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(Circulation. 2001;103:889.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Interactions Between Phospholamban and -Adrenergic Drive May Lead to Cardiomyopathy and Early Mortality

Rajesh Dash, BS; Vivek J. Kadambi, PhD; Albrecht G. Schmidt, MD; Nicole M. Tepe, PhD; Danuta Biniakiewicz, PhD; Michael J. Gerst, BS; Amy M. Canning, BS; William T. Abraham, MD; Brian D. Hoit, MD; Stephen B. Liggett, MD; John N. Lorenz, PhD; Gerald W. Dorn, II, MD; Evangelia G. Kranias, PhD

From the Department of Cardiovascular Biology (V.J.K.), Millennium Pharmaceuticals Inc, Cambridge, Mass; the Department of Medicine (B.D.H.), Case Western Reserve University, Cleveland, Ohio; and the Departments of Pharmacology (R.D., A.G.S., N.M.T., M.J.G., S.B.L., G.W.D., E.G.K.), Physiology (J.N.L., E.G.K.), and Medicine (D.B., A.M.C., W.T.A., S.B.L., G.W.D.), University of Cincinnati College of Medicine, Cincinnati, Ohio.

Correspondence to E.G. Kranias, PhD, Department of Pharmacology and Cell Biophysics, University of Cincinnati, 231 Bethesda Ave, Cincinnati, OH 45267-0575.

	Abstract

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Background—Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of -adrenergic stimulation. Chronic -adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental.

Methods and Results—Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality.

Conclusions—The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.

Key Words: aging • calcium • catecholamines • sarcoplasmic reticulum

	Introduction

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Heart failure is a complex syndrome, characterized by myocardial remodeling, left ventricular dysfunction, and impaired myocyte calcium handling. Concurrent with a progressive deterioration in cardiac performance are graded adjustments in the regulation of myocardial contractility, vascular tone, and intravascular volume, which stem primarily from an increased neurohormonal influence over the cardiovascular system.¹ One such neurohormonal adjustment is increased plasma norepinephrine concentrations and/or depleted myocardial norepinephrine stores, which reflect enhanced adrenergic stimulation to correct systolic and diastolic dysfunction in failing myocardium.² However, chronic elevation of sympathetic drive can exert deleterious effects on the myocardium, promoting cardiomyocyte hypertrophy and left ventricular dysfunction,³ while serving as a powerful prognostic indicator of heart failure mortality.⁴ Despite these observations, it is unknown whether an isolated physiological increase in adrenergic stimulation may also lead to cardiomyopathy.

At the subcellular level, -adrenergic signaling is associated with phosphorylation of several downstream targets, which mediate both inotropic and lusitropic effects, composing a generalized compensatory response to diminished cardiac output.⁵ The most critical functional substrate of the cardiac -adrenergic pathway is phospholamban (PLB), the inhibitor of sarcoplasmic reticular (SR) calcium sequestration. Several reports have shown that increases in the activity of PLB may contribute to depressed SR function and contractility in failing human hearts.^{6 7 8} However, the activity of PLB can be negated, and contractility augmented, on the phosphorylation of the protein by protein kinase A during -adrenergic stimulation.^{9 10} Indeed, transgenic mouse models, which overexpressed upstream adrenergic signaling effectors (ie, the ₂-receptor and G_s subunit), induced marked increases in PLB phosphorylation¹¹ and even decreases in total PLB protein¹² that were linked to enhanced cardiac performance.

Although the forward cascade leading to PLB phosphorylation has been well documented, a feedback regulatory mechanism by which increased PLB inhibition may modulate its own neurohormonal axis has not yet been explored. Thus, the present study examined whether compensatory increases in adrenergic drive could be elicited by increased PLB inhibition of cardiac output and whether this physiologically-induced adrenergic response could lead to cardiomyopathy on its own. By use of transgenesis, an animal model with high expression levels (4-fold) of PLB in the heart was created to maximally inhibit SR and cardiac function. The overexpressed PLB was highly phosphorylated in vivo because of increased adrenergic drive, an important compensatory response in young mice. However, with aging, PLB phosphorylation diminished as transgenic hearts developed a desensitization of adenylyl cyclase and hypertrophy, accompanied by deteriorating left ventricular function and premature mortality. In this regard, the influence of PLB on its neurohormonal axis evoked a heightened sympathetic tone that eventually produced cardiomyopathy and failure in transgenic hearts.

