverse effects, including muscle tremor, headache, and chest pains (8, 40). The results from the present study suggest that some of the most important effects of formoterol are only observable after cessation of chronic β2-AR stimulation and are attributed to cardiac relaxation. Thus, future studies examining the (relative) safety of formoterol (or other β2-AR agonists) should consider these findings. Furthermore, it will be interesting to determine whether these changes in cardiac function are reversible after an extended period of formoterol withdrawal. We have demonstrated previously that the cardiac hypertrophy associated with chronic formoterol treatment is reversible after 4 wk of formoterol withdrawal (41). Further studies are required to determine whether the changes in cardiac relaxation are similarly reversible.

It should be noted that in the present study, formoterol was administered via intraperitoneal injection, whereas formoterol is usually administered orally or via inhalation for treatment of asthma (37). Previous studies have found that the efficacy of β2-AR agonists is related to their mode of administration, with intraperitoneal injection found to be the most efficacious for eliciting systemic effects, such as striated muscle hypertrophy (38).

In conclusion, we have shown that while chronic β2-AR stimulation can have beneficial effects on skeletal muscle (24, 25, 42), its clinical application may be limited by potentially deleterious effects on cardiac function, particularly those related to cardiac relaxation. Formoterol is used clinically as a preventive drug and taken on a daily basis at a similar dose to that used in the present study. To our knowledge, these are the first data to demonstrate, in vivo, impaired cardiac relaxation as a result of chronic β2-AR stimulation. We also show that these changes are associated with alterations in ventricular SR Ca2+ handling. Importantly, these results also indicate a number of important differences of acute vs. chronic β2-AR stimulation on cardiovascular function.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported under the Australian Research Council's Discovery-Project funding scheme (Project No. DP0665071). J. G. Ryall was supported by a postgraduate scholarship from the National Heart Foundation of Australia.


   ACKNOWLEDGMENTS
 
We thank Fiona Colarossi for help in the preparation of Fig. 3.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. S. Lynch, Dept. of Physiology, Univ. of Melbourne, Victoria, 3010 Australia (e-mail: gsl{at}unimelb.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
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