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Am J Physiol Heart Circ Physiol 291: H61-H70, 2006; doi:10.1152/ajpheart.01353.2005
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Phosphorylation of phospholamban at threonine-17 reduces cardiac adrenergic contractile responsiveness in chronic pressure overload-induced hypertrophy

Geoffrey D. Mills, Hajime Kubo, David M. Harris, Remus M. Berretta, Valentino Piacentino, III, and Steven R. Houser

Temple University School of Medicine, Philadelphia, Pennsylvania

Submitted 21 December 2005 ; accepted in final form 21 February 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Physiological hemodynamic stress, such as aerobic exercise, is intermittent and requires an increase in Ca2+-dependent contractility through sympathetic nervous system activation. Pathological hemodynamic stress, such as hypertension, is persistent and requires sustained increases in cardiac function. Over time, this causes left ventricular hypertrophy (LVH)-reduced responsiveness to sympathetic stimulation. In this study, we examined the hypothesis that blunted in vivo adrenergic contractile responsiveness in pressure overload (PO)-induced cardiac hypertrophy is caused by abnormalities in the abundance and/or basal phosphorylation state of Ca2+ regulatory proteins. PO, induced by aortic constriction, caused concentric LVH or dilated LVH. Only animals with dilation exhibited a decrease in baseline left ventricle function [fractional area change (FAC); measured with echocardiography]. All PO animals had a reduced contractile response to adrenergic agonists (increase in FAC with 40 µg·kg–1·min–1 dobutamine, control 0.30 ± 0.04, n = 5 vs. banded 0.10 ± 0.03, n = 10; P < 0.01). PO animals had reduced phospholamban (PLB) protein abundance (P = 0.07, not significant) and increased PLB phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLB-Thr17, P < 0.05) but not at the protein kinase A-specific site (PLB-Ser16). PLB-Thr17 phosphorylation was inversely correlated with dobutamine-induced increases in contractility in PO animals (r2 = 0.81, P < 0.05). Continuous induction of Ca2+ transients in isolated ventricular myocytes for 24 h increased phosphorylation at PLB-Thr17 and diminished inotropic responsiveness and PLB-Ser16 phosphorylation after exposure to isoproterenol (P < 0.05). These data show that reduced adrenergic responsiveness in feline PO hypertrophy and failure involves increases in basal PLB-Thr17 phosphorylation, suggesting that activation of CaMKII in PO hypertrophy contributes to defective adrenergic reserve in compensated LVH and early heart failure.

dobutamine; excitation-contraction coupling; calcium

ACTIVATION OF ADRENERGIC SIGNALING pathways in cardiac myocytes is an essential mechanism to increase cardiac contractility during physiological hemodynamic stress, such as occurs during exercise. Cardiovascular disorders such as hypertension and valvular disease cause persistent hemodynamic overload, requiring that contractility and its regulators (sympathetic nervous system) are also persistently activated (2, 18). Persistent activation of cardiac adrenergic signaling pathways is associated with depression of additional sympathetic inotropic responses to further increase contractility. This blunted adrenergic contractile reserve is a characteristic feature of human heart failure (HF; see Ref. 14) and is an important indicator of the viability of diseased myocardium (36). Persistent sympathetic activation in HF patients correlates with myocyte apoptosis (3), increased mortality, and advanced disease status (38), and beta-receptor (sympathetic) antagonists reduce hospitalizations and improve survival (10).

Studies to define the basis of abnormal adrenergic regulation of contractility in HF have shown that beta1-receptors are downregulated and downstream signaling cascades of both beta1- and beta2-adrenergic receptors are altered (41, 51). These changes should reduce the activation of protein kinase A (PKA), which regulates contractility via phosphorylation of Ca2+ handling (42) and thin filament Ca2+-binding proteins (13). Although alterations in molecules within the adrenergic signaling cascade are established in left ventricular (LV) hypertrophy (LVH) and HF, changes in the abundance and phosphorylation state of PKA target proteins and their contribution to reduced contractility reserve are not yet clearly defined and are the topic of this study.

Phospholamban (PLB) is a key Ca2+ regulatory protein involved in adrenergic-mediated increases in cardiac contractility (48). Phosphorylation of PLB eliminates its inhibitory effect on the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA; see Ref. 39), thereby accelerating the rate of Ca2+ uptake in the SR (and thus speeding the rate of relaxation) of cardiac ventricular myocytes and increasing SR load (28). This increases the size of the systolic Ca2+ transient, ultimately increasing contractility. Changes in PLB abundance (e.g., PLB knockout) and phosphorylation state increase contractility and can rescue certain mouse myopathy models from HF (43), but mutations of PLB that increase SERCA activity in humans induce cardiomyopathies (44). Thus the role of PLB abundance and phosphorylation state in the cardiac dysfunction seen in large mammals with hemodynamic overload is not yet clearly defined.

