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Cardiovascular Research 2002 56(2):248-259; doi:10.1016/S0008-6363(02)00541-2
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Structural and functional implications of the phospholamban hinge domain: impaired SR Ca2+ uptake as a primary cause of heart failure

Albrecht G Schmidta,1, Jing Zhaia,1, Andrew N Carra, Mike J Gersta, John N Lorenzb, Piero Polleselloc, Arto Annilad, Brian D Hoite and Evangelia G Kraniasa,*

aDepartment of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, USA
bDepartment of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
cCardiovascular Research, Orion Pharma, FIN-02101 Espoo, Finland
dVTT Biotechnology, FIN-00014 Helsinki, Finland
eDivision of Cardiology, Case Western Reserve University, Cleveland, OH 44106, USA

* Corresponding author. Tel.: +1-513-558-2327; fax: +1-513-558-2269. litsa.kranias{at}uc.edu

Received 20 March 2002; accepted 17 June 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The role of sarcoplasmic reticulum (SR) in the onset and progression of heart failure is controversial. We tested the hypothesis that impairment of SR Ca2+ sequestration may be a primary cause for progressive left ventricular (LV) dysfunction and the phospholamban hinge domain may be critical in this process. Methods: A phospholamban hinge domain mutant (PLB/N27A) was introduced in the cardiac compartment of the phospholamban null mouse. An integrative approach was used to characterize the resulting cardiac phenotype at a structural, cellular, whole organ and intact animal level. Results: NMR analysis revealed a defined alteration in the {alpha}-helical configuration between residues Q22 to F35 in mutant phospholamban. Transgenic lines expressing similar levels of mutant compared to wild-type phospholamban exhibited super-inhibition of the SR Ca2+ ATPase affinity for Ca2+ (EC50 0.52 µM) in oxalate-supported Ca2+ uptake measurements, which translated into impaired relaxation and attenuated responses to β-adrenergic stimulation. Importantly, a blunted force–frequency relation was observed in mutant hearts preceding left ventricular dilation. Upon aging to 10 months, the predominantly diastolic dysfunction progressed to congestive heart failure, characterized by induction of a fetal gene program, cardiac remodeling, lung congestion, depressed systolic function and early mortality. Conclusion: Increased inhibition of Ca2+ sequestration may be a causative factor in the development of left ventricular dysfunction and myocyte remodeling leading to heart failure. Furthermore, the hinge domain may play an important role in transmitting PLB’s regulatory effects on SERCA.

KEYWORDS Ca-pump; Calcium (cellular); Contractile function; Heart failure; SR (function)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Dilated cardiomyopathy represents a final common pathway in response to a variety of different pathologic stimuli and reflects the complex interaction of cascades that drive the onset and progression of the disease. At the functional level, end-stage heart failure is commonly associated with depression of systolic performance [1] and marked diastolic dysfunction [2]. Although there is now evidence that diastolic dysfunction may represent the early phase of heart failure preceding systolic impairment [2], the effectors and cellular mechanisms that underlie impaired myocardial relaxation and its transition to congestive heart failure have not been well defined.

Studies in human and animal models of heart failure have suggested that the elevated resting [Ca2+] and prolonged duration of the Ca2+ transient [3–5] are primarily due to impaired Ca2+ sequestration by the SR. However, other Ca2+ regulatory mechanisms, such as blunted myofilament activation [6], impaired ability of L-type Ca-channel to activate SR Ca2+ release [7], dysfunction of the ryanodine receptor [8], or abnormalities in myocyte cytoskeleton [9] may contribute to the disturbed Ca homeostasis. Furthermore, it has been postulated that depressed SR Ca function may be an important compensatory response to minimize contractile depression and preserve energy expenditure [6]. Unfortunately, the pleiotropic factors leading to heart failure and the complex phenotype characterizing the end stages, limit our ability to distinguish between primary causes and secondary adaptive responses.