	Methods

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Generation of 4-Fold PLB TG Mice
Hemizygous transgenic mice (2-fold PLB overexpression)¹³ were mated to generate homozygous transgenic (TG) mice. Homozygosity was confirmed with wild-type (WT) backcross matings, from which all offspring were transgene positive by polymerase chain reaction analysis of tail DNA.¹³ Also, two hemizygous lines (18 and 23) were mated to generate offspring (No. 418) possessing transgenes from each parent. All mice used in the present study were FVB/N males.

Quantitative Immunoblotting
Cardiac homogenates and SR-enriched membranes were subjected to quantitative immunoblotting.¹³ Primary antibody was detected by peroxidase-conjugated secondary antibody (ECL, Amersham). Protein concentration was determined by the Bradford method with a BSA standard.

Ventricular Cardiomyocyte Parameters and Calcium Transients
Calcium-tolerant, unloaded, left ventricular myocytes were isolated¹³ and paced at 0.5 Hz with or without 2-minute isoproterenol preincubation (400 nmol/L). Calcium transients were measured with the use of fura 2 (emission 510 nm) and reported as the 340/380-nm fluorescence ratio.

Closed-Chest Catheterization Measurements
Closed-chest anesthetized mice were catheterized as described.¹⁴ Ten-minute baseline recordings were obtained before propranolol (150 µL of 100 ng/µL stock) administration, and propranolol treatment was compared with baseline. The time constant for isovolumic relaxation, tau (), was calculated as described.¹⁵ Left ventricular pressure tracings were fit to P_t=P_oe^-t/, where P is pressure at time t (P_t) and at dP/dt_min (P_o).

Adenylyl Cyclase Activity and -Adrenergic Receptor Density
Adenylyl cyclase was determined in cardiac membranes¹⁶ by using 100 µmol/L forskolin for maximal activity. Total -receptor density used 400 pmol/L [¹²⁵I]cyanopindolol binding.¹⁶

In Vivo Echocardiography
Two-dimensional M-mode echocardiography and color Doppler echocardiography were performed as described.¹⁷

-Adrenergic Blockade
Eight-week-old WT and TG mice were given propranolol (0.5 g/L) in their drinking water¹⁸ for 10 weeks. This dose of propranolol was previously determined to blunt the stimulation of cardiac function by isoproterenol.¹⁸

Tissue and Plasma Catecholamine Levels
Catecholamine levels were determined by use of a high-performance liquid chromatographic pump (model 580, HR-80 [C18], 4.6x80-mm column; ESA, Inc) and an electrochemical detector 5200A (Coulochem II, ESA) with a mobile phase (Cat-A-Phase II, ESA) at a 1.2-mL/min flow rate.¹⁹ Mice were anesthetized with 2.5% tribromoethanol (15 µL/g) for 45 minutes before left ventricular puncture and blood/heart extraction.

Histopathologic and Dot-Blot Analyses
Standard techniques were used for histological examination (Masson’s trichrome sections) and dot-blot total RNA analysis of the cardiac left ventricles.²⁰

Materials
Type II collagenase (Worthington Biochemical) was used. Antibodies were as follows: PLB-monoclonal, calsequestrin-polyclonal (Affinity BioReagents), PS-16- and PT-17-polyclonal (PhosphoProtein Research), and SERCA-polyclonal.²¹

Statistical Analysis
Data are presented as mean±SEM. Statistical analyses were performed by use of the Student t test or 2-way ANOVA.

	Results

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Generation and Identification of Homozygous TG Animals
Mice with cardiac-specific overexpression of PLB (hemizygous) were generated previously.¹³ Despite 2 pronuclear microinjections of the -myosin heavy chain promoter–driven PLB cDNA construct, 2-fold PLB overexpression was the highest expression level achieved. To further increase cardiac PLB levels, hemizygous transgenic mice¹³ were interbred. Approximately one fourth of the offspring exhibited twice the transgene level (TG mice) compared with the level in hemizygous transgenic animals, via Southern blotting (Figure 1A). In addition, 2 separate hemizygous transgenic lines were crossbred to generate offspring expressing transgenes from each parent.