PLB is phosphorylated by PKA at Ser16 subsequent to adrenergic activation and by calmodulin (CaM)-dependent kinase II (CaMKII) at Thr17 when pacing rate is increased (16) or during acidosis (33). Phosphorylation at either site is sufficient to remove the inhibitory effect of PLB on SERCA (20) and results in increased SR Ca2+ uptake with enhanced myocyte contractility and relaxation. Therefore, PLB acts as an integrator of beta-adrenergic and Ca2+-dependent signaling pathways to promote increased contractility. PLB-Ser16 phosphorylation occurs rapidly after adrenergic stimulation and is followed by PLB-Thr17 phosphorylation, subsequent to increased intracellular Ca2+ (25, 52). PLB-Thr17 phosphorylation can occur independently of PLB-Ser16 phosphorylation and is thought to be responsible for the gradual increase in relaxation rate with increased heart rate (HR). However, the role of CaMKII-mediated PLB-Thr17 phosphorylation in the regulation of contractility in the normal and diseased heart is controversial (6, 27).

There is evidence for significant interactions between Ca2+-mediated and beta-adrenergic signaling pathways, some suggesting that increases in intracellular Ca2+ lead to reduced beta-adrenergic effects on contractility (39, 47). Although the basis of these interactions is not well understood, it is clear that both adrenergic and Ca2+-dependent signaling are altered in hypertrophy and failure and that both signaling pathways influence myocyte survival, death, hypertrophy, and failure (37). PLB is a useful molecule to explore the alterations in these two important signaling cascades in hemodynamic overload because it has phosphorylation sites specific for both PKA and CaMKII.

The objective of the present study was to define the respective contributions of altered Ca2+ regulatory protein abundance and phosphorylation state to depressed sympathetic inotropic reserve in a large (feline) mammalian model of hypertrophy and early HF. Our results show that depressed adrenergic responsiveness is present in both compensated LVH and early HF and is significantly related to increased basal phosphorylation of PLB-Thr17, suggesting that activation of Ca2+-dependent signaling pathways is centrally involved in the depressed contractile reserve that characterizes the diseased heart.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aortic banding. Slow progressive pressure overload (PO) was induced by aortic constriction in 10 mongrel cats, as previously described (5). All animal work was done with the approval and under the supervision of the Temple University School of Medicine International Animal Care and Use Committee. Briefly, at 6–8 wk of age (0.9–1.1 kg), animals were sedated with ketamine and acepromazine (35 and 0.5 mg/kg, respectively), intubated, and maintained on 1.0% isoflurane and 100% O2. Through a right thoracotomy at the third or fourth intercostal space, the pericardium was entered, and a fixed, stationary band (12 mm circumference) was placed on the ascending aorta. This technique causes modest PO initially. However, as the animals grow, the band causes slow, progressive PO.

Echocardiography. Banded (n = 10) and control (n = 5) cats were sedated with ketamine (15–20 mg/kg). Six doses of dobutamine (0–40 µg·kg–1·min–1) were infused through a forelimb vein at 5-min stages. With the use of a Sonos 5500 (Hewlett-Packard, Andover, MA) echocardiography system, two-dimensional short-axis views and M-mode images of the LV were recorded at steady state. All short-axis and M-mode images were obtained at the level of the papillary muscles. Fractional area change (FAC) is expressed as (diastolic chamber area – systolic chamber area)/diastolic chamber area. HR was recorded during the echocardiogram for each animal using the Sonos 5500 electrocardiogram module.

Protein abundance and phosphorylation state. Animals were killed 2 days after dobutamine stress echocardiography, and protein abundance and phosphorylation state in the myocardium were analyzed using Western analysis (24). Target proteins were probed with antibodies for PLB (A1; Upstate, Lake Placid, NY), ryanodine receptor (RyR; Research Diagnostics, Flanders, NJ), and site-specific, phosphorylation-specific antibodies to PLB-Ser16, PLB-Thr17, and RyR-Ser2809 (gifts from Dr. J. Colyer, University of Leeds, Leeds, UK). SERCA was from Sigma (St. Louis, MO), L-type Ca2+ channel (Cav1.2 {alpha}1C) was from Chemicon (Temecula, CA), and Na+/Ca2+ exchanger (NCX) was from Swant (Bellinzona, Switzerland). Actin (Sigma) was used as an internal control. Target antigens were visualized with enhanced chemiluminescence (NEN Life Science), and band intensities were quantified with densitometric analysis using NIH Image 1.62f.