Cardiac relaxation is critically dependent on the activity of the SR Ca2+ ATPase (SERCA), which is under reversible regulation by phospholamban (PLB) [10,11]. Dephosphorylated PLB inhibits SERCA’s Ca2+ affinity, whereas phosphorylation by cAMP-dependent and Ca2+/calmodulin-dependent protein kinases, relieves this inhibition [12]. The inhibitory effects of PLB involve amino acids (AA) in the hydrophobic transmembrane domain II (AA 31–52) and in cytosolic domain Ia (AA 1–20) [13,14]. Domain Ib (AA 21–30), a hinge region in the cytosolic portion of PLB, has been suggested to regulate a long range coupling between domains Ia and II [15], but its functional significance is not clear. The role of PLB in the regulation of cardiac function has been elucidated through the characterization of genetically altered mouse models. Ablation of PLB resulted in hyperdynamic contractile parameters, while PLB overexpression led to significantly depressed function [10,11]. Furthermore, overexpression of PLB or mutants, which act as gain-of-function inhibitors, indicated that depressed SR Ca2+ cycling was associated with hypertrophy [16,17]. Thus, it is interesting to postulate that PLB may be a control point in the regulation of intracellular Ca2+ dynamics and myocyte adaptive responses to compensatory hypertrophy. However, interpretation of the PLB overexpression studies is rather limiting since they are done in the presence of the endogenous wild-type molecule, and it is difficult to discern between the effects of mass action or mutation. Furthermore, the overexpressed mutant may either coexist with the endogenous protein or may disrupt the regulatory effects of the wild-type molecule, leading to the observed phenotype.

Thus, we introduced an Asn27 to Ala (N27A) PLB mutant, which acts as a superinhibitor of SERCA while maintaining its native pentameric structure, in the cardiac compartment of the null background. In the present study, we have characterized structural alterations and the primary functional effects of this hinge region mutant on SERCA2a activity, basal contractility, Ca2+ handling, β-adrenergic responsiveness and contractile reserve in vitro and in vivo. Furthermore, we have used this animal model to assess the pathophysiological significance of chronic impairment of SR Ca2+ resequestration for the onset and progression of heart failure.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Introduction of the PLB/N27A mutant into the PLB null background
Transgenic mice, overexpressing the PLB/N27A mutant (FVB/N) [17] were crossbred with the PLB knockout mice (129X1/SvJxCF-1) [10]. PCR methodology was employed to identify offspring expressing both the PLB/N27A transgene and the neo gene [10,17]. The transgenic construct was also subjected to sequencing to confirm the presence of the PLB Asn27 to Ala mutation (AAT to GCT) and the absence of additional mutations in the coding region of the PLB cDNA. Characterization studies utilized mice from the F4 generation. Wild-type mice with 129X1/SvJxCF-1 mixed background were used as controls. The investigation conforms 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).

2.2 Biochemical assays
Crude cardiac homogenates and SR enriched microsomes [17] were subjected to quantitative immunoblotting [18]. Phosphorylation assays were conducted as previously described [19]. Oxalate-supported Ca2+ uptake in SR was measured by a modified Millipore filtration technique, and data were analyzed by nonlinear regression using Origin software [17].

2.3 Structural NMR-analysis
Wild-type and mutant N27A PLB peptides (AA 1–36) were synthesized, purified [20], and two-dimensional proton correlation NMR spectra were acquired from 1 mM samples dissolved in 30% TFE at pH 3.0, 17 °C, using an 800 MHz NMR spectrometer [21]. Structures were generated by torsion angle simulated annealing (DYANA 1.5) [22]. Families of structures were analyzed for restraint violation, backbone dihedral (Ramachandran plot), energy and root mean square deviation.

2.4 Ventricular cardiomyocyte mechanical parameters and Ca2+ transient measurements
Ca2+-tolerant, isolated cardiomyocytes were loaded with 3–5 µM Fura 2-AM for 20 min at room temperature. Cardiomyocytes were perfused with 1.8 mM Ca2+-Tyrode and field-stimulated at 0.5 Hz in the absence or presence of isoproterenol (300 nM). Ca2+ transients were measured as the 340/380 nm fluorescence ratio and reported in arbitrary units [18].

2.5 Langendorff heart perfusion
Retrograde aortic Langendorff perfusion was performed using modified Krebs buffer saturated with 95% O2–5% CO2 at 37 °C [19]. Hearts were frozen upon maximal isoproterenol stimulation (1.0 µM), and subjected to Western blot analysis or SR Ca2+ uptake measurements.

2.6 Closed-chest catheterization and force–frequency relation
Left ventricular (LV) catheterization was performed using a 1.4 Fr Millar high fidelity catheter via the right carotid artery [23,24]. Pacing was initiated above intrinsic heart rates using bipolar pacing electrodes in the right atrium, and was stepwise increased up to 600 bpm. The time constant for isovolumic relaxation, {tau}, was calculated as previously described [23] and left ventricular pressure tracings were fit to Pt=Poe–t/{tau}, where P is pressure at time t (Pt) and at dP/dtmin (Po).