View larger version (34K): [in this window] [in a new window]   Figure 1. Generation of 4-fold PLB-overexpressing mice. A, Southern blot for PLB transgene. Hemizygous transgenic lines were mated, and progeny tail DNA was digested with EcoRI and screened with polymerase chain reaction–labeled 32P-labeled PLB cDNA probe. HZ indicates hemizygous transgenic; 6.8 kb and 2.5 kb, fragment sizes of endogenous PLB and PLB transgene, respectively. B, Immunoblots of SERCA, calsequestrin (CSQ), and PLB from cardiac homogenates (top) and SR-enriched preparations (bottom). C, Relative PLB/SERCA ratios in TG compared with WT homogenates. Data represent mean±SEM of 4 determinations per group of 4 pooled hearts.

Quantitative immunoblotting of cardiac homogenates or SR-enriched membranes revealed no alterations in TG SERCA or calsequestrin protein levels compared with WT levels. However, the protein levels of PLB and the relative PLB/SERCA ratio were 4-fold higher in TG hearts (line 415), with the overexpressed PLB inserted into the SR membrane (Figure 1B and 1C).

Isolated Cardiomyocyte Analysis
To determine the functional effects of 4-fold PLB overexpression, mechanical parameters were assessed in isolated myocytes, which represent a load-independent preparation, under identical pacing conditions. Myocytes from 2-fold PLB-overexpressing hearts were also analyzed to compare the present results with previous findings.¹³ The extent of shortening (percent shortening) and the maximal rates of both relengthening (-dL/dt) and shortening (+dL/dt) were significantly reduced in TG myocytes compared with WT myocytes or 2-fold PLB-overexpressing myocytes (percent shortening 72±7% versus 52±9%, +dL/dt 68±10% versus 39±7%, and -dL/dt 60±8% versus 33±7% in 2-fold versus 4-fold overexpressing cells, respectively; WT 100%) (Figure 2A through 2C). Myocyte mechanical function was also analyzed in a second line (No. 418), generated by crossing 2 hemizygous transgenic lines, as described above. There were 3.3-fold increases in cardiac PLB levels, which resulted in impaired mechanical parameters equaling 46±5% for percent shortening, 38±5% (57±6 µm/s) for +dL/dt, and 30±8% (38±3 µm/s) for -dL/dt compared with WT values (100%). Thus, the observed PLB gene dosage effect indicated that the depressed function in homozygous transgenic hearts was not due to transgene insertional position in the genomic DNA. All subsequent analyses were performed with use of the 4-fold PLB overexpression line (TG mice).

View larger version (28K): [in this window] [in a new window]   Figure 2. Cardiomyocyte mechanics and calcium transients. Basal (-) and isoproterenol (ISO)-stimulated (+, 400 nmol/L) contraction (A, +dL/dt; C, percent shortening) and relaxation (B, -dL/dt; D, t80) parameters in WT (solid bars) versus TG (shaded bars) myocytes paced at 0.5 Hz. t80 represents the time for 80% of peak calcium amplitude decay. Values are mean±SEM of 4 hearts with 7 to 10 myocytes analyzed per heart. *P<0.05 vs WT.

The functional impairment in myocytes overexpressing 4-fold PLB pointed to an alteration in calcium handling. Fura 2–loaded WT and TG myocytes revealed that the time required for calcium to return to 80% of baseline (t₈₀) was significantly longer in TG myocytes (Figure 2D), although the systolic calcium amplitude was not significantly depressed (340/380 nm ratio was 1.02±0.13 for WT and 0.82±0.17 for TG; P=0.39). Notably, this prolongation of the calcium transient (54%) was greater than that observed in 2-fold PLB-overexpressing myocytes (30%). Because PLB has been proposed to mediate a major portion of the cardiac response to -agonists, cells were maximally stimulated with 400 nmol/L isoproterenol. Isoproterenol-stimulated mechanical and calcium kinetic parameters were similar between TG and WT myocytes, consistent with removal of the inhibitory effects of PLB (Figure 2A through 2D).