Myocyte isolation and culture. LV myocytes were isolated as previously described (50). After being washed with Krebs solution (in mmol/l: 12.5 glucose, 5.4 potassium chloride, 1.0 lactic acid, 1.2 magnesium sulfate, 130 sodium chloride, 1.2 sodium phosphate, 2.5 sodium bicarbonate, and 2.0 sodium pyruvate, pH 7.4) containing 1% (wt/vol) BSA, 10 mmol/l taurine, and 0.20 mmol/l calcium chloride, cells were resuspended in L-glutamate-free medium 199 (HEPES modification; Sigma Chemical) with antibiotics and plated on laminin-coated dishes (Nuclon 4-well dish, 1,600 mm2). Cells were paced at 1 Hz for 24 h (C-Pace 100; IonOptix, Milton, MA) or remained unpaced. After 4 h, the medium was changed, and, at 24 h, cells were either scraped for protein measurements or moved to an experimental chamber for cell contractility studies.

Cell physiology. All experiments were performed on single rod-shaped myocytes with clear sarcomeric cross striations. Suction-type patch pipettes were prepared from borosilicate glass (1B150F; World Precision Instruments, Sarasota, FL) using a two-stage pipette puller (model P-87; Sutter Instruments, Novato, CA). The pipette tip was heat polished before use and had a tip resistance of 4–8 M{Omega}. The pipette was attached to a patch-clamp amplifier (Axoclamp II; Axon Instruments, Foster City, CA). Cells were studied in a water-jacketed chamber at 37°C mounted on the stage of an inverted microscope (Axiovert 10; Zeiss, Thornwood, NY). The experimental chamber was perfused with normal Tyrode (NT) solution (in mmol/l: 150 NaCl, 5 HEPES, 2 sodium pyruvate, 5.4 KCl, and 1.2 magnesium chloride, pH 7.4) with 1 mmol/l calcium chloride.

The perforated patch technique was used to gain electrical access to the cell and induce contraction (9). The pipette solution contained (in mmol/l) 10 HEPES, 110 potassium aspartate, 5 dipotassium-ATP, 0.001 Tris-GTP, 2 magnesium chloride, 20 potassium chloride, 10 sodium chloride, and 1 calcium chloride and 240 µg/ml amphotericin B (in DMSO), pH 7.2 with potassium hydroxide. Amphotericin B caused perforations in the cell membrane, permitting electrical access to the cell while preventing the dialysis of cellular components that could alter cell function. After seal formation, increases in the capacitive current response to a –10-mV step were monitored to determine cellular access (3–5 min). To initiate action potentials, a square current pulse was applied at 1.0 Hz. After 5 min of recorded contractions, the bath solution was changed to NT with 0.01 µmol/l isoproterenol (ISO), and contractions were recorded for another 5 min. Contractions were recorded by video edge detection (VED-104; Crescent Electronics, Sandy, UT). pCLAMP 8.2 software (Axon Instruments) was used for data acquisition and analysis.

Statistical methods. All data are presented as means ± SE, and Figs. 1–4 are reported as either box plots (mean, median, quartiles, and extremes) or as histograms (means ± SE). Statistical difference between the mean values for two groups was evaluated using paired or unpaired Student's t-test, and one-way ANOVA was used to compare means between more than two groups. Linear regression analysis was used to test the relationship between two variables.


Figure 1
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  		Fig. 1. Dobutamine stress echocardiography in banded and control cats. A: M-mode images of the left ventricle (LV) at steady state in a control animal before and after 40 µg·kg–1·min–1 dobutamine M-mode recorded at steady state in the same animal. Fractional area change (FAC), derived from 2-dimensional (2-D) images (not shown), increased by 0.30 ± 0.04 (n = 5) in control animals. M-mode images of the LV at steady state in a representative banded animal before and after dobutamine are shown on bottom. FAC increased by 0.10 ± 0.03 (n = 10) in banded animals. SW, septal wall; FW, free wall. B–E: dobutamine stress echocardiography in banded (hatched boxes, n = 10) and control (open boxes, n = 5) cats. B: mean baseline FAC in banded and control animals. NS, not significant. C: FAC increase from baseline with 40 µg·kg–1·min–1 dobutamine from 10 individual banded animals ({square}) and pooled controls ({blacksquare}, n = 5). Some banded animals had higher baseline FAC than control, and others had lower baseline FAC than control. D: absolute increase in FAC with peak dobutamine. E: relative increase in FAC with peak dobutamine. Box plots show the median (central line), mean (open square), quartiles (outer box limits), and extremes. *P < 0.05 and **P < 0.01.

 

Figure 4
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  		Fig. 4. PLB phosphorylation state and isoproterenol (ISO) response in paced and unpaced cultured control feline LV myocytes. A: representative Western blot analysis of PLB and RyR phosphorylation state and SERCA abundance in quiescent cells, cells continuously paced for 24 h, or quiescent cells (for 24 h) paced for 20 min at 0.5 and 1.0 Hz, before and after 0.01 µmol/l ISO. B–D: data from quiescent cells and cells paced for 24 h. PLB-Thr17 (B) and PLB-Ser16 (C) phosphorylation state in paced and unpaced cells before and after ISO. D: representative contractions from paced and unpaced cells before and after ISO. E: mean data ± SE of fractional shortening [FS, %resting cell length (RCL)] of paced and unpaced cells before and after ISO. ND, not detectable. *P < 0.05 and **P < 0.01 vs. unpaced. #P < 0.05 and ##P < 0.01 vs. before ISO (within group); n = 9 for both groups.

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Baseline in vivo structure and functional data. Aortic constriction (banding) caused LVH and, in a subset of animals, LV dilation (Table 1). Banded animals with LV dilation [end- diastolic diameter (EDD) >1.5 cm] exhibited signs of early congestive HF (shortness of breath upon exertion and pulmonary effusions and ascites at death), whereas those with concentric hypertrophy (EDD < 1.5 cm) had no signs or symptoms of HF. Banded animals had a higher HR under baseline conditions (285 ± 19 vs. 222 ± 10 beats/min in controls, P < 0.05, Table 1), LV free wall thickening (0.61 ± 0.04 vs. control 0.43 ± 0.04 cm, P < 0.05, Table 1), and larger EDD [1.43 ± 0.07 vs. control 1.30 ± 0.05 cm, not significant (NS)] and end-systolic chamber diameter (0.82 ± 0.09 vs. control 0.64 ± 0.02 cm, P < 0.05). Figure 1A, left, shows representative M-mode echocardiographic images representing baseline LV contractions from which FAC was derived. Systolic function was evaluated by measuring the baseline FAC, an estimate of ejection fraction. Data in Fig. 1B are represented as box plots with mean, median, and quartiles to visually represent the spread of the data. This parameter was not different between banded and control groups at baseline. However, there was a broad range of FAC with a significant inverse correlation between FAC and EDD (data not shown). Animals with EDD <1.5 cm had a trend toward an increased baseline FAC when compared with animals with EDD >1.5 cm (65 ± 5%, n = 6 vs. 52 ± 8%, n = 4, NS; Fig. 1B).


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  		Table 1. Echocardiography data of LV geometry and function in pressure overload hypertrophy and control animals

 
Dobutamine stress echocardiography. Banded and control animals had similar HR at maximal dobutamine concentrations (294 ± 15 vs. 292 ± 9 beats/min in controls, NS; Table 1). Figure 1A, right, shows representative M-mode images from control and banded animals after 40 µg·kg–1·min–1. Dobutamine caused a dose-dependent increase in FAC in all control animals and in 8 of 10 banded animals (Fig. 1C). Note that in two animals shown in Fig. 1C, FAC was reduced when stimulated with dobutamine. These animals exhibited a bell-shaped inotropic response to increasing doses of dobutamine with a slight reduction in FAC at the peak dose. In both cases, contractility increased between 0 and 20 µg·kg–1·min–1 dobutamine (data not shown) but diminished at 40 µg·kg–1·min–1. The peak effect of dobutamine (40 µg·kg–1·min–1) on contractility was significantly smaller in banded vs. control animals (increase in FAC of 10 ± 3 vs. 30 ± 4%, P < 0.01, Fig. 1D). Figure 1E shows that the increase in FAC relative to baseline FAC is also significant (45 ± 3 vs. 17 ± 4%, P < 0.05), showing that the response to adrenergic stimulation was greater in controls when normalized to baseline FAC. The mean increase in FAC in banded animals with concentric hypertrophy (EDD < 1.5 cm) was not significantly different than in animals with LV dilation (EDD >1.5 cm; 11 ± 3%, n = 6 vs. 7 ± 1%, n = 4, NS). However, both groups had reduced dobutamine-induced increases in FAC compared with control (P < 0.05 compensated LVH vs. control and P < 0.01 dilated LVH vs. control). The reduced dobutamine effect in banded animals did not correlate significantly with baseline FAC or ventricular dimensions (data not shown). These experiments showed that reduced adrenergic responsiveness was similar in animals with compensated LVH and in those with dilation and early HF. Therefore, those aspects of the study that examined the cellular and molecular bases of the depressed in vivo contractile response to dobutamine combined data from both groups of banded animals (concentric and dilated LVH).