In Vivo Cardiac Function
To determine whether the depressed myocyte function on PLB overexpression reflected similarly impaired function in vivo, left ventricular contraction and relaxation indices were assessed by use of invasive solid-state micromanometer catheters. TG mice exhibited no alterations in developed pressure; however, a significant depression was observed in the maximal rate of pressure development (+dP/dt, Figure 3A and 3B). Furthermore, TG ventricles displayed elevated end-diastolic pressure (Figure 3C), a significant (P<0.05) depression in -dP/dt (8.3±0.2 versus 7.5±0.3 mm Hg/sx10^-3 for WT versus TG), and a prolongation in the time constant for isovolumic left ventricular relaxation, or (Figure 3D).

View larger version (26K): [in this window] [in a new window]   Figure 3. In vivo cardiac function. Left ventricular inotropic indices are systolic pressure (A) and rate of pressure development (+dP/dt, B); lusitropic indices are left ventricular end-diastolic pressure (C) and time constant for left ventricular relaxation (tau, D). Values represent mean±SEM in WT (solid bars, n=6) and TG (shaded bars, n=6) mice. *P<0.05 vs WT.

Compensatory Mechanisms
To determine whether phosphorylation of the overexpressed PLB was serving as compensation for its increased inhibition of SERCA, quantitative immunoblotting with phosphorylation site–specific antibodies was used. Figure 4A shows that basal PLB phosphorylation was substantially higher in TG than in WT hearts at both Ser16 (8.9±0.7-fold) and Thr17 (5.1±1.3-fold), representing an important compensatory mechanism to relieve the inhibitory effects of PLB on SR function. These increases were specific to hearts expressing high (4-fold) PLB levels, inasmuch as hearts expressing 2-fold PLB did not exhibit any phosphorylation increases (mol P_i/mol PLB data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 4. PLB phosphorylation status. Representative immunoblots probed with site-specific PLB phosphorylation antibodies to P-Ser16 (PS-16) and P-Thr17 (PT-17). A, Three-month-old hearts. High-molecular-weight phosphorylated PLB (PLBH) and low-molecular-weight phosphorylated PLB (PLBL) are shown. B, Hearts at 18 weeks of age, without (-) or with (+) 10-week propranolol (prop) treatment (0.5 g/L).

The phosphorylation of PLB is mediated predominantly through -adrenergic signaling. To examine whether the increased phosphorylation was due to physiological increases in catecholamine stimulation, in vivo cardiac function was assessed in the presence and absence of a -receptor antagonist, propranolol. In catheterized mice, acute administration of propranolol had no significant effect on the contractile parameters of WT hearts (for respective baseline versus propranolol values, +dP/dt was 10.3±0.7 versus 9.1±0.4 mm Hg/sx10^-3, and was 5.5±0.1 versus 5.9±0.3 ms). Conversely, propranolol was able to significantly depress the contraction rate and prolong the relaxation phase in TG hearts (for respective baseline versus propranolol values, +dP/dt was 10.2±0.4 versus 7.4±0.3 mm Hg/sx10^-3 [P<0.05], and was 7.2±0.2 versus 7.7±0.2 ms [P<0.05]). To observe the effects of a more prolonged -blockade treatment, propranolol was administered in the drinking water (0.5 g/L) of 8-week-old animals for 10 weeks. There was no effect of -blockade on WT left ventricular function, as determined by echocardiography. However, treated TG hearts exhibited significantly depressed fractional shortening and circumferential fiber shortening velocities (Vcf_c) compared with Vcf_c values in untreated TG hearts (Table 1). These functional effects appeared to be due to reduced phosphorylation of the overexpressed PLB (Figure 4B) and, thus, increased inhibition of SR function.