Protein abundance and phosphorylation state in hypertrophy. Western blot analysis was used to determine the protein abundance of PLB, RyR, SERCA, CaV1.2 {alpha}1C, and NCX and the abundance of phophorylated PLB and RyR (Table 2). Representative Western blot examples of baseline tissue samples taken from individual control and banded myocardium are shown in Fig. 2A. Protein abundance was normalized to actin (shown in Fig. 2A). PLB abundance was smaller in banded animals vs. controls, but this difference just failed to reach statistical significance (Fig. 2B, P = 0.07) and is shown as a box plot to graphically represent the spread of the data. Total PLB phosphorylation (combined PLB-Ser16 and PLB-Thr17; Fig. 2C) was significantly greater in banded animals (P < 0.05). Because there was no significant difference between control and banded PLB-Ser16 phosphorylation (Fig. 2D), this increase was largely the result of increased PLB-Thr17 phosphorylation (Fig. 2E, P < 0.05). These experiments show that there is a significant increase at the PLB phosphorylation at the CaMKII site in banded animals with persistent PO.


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  		Table 2. Western blot analysis of baseline abundance and phosphorylation state of molecules involved in calcium handling in LV myocardium from control and pressure-overload hypertrophy animals

 

Figure 2
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  		Fig. 2. A: Western blots of representative control and banded LV myocardial samples. RyR, ryanodine receptor; {alpha}1C, L-type Ca2+ channel {alpha}1C-subunit; NCX, Na+/Ca2+ exchanger; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; PLB, phospholamban; MW, mol wt marker. B–E: PLB abundance and phosphorylation state in control (open boxes, n = 5) and banded (hatched boxes, n = 10) animals. B: Western blot analysis of total PLB abundance (P = 0.07). RU, relative units. C: total phosphorylation using phosphorylation-specific, site-specific antibodies (Ser16- and Thr17-site phosphorylation, normalized to total PLB). D: PLB-Ser16 phosphorylation state-specific band intensity, normalized to PLB abundance. E: PLB-Thr17 phosphorylation state-specific band intensity, normalized to PLB abundance. *P < 0.05.

 
RyR abundance was increased in banded animals (P = 0.07), but RyR-Ser2809 phosphorylation was not significantly different in control vs. banded animals (Table 2). Cav1.2 {alpha}1C-subunit was significantly reduced in banded animals (P < 0.01), but NCX and SERCA abundance were not significantly different between groups. In a previous study from our laboratory in this model, we showed that Ca2+ current (ICa) is reduced in banded animals with LVH and even further reduced in animals with HF compared with control animals (17). Combined, these data strongly support the idea that ICa is reduced in this model and that its reduction relates directly to the degree of functional derangement.

In vivo adrenergic responsiveness and baseline PLB phosphorylation. To test the hypothesis that abnormal adrenergic contractility reserve in LVH is related to the baseline phosphorylation state of PLB, we correlated in vivo contractile responses to 40 µg·kg–1·min–1 dobutamine in each animal with the basal in situ PLB phosphorylation state (Fig. 3). These analyses showed that the amount of Thr17 phosphorylation was inversely related to the increase in FAC with dobutamine (r2 = 0.81, P < 0.05). Neither total PLB phosphorylation (Fig. 3B) nor PLB-Ser16 phosphorylation (Fig. 3C) was significantly correlated with the increase in FAC with dobutamine. Pooled controls are shown for comparison. These data show an association between the baseline phosphorylation state of PLB, a key effector molecule for beta-adrenergic and Ca2+-dependent signaling, and adrenergic contractile reserve with a significant correlation between the magnitude of reduced adrenergic reserve in feline LVH and PLB-Thr17 phosphorylation. Adrenergic contractile responses did not correlate with the abundance or phosphorylation state of any other measured Ca2+ regulatory protein (data not shown).


Figure 3
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  		Fig. 3. Correlation between in vivo adrenergic reserve and PLB phosphorylation state in control ({blacksquare}, pooled, n = 5) and banded ({square}, n = 10) animals. A: linear regression analysis of the relationship between the increase in FAC with 40 µg·kg–1·min–1 dobutamine and phosphorylation state of PLB-Thr17 for 10 banded animals. Pooled control (n = 5) is shown for comparison. The correlation coefficient (r2) is 0.81 (P < 0.05), and 95% confidence intervals are shown for the linear regression analysis. The increase in FAC with 40 µg·kg–1·min–1 dobutamine did not correlate with total phosphorylation (PT; B) or PLB-Ser16 phosphorylation (C).

 
Phosphorylation state of PLB and ISO response in isolated myocytes. Our in vivo studies suggest that, in PO hypertrophy, the reduced contractile effects of catecholamines are related to increased PLB-Thr17 phosphorylation, possibly through the elevation in cell Ca2+ needed to maintain cardiac output in the face of persistent hemodynamic overload. To further explore this idea, the effects of ISO on contractility and PLB phosphorylation were measured in isolated myocytes that had been either quiescent for 24 h or continuously paced to induce Ca2+ transients and contractions. Figure 4A shows a representative Western blot of proteins extracted from paced and unpaced control cells. Continuously paced cells had greater PLB-Thr17 phosphorylation than unpaced cells (Fig. 4B). Short durations of pacing (20 min) in cells that were quiescent for 24 h also caused an increase in PLB-Thr17 and RyR-Ser2809 phosphorylation, and the magnitude of the effect was rate dependent (Fig. 4A). Subsequent exposure to ISO caused smaller increases in PLB-Ser16 phosphorylation in myocytes that had been paced. Importantly, PLB-Ser16 was not detected in either paced or unpaced myocytes in the absence of ISO, showing no basal activation of PKA signaling under these conditions. Exposure to 0.01 µmol/l ISO caused a significantly smaller increase in PLB-Ser16 phosphorylation in paced cells relative to unpaced cells (Fig. 4C, P < 0.05). Paced and unpaced cells had similar contraction magnitudes before ISO application (2.1 ± 0.3% unpaced vs. 2.1 ± 0.2% paced, Fig. 4, D and E). However, the effect of ISO on contractility was significantly smaller in paced vs. unpaced cells (increase in fractional cell shortening, 5.1 ± 0.05%, n = 9 vs. 1.5 ± 0.04%, n = 9, P < 0.01). These experiments demonstrate that the basal phosphorylation state of PLB at Thr17 is greater in paced cells with Ca2+ transients and that these cells have a smaller response to beta-agonists than quiescent myocytes. These effects can occur within minutes and are maintained when pacing is continued for up to 24 h. These data suggest a direct relationship between persistent activation of Ca2+-dependent signaling and blunted adrenergic regulation of contractility.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Chronic hemodynamic stress increases the contractile demands on cardiac myocytes. Physiological hemodynamic stress, such as during aerobic exercise, is transient and induces beneficial changes in cardiac function. Pathological hemodynamic stress, such as hypertension or aortic stenosis, is persistent and induces dysfunctional changes in cardiac performance. The initial cardiovascular response to PO involves activation of the sympathetic nervous system to increase myocyte contractility and maintain cardiac output (23, 26). With time the myocytes hypertrophy and develop alterations in their physiological properties. Sustained sympathetic activity to the heart is known to induce alterations in adrenergic signaling, including reduced beta-adrenergic receptor density (26), abnormal coupling of agonist-activated receptors to G proteins and downstream enzymes such as adenylate cyclase, alterations in the phosphorylation of PKA target proteins (8, 31), and the induction of apoptosis (3). All of these changes could be involved in the reduced contractile reserve of the diseased heart and in the transition from compensated hypertrophic states to congestive HF.

In the present investigation, we studied the idea that the persistent increases in Ca2+ needed to maintain cardiac output in the face of increased hemodynamic stress contribute to abnormal sympathetic regulation of contractility. We correlated abnormalities in in vivo sympathetic contractility reserve in an animal model of progressive PO with either compensated hypertrophy or early HF with alterations in the abundance and phosphorylation state of Ca2+ regulatory proteins. We then determined adrenergic responsiveness in paced vs. unpaced isolated myocytes with demonstrated alterations in Ca2+ regulatory protein phosphorylation state to more clearly define the mechanisms for our in vivo findings. Our major findings are that 1) sympathetic contractile reserve is reduced in animals with compensated LVH and those with LVH dilation and early HF; 2) there were no significant changes in SERCA, NCX, or RyR abundance or RyR phosphorylation in these hypertrophied hearts; 3) PLB-Thr17 phosphorylation was increased and L-type Ca2+ channel (CaV1.2 {alpha}1C) protein abundance was reduced in LVH hearts; however, only altered PLB-Thr17 phosphorylation was correlated with blunted adrenergic effects on contractility; and 4) chronic pacing of isolated myocytes for 24 h increased PLB-Thr17 phosphorylation, and these myocytes had reduced adrenergic contractile responsiveness. These results strongly support the idea that activation of Ca2+-dependent signaling pathways can induce PLB-Thr17 phosphorylation and that this effect is sufficient to produce blunted adrenergic effects on contractility. Our findings also show that, in this model, the loss of adrenergic contractility reserve occurs before the transition from compensated hypertrophy to HF and therefore might be a critical factor in this transition.