View this table: [in this window] [in a new window]   Table 1. Effects of -Adrenergic Blockade on Left Ventricular Function

Further verification of increased sympathetic drive was observed on high-performance liquid chromatographic analysis of cardiac tissue and plasma catecholamine levels. Significant (P<0.05) reductions in cardiac norepinephrine (968±58 pg/g for WT, 790±19 pg/g for TG), epinephrine (133±37 pg/g for WT, 19±3 pg/g for TG), and dopamine (63±4 pg/g for WT, 38±1 pg/g for TG) levels were found in TG hearts compared with WT hearts, consistent with previous findings of sympathetic nerve terminal depletion in hyperadrenergic mice and failing human hearts.^{22 23} The depletion of tissue catecholamine stores was accompanied by significant increases in plasma norepinephrine levels (3.63±0.48 ng/mL for WT, 5.55±0.49 ng/mL for TG; P=0.028), which more directly reflected the elevated adrenergic drive in TG animals. However, the degree of increased catecholamine stimulation in young TG animals did not significantly alter cardiac -adrenergic receptor density (20.2±1.0 versus 23.6±1.4 fmol/mg for WT versus TG, n=3) or adenylyl cyclase activity (Figure 7A).

View larger version (33K): [in this window] [in a new window]   Figure 7. Adenylyl cyclase and SR proteins. A, Adenylyl cyclase activity in 3-month (left) and 15-month (right) WT (open circles, n=3) and TG (closed circles, n=3) hearts, measured with increasing concentrations of isoproterenol (Iso). Data are expressed as percentage of forskolin-stimulated activity (average forskolin response was 515±27 pmol · min-1 · mg for WT and 507±20 pmol · min-1 · mg for TG). *P<0.05 for significantly lower maximum cyclase activity, and **P<0.05 for significantly higher EC50 compared with age-matched WT. B, SERCA, PLB, and PLB phosphorylation (PS-16) in 15-month-old WT (n=5) and TG (n=5) hearts. Protein levels for SERCA and PLB were normalized to calsequestrin quantified on same immunoblots. Data are expressed as fold±SEM of 3-month counterparts for WT and TG hearts.

Effects of Age
Because of impaired myocardial function and increased sympathetic tone, we examined whether TG hearts exhibited any associated cardiac pathology. At the morphological level, there were no alterations in young (3-month) TG hearts, as evaluated by light microscopy. Furthermore, analysis of left ventricular dimensions and left ventricular mass–to–body mass ratios indicated no significant differences (Table 2), and no alterations were detected in the expression levels of -myosin heavy chain, atrial natriuretic factor, or -skeletal actin, which are markers of hypertrophy (data not shown). Despite the absence of cardiac pathology at 3 months, TG mice displayed an early mortality between 15 and 18 months (Figure 5A). Therefore, we examined TG animals at the beginning of this time period. Fifteen-month-old WT mice displayed contractile parameters that were similar to those of their 3-month-old counterparts (Figure 5B, Table 2). Conversely, 15-month-old TG mice exhibited substantial deterioration in fractional shortening and Vcf_c compared with those values in 3-month-old TG or age-matched WT mice. In addition, 3-month TG Vcf_c values, which were corrected for heart rate differences, were significantly lower than 3-month WT values, similar to the closed-chest catheterization results. Besides the deterioration in left ventricular function, all aging TG mice (n=5) demonstrated dilated left ventricular chambers (13.5% increase in end-diastolic dimension) and increased left ventricular mass–to–body mass ratios (38%, Table 2). Histopathologic and gross examination revealed extensive interstitial fibrosis and hypertrophic myocytes in these hearts (Figure 6D and 6F) compared with minimal changes in aging WT hearts (Figure 6C and 6E). Increased atrial natriuretic factor (6.9±1.2-fold) and -myosin heavy chain (3.8±0.8-fold) gene expression, which may contribute to depressed function, were also detected in aging compared with young TG hearts, whereas no alterations occurred in aging WT hearts.

View this table: [in this window] [in a new window]   Table 2. Echocardiographic Parameters of Left Ventricular Function

View larger version (41K): [in this window] [in a new window]   Figure 5. Early mortality and depressed left ventricular function in aging TG mice. A, Kaplan-Meier survival curves comparing WT (dashed line, n=21) and TG (solid line, n=14) mice over a 20-month period. B, Representative M-mode echocardiograms from 15-month-old WT (top) and TG (bottom) left ventricles. Arrows mark interventricular septal wall (SW) and posterior wall (PW) of the left ventricle. Black lines denote scale.

View larger version (101K): [in this window] [in a new window]   Figure 6. Histopathology in WT and TG hearts: representative Masson’s trichrome staining for fibrosis (blue) in horizontally sectioned hearts from young and aging WT and TG mice. LV indicates left ventricle; RV, right ventricle. A and B, Three-month WT and TG hearts, respectively, are shown. Note relative absence of fibrosis in these sections. C through F, Aging WT (C; E, original magnification x200) hearts exhibited minimal changes, whereas aging TG (D; F, original magnification x200) hearts revealed extensive interstitial fibrosis (arrowheads) and enlarged cardiomyocytes. Coronary arteries (*) are shown in panels E and F as positive controls for fibrosis.

We then determined whether aging TG hearts had undergone any subcellular alterations. -Receptor densities were not significantly different (19±1 fmol/mg for WT [n=3], 27±4 fmol/mg for TG [n=3]; P=0.12), but the adenylyl cyclase activity from aging TG hearts revealed a blunted response to isoproterenol compared with activity from aging WT hearts (Figure 7A). Furthermore, protein levels of SERCA were reduced by 26%, total PLB was unchanged, and PLB phosphorylation was reduced at both Ser16 (44%) and Thr17 (50%) in aging compared with 3-month-old TG hearts (Figure 7B). Meanwhile, aging WT hearts exhibited no alterations in SERCA, PLB, or PLB phosphorylation (Figure 7B). Thus, the proportion of active (dephosphorylated) PLB and the relative PLB/SERCA ratio were increased, whereas -adrenergic responsiveness decreased in aging TG hearts, which may reflect the deterioration in left ventricular function.

	Discussion

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The contractile benefits derived from increased adrenergic stimulation, over time, undergo a poorly understood maladaptive transition, the clinical implications of which are still heavily debated.³ Although the cardiotoxic effects of an isolated increase in sympathetic drive have been illustrated in several animal models,^{24 25 26 27} these studies used either direct infusions of catecholamines²⁴ or increased expression of upstream signaling components in the adrenergic cascade. Four such models, 1 that overexpressed the active G_s subunit²⁵ and 3 that overexpressed the ₁-adrenergic^{27 28} and ₂-adrenergic receptors,²⁶ exhibited normal or even increased left ventricular performance in young mice, with eventual transition to cardiomyopathy and/or mortality on aging. However, apart from specific disease states that exhibit direct increases in catecholamines, such as pheochromocytoma,²⁹ chronic enhancement of sympathetic tone most commonly derives from primary decreases in cardiac output that elicit graded physiological increases in adrenergic drive to preserve function.¹ In the present study, we increased the levels of an inhibitory protein, PLB, which is a downstream substrate of protein kinase A and a major mediator of the -adrenergic response in the heart.¹⁰ A resulting threshold inhibition of SR and cardiomyocyte function triggered a compensatory increase in the sympathetic drive of this model, independent of any apparent cardiac remodeling, to relieve the inhibitory effects of PLB. On aging, TG mice exhibited a progression to cardiac hypertrophy, left ventricular chamber dilatation, and heart failure/sudden death, which resemble human disease.^{4 30}

Increased levels and/or activities of PLB^{6 13} have been implicated in the altered calcium homeostasis during excitation-contraction coupling of end-stage heart failure.^{31 32} However, increased PLB inhibition has never been implicated as a direct initiator of cardiomyopathy, inasmuch as a previously characterized 2-fold PLB overexpression model displayed no morphological alterations.¹³ Therefore, we used transgenic mice with high cardiac PLB levels (4-fold) as a mechanism to diminish SR and cardiac performance without directly inducing cardiac hypertrophy or failure. Four-fold PLB overexpression did produce a greater inhibition of calcium kinetics and mechanical function than did 2-fold PLB overexpression in isolated cardiomyocytes. The substantial in vitro depression translated into a significant but mild depression in vivo, as high PLB phosphorylation levels at both the Ser16 (9-fold) and Thr17 (5-fold) sites attenuated the inhibitory effects of PLB. Although stress/exercise tolerance experiments might have revealed deficits, young TG animals displayed no signs of heart failure and a full response to isoproterenol in vivo (data not shown). Moreover, the observed increase in PLB phosphorylation was specific to hearts with 4-fold overexpression, inasmuch as mice with a PLB/SERCA ratio of 2:1 did not exhibit increased phosphorylation compared with WT mice (data not shown). These findings suggested that 4-fold PLB overexpression might be associated with a "threshold" inhibition of SR and cardiac function, thus triggering an enhanced in vivo catecholaminergic drive as a compensatory mechanism to phosphorylate and inactivate the overexpressed PLB. In support of this hypothesis, increases in catecholamine signaling were evident in TG hearts, as -receptor blockade was able to reduce PLB phosphorylation and significantly depress transgenic contractile parameters. Neither acute nor 10-week propranolol administration had an effect on WT contractility, a finding that is consistent with results in nonfailing patients undergoing -adrenergic blockade³³ and that illustrates the lack of catecholaminergic support in WT hearts. Moreover, significant increases in TG plasma norepinephrine, which was associated with depletion of cardiac norepinephrine content, reflected the enhanced adrenergic stimulation of the TG myocardium.^{2 34}