Aortic banding creates LVH with and without cardiac dysfunction and dilation. In our model of PO hypertrophy, a fixed constriction in young animals creates a slowly increasing PO as animals mature and grow. A subset of these animals shows clinical signs of HF such as labored breathing and lethargy with activity. Echocardiographic evaluation documented LV dilation with mitral regurgitation and left atrial enlargement in this subset of PO animals. The animals with a dilated phenotype typically had weaker contractions and a smaller baseline FAC. PO most often induced concentric hypertrophy, with well-maintained contractile function and, in most cases, high baseline FAC. A major goal of this study was to determine if and why adrenergic contractility reserve is altered in PO LVH with or without LV dilatation and to determine if there are differences between LVH with and without dilation and early HF. Dobutamine stress echocardiography revealed that banded animals have a blunted response to adrenergic stimulation compared with controls. Importantly, contractile responses to dobutamine were uniformly depressed and were not exacerbated in the animals with dilation and early HF. These findings show that, in this model, alterations in adrenergic signaling are not exclusive to HF but also occur earlier in hearts with well-compensated baseline function. These results suggest that animals with compensated baseline LV function have an observable defect in contractility reserve that may predispose them to subsequent failure.

We also found that some banded animals had a bell-shaped dose response to dobutamine and, in two animals, reduced contractility at the peak dose of dobutamine. It is possible that these animals experienced Ca2+ overload with this high inotropic state that can reduce systolic force generation by causing heterogeneous spontaneous SR Ca2+ release, an effect well known with cardiac glycosides (11). These animals were included in the study in spite of this confounding observation, and we found that the animal with the greatest drop in FAC with dobutamine had the greatest phosphorylation at PLB-Thr17 by Western blot analysis.

PLB abundance and phosphorylation state are altered in hypertrophy. Sympathetic activation increases contractility by increasing Ca2+ influx (phosphorylation of the L-type Ca2+ channel) and by increasing the rate of SR Ca2+ uptake and storage (secondary to PLB phosphorylation), thereby increasing the size of the systolic Ca2+ transient. Blunted adrenergic responsiveness in LVH and HF is likely to involve changes in the abundance of Ca2+ regulatory proteins and their phosphorylation state (42). Ca2+ homeostasis is altered in end-stage human HF (19), with reduced SR Ca2+ stores, especially at fast pacing rates, being largely responsible for these changes (40). Reduced SERCA abundance (24), decreased PLB phosphorylation (46), and increased RyR (30) and L-type Ca2+ channel phosphorylation (12) appear to underlie these end-stage defects. Ca2+ regulation in earlier stages of the cardiac response to stress is less well understood. The few studies that have addressed this issue suggest that basal Ca2+ regulation is near to or above normal in compensated LVH (49). Our results are consistent with these findings but expand these ideas by showing that derangements of adrenergic contractile reserve are present. Therefore, although basal contractile function is well preserved in PO-induced concentric hypertrophy, contractile reserve is significantly compromised.

We found further evidence supporting reduced ICa in this model of LVH and early HF. Although the functional significance of this alteration has not been fully documented in this model, reduced ICa could reduce trigger Ca2+ (possibly affecting excitation-contraction coupling) and reduce SR load. In the context of higher basal PLB phosphorylation at Thr17, there may be reduced trans-sarcolemmal Ca2+ fluxes from beat to beat and enhanced reliance on the SR as a source of Ca2+. Related studies in human HF have shown increased phosphorylation of the L-type Ca2+ channel but preserved current density in vitro, likely because of dysfunctional phosphatase activity (12, 45). Similarly, we found reduced Ca2+ channel density but also found reduced current density in vitro in this model of hypertrophy and early HF (17). L-type Ca2+ channels may be hyperphosphorylated in our feline model, and it is possible that this may also contribute to reduced adrenergic contractile responsiveness. In consideration of our findings with PLB, an interesting hypothesis is that CaMKII activation contributes to this L-type Ca2+ channel phosphorylation. This topic may be examined in future studies.

In the present experiment, we found that basal PLB phosphorylation was increased in PO feline hearts, a finding at odds with what we and others (24, 32) have observed in human end-stage HF. We suggest that this is likely to reflect an earlier stage in the response to persistent hemodynamic overload, at least in this animal model.

Adrenergic responsiveness is inversely related to PLB phosphorylation. The most important new finding in this study is that loss of adrenergic contractile reserve is related to increased PLB-Thr17 phosphorylation. Phosphorylation at PLB-Thr17 was significantly increased in PO hypertrophy, whereas phosphorylation at Ser16 was elevated but not statistically so. Because PLB-Thr17 phosphorylation, but not PLB-Ser16 phosphorylation, was significantly correlated with the inotropic response to infused dobutamine in PO hypertrophy animals, we conclude that CaMKII-mediated phosphorylation of PLB is directly involved in the reduced adrenergic reserve seen in this model. These observations are consistent with recent human studies that show that dobutamine stress echocardiography can be used to assess and predict myocardial reserve in hypertrophy and HF (22, 36). In addition, the positive predictive value of preserved adrenergic reserve in unloading procedures, like valve repair and LV mass reduction, suggests that the status of adrenergic contractile reserve may be a sensitive indicator of the overall status of the ability to maintain cardiovascular homeostasis. The relationship between blunted adrenergic responsiveness and increased phosphorylation at the CaMKII site on PLB suggests a central role for Ca2+-mediated signaling pathways in the myocardial response to pathological hemodynamic stress. We hypothesize that abnormal dobutamine stress echocardiography in patients with cardiovascular disease may be predictive of the level of activation of Ca2+-dependent signaling pathways that drive both dysfunctional cardiac contractility and pathological hypertrophy.