The heightened adrenergic signaling in young TG animals may constitute an important physiological adjustment early in life, in an attempt to relieve the inhibition of SR function by PLB. Interestingly, the degree of catecholamine elevation in young TG hearts was below the level required to desensitize adenylyl cyclase responses or to downregulate -receptor density. However, aging TG animals displayed cardiac hypertrophy, fibrosis, and deteriorating left ventricular function, which are indicative of the progressive nature of this adrenergic response and which are consistent with previously described maladaptive consequences of chronically elevated sympathetic tone.^{3 35} In concurrence with this pathology, these mice also displayed a desensitized adenylyl cyclase response, which may have precluded adequate compensatory phosphorylation of PLB and thereby promoted depressed myocardial function. The desensitized adenylyl cyclase isoproterenol response (27% reduction in maximal activity, 3-fold increase in EC₅₀) was independent of any alterations in -receptor density, similar to previous observations in other animal models with adrenergic signaling–induced and hypertension-induced cardiac hypertrophy.^{20 36} Moreover, the diminished responsiveness to isoproterenol was specific to aging TG hearts, inasmuch as aging WT mice showed no such alterations in adenylyl cyclase compared with 3-month WT mice. It is critical to note that the Ser16 residue of PLB is phosphorylated exclusively through the -adrenergic cascade and that the reduction in its phosphorylation on aging coincided with the deterioration in left ventricular function. Therefore, the dominant neurohormonal effect, resulting from increased PLB expression, appears to be centered on the -adrenergic system. However, long-term studies with -blockade or catecholamine infusion may dissect the contributions of the -adrenergic cascade and other nonadrenergic neurohormonal pathways in this TG model. In this regard, increases in agonists such as angiotensin II and endothelin have been shown to induce cardiac hypertrophy³⁷ and may also be involved in PLB-overexpressing mice, thereby contributing to the pathology observed on aging.

The observed alterations in adrenergic signaling have suggested that SR calcium-handling proteins might also be altered with aging. Aging TG hearts displayed no change in total PLB but significant decreases in SERCA protein levels (26%) and in the phosphorylation status of PLB (44% to 50%), which might reflect the desensitized adenylyl cyclase response. Together, the selective downregulation of SERCA and decreased PLB phosphorylation would lead to greater inhibition of SR function, similar to previous observations in catecholamine-induced hypertrophy models and explanted failing human hearts.^{6 8 38}

In summary, the present study provides the first evidence that increased PLB expression can induce a physiologically enhanced sympathetic drive to attenuate its inhibitory effects. Over the long term, this hyperadrenergic state appears to progress from an early compensatory mechanism to a maladaptive one, which leads to the development of fibrotic cardiomyopathy. Future studies may be designed to elucidate the physiological and molecular mechanisms that connect SR calcium handling, neuroendocrine activation, and the progressive development of hypertrophy and heart failure.

	Acknowledgments

 
This work was supported by an American Federation for Aging Research fellowship (R. Dash) and National Institutes of Health grants HL-26057 and P40 RR-12358 (Dr Kranias), HL-52318 (Drs Kranias, Abraham, Hoit, Dorn, and Liggett), and HL-07527 (David Y. Hui). We are grateful to Michelle Nieman and Sarah McDonald for their technical assistance.

Received May 18, 2000; revision received August 17, 2000; accepted August 24, 2000.

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