Ca2+-mediated signaling and adrenergic signaling interactions. Increases in intracellular Ca2+ alter adrenergic signaling pathways (29, 34, 35, 39). Ca2+ is thought to have a negative feedback effect on sympathetic stimulation, where the increase in Ca2+ stimulated by PKA activates CaM, which in turn activates the phosphatase calcineurin (CN). Ca2+ and CaM/CN can directly inhibit the formation of cAMP to modulate PKA-mediated protein phosphorylation (39). This mechanism, in combination with inactivation of the beta1-adrenergic receptor via receptor phosphorylation (23), could limit elevations in cytosolic Ca2+ in times of hemodynamic stress and prevent cellular Ca2+ overload and its sequelae. The literature suggests that persistent increases in intracellular Ca2+, such as those present in response to hemodynamic overload states, negatively regulate adrenergic signaling (29, 34, 35, 39). Recent studies have shown that increased CaMKII activity in HF may contribute to reduced SR Ca2+ content through enhanced leak through the RyR, contributing also to systolic dysfunction (1). We hypothesize that the persistent increases in Ca2+ transients that are required to maintain forward cardiac output in PO contribute to the abnormal adrenergic responsiveness we observed in vivo.

Why use myocytes from large mammals to study Ca2+-mediated cell signaling? In the present experiments, we used adult feline LV myocytes in short-term primary culture to test the effects of activation of Ca2+ transients on the basal phosphorylation state of PLB and subsequent phosphorylation caused by ISO. Our results show that continuously paced myocytes (with activation of Ca2+ release and uptake) have greater RyR phosphorylation at Ser2809 and PLB phosphorylation at Thr17 than unpaced cells and that paced myocytes have a reduced response to ISO. These types of studies are not easily performed in adult myocytes from small mammals (rats and mice) because these myocytes have higher cytosolic Na+ concentration ([Na+]) than myocytes from large mammals (15). The high cytosolic [Na+] raises cytosolic Ca2+ via the NCX, which promotes Ca2+ uptake in the SR via SERCA (7), thereby causing SR Ca2+ overload and spontaneous SR Ca2+ release (21). Therefore, unpaced mouse and rat ventricular myocytes have high time-averaged cytosolic Ca2+ concentration ([Ca2+]) that are likely to fully activate Ca2+-dependent signaling pathways under baseline conditions. Unpaced myocytes from large mammals (felines and humans) have lower cytosolic [Na+], which causes robust forward-mode Na+/Ca2+ exchange, low cytosolic [Ca2+], and low SR Ca2+ stores (4). With pacing, time-averaged cytosolic [Ca2+] increases in large mammalian myocytes, allowing exploration of Ca2+-mediated signaling over a broad range of cytosolic [Ca2+]. Our results show very low basal phosphorylation of either PLB-Ser16 and -Thr17 and, with pacing, a selective increase in PLB-Thr17 and RyR-Ser2809 phosphorylation with reduced adrenergic effects on contractility. These findings support the idea that a persistent increase in Ca2+-mediated signaling reduces adrenergic responsiveness by limiting PKA-mediated protein phosphorylation.

In summary, our study shows that blunted adrenergic responsiveness in PO hypertrophy occurs during "compensated" LVH, continues into early HF, and is directly related to the phosphorylation state of PLB at Thr17. Moreover, we demonstrate that Ca2+-dependent signaling mechanisms are modulators of adrenergic responsiveness and hypertrophy. Our results suggest that activation of Ca2+-mediated signaling pathways directly modulates adrenergic responsiveness through downstream effects on PLB, an integrator of PKA and CaMKII signaling cascades. We further suggest that these mechanisms are centrally involved in the alterations in inotropic responsiveness that characterize the hypertrophied and failing heart.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by American Heart Association postdoctoral fellowship 01205021U (H. Kubo), by National Heart, Lung, and Blood Institute Grants HL-33921 and HL-61495, and by the Temple University School of Medicine MD/PhD Program (G. D. Mills).


   ACKNOWLEDGMENTS
 
We thank Rachel Wilson and Dr. Xiongwen Chen for technical guidance and for assistance in manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. R. Houser, Cardiovascular Research Center, MRB 204, Temple Univ. School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140 (e-mail: srhouser{at}temple.edu)

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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