2.7 In vivo echocardiography
2D guided M-mode echocardiography (9 MHz) and color-flow directed Doppler (5–7 MHz) were performed using an Interspec Apogee CX-200 ultrasonograph (Interspec-ATL, Ambler, PA) [24]. Mice were lightly anesthetized with 2.5% avertin (0.01 ml/g i.p.) and studies performed at baseline and after administration of isoproterenol (2.0 µg/g i.p.) [25].

2.8 Histopathologic and dot-blot analyses
Standard techniques were used for histological examination (Mason’s trichrome sections) and dot-blot analysis of total RNA from left ventricles [18].

2.9 Materials
Type II collagenase (Worthington Biochemical). Antibodies were: PLB-monoclonal and calsequestrin-polyclonal (Affinity BioReagents); PS-16- and PT-17-polyclonal (PhosphoProtein Research); SERCA-polyclonal, which was generated in our laboratory.

2.10 Statistical analysis
Data are presented as mean±S.E.M. Statistical analysis was performed by Student’s t-test for comparisons between two groups, and one-way or two-way ANOVA, followed by Student–Newman–Keuls’ test, for multiple comparisons. P<0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Generation and identification of transgenic mice expressing mutant PLB/N27A in the phospholamban null background
Cardiac-specific expression of the PLB N27A mutant in the absence of endogenous wild-type PLB was achieved by mating transgenic mice overexpressing the mutant PLB/N27A with the PLB knockout mouse, previously generated in our laboratory. PCR analysis of tail genomic DNA was employed to identify the selected genotype (characterized by the presence of both the mutant PLB/N27A transgene and the neo gene, as well as the absence of endogenous PLB gene) using different sets of primers (Fig. 1). The presence of the mutant transgene was detected using a 5'-end {alpha}-MHCp-1 primer and a 3'-end PLB-RC primer corresponding to part of the transgenic construct to amplify an ~180 bp fragment. The presence of the neo gene, which was successfully used to replace part of the PLB gene in generating the PLB knockout mice in our laboratory, was identified using a 5'-end HLT7 primer corresponding to part of the PLB genomic sequence and a 3'-end neo 3 primer corresponding to part of the neo gene to amplify an ~550 bp fragment. Ablation of endogenous PLB was confirmed by absence of an ~650 bp PCR product using 5'-end HLT7 and 3'-end JS 940 primers corresponding to part of the PLB genomic sequence.


Figure 1
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  		Fig. 1 Identification of transgenic mice expressing mutant PLB/N27A in the phospholamban null background. The presence of the transgene (TG) was identified by a 180-bp PCR product. The neo gene (PLBKO) was recognized by a 500-bp PCR product. A 650-bp PCR product represents the endogenous PLB gene (PLBendo). The selected genotype (PLB/N27A) is characterized by the presence of TG and PLBKO genes, as well as the absence of PLBendo. NS indicates the non-selected genotype.

 
Quantitative immunoblotting of cardiac homogenates or SR-enriched membranes from mice expressing the mutant PLB/N27A in the PLB knockout background revealed no alterations in SERCA or calsequestrin protein levels in transgenic compared to wild-type hearts (Fig. 2A). The mutant PLB predominantly migrated as pentamers on SDS–PAGE, and dissociated to monomers upon boiling, similar to wild-type PLB. The levels of the mutant PLB were 1.11±0.11 in transgenic compared to wild-type (1.00) hearts, resulting in a similar apparent PLB/SR Ca2+ ATPase ratio (1.08±0.16 in transgenic and 1.00 in wild-type). Furthermore, similar ratios of PLB/SR Ca2+ ATPase were observed in SR-enriched microsomal fractions from transgenic (0.84±0.13) and wild-type (1.00) hearts (Fig. 2A), indicating that the mutant PLB was incorporated into the SR membrane. 32P-phosphorylation experiments by PKA catalytic subunit or Ca2+/calmodulin-dependent protein kinase, revealed that the mutant PLB could be also phosphorylated to the same extent as wild-type PLB (data not shown).


Figure 2
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  		Fig. 2 Biochemical characterization of PLB/N27A hearts. (A) Representative quantitative immunoblots of cardiac SR enriched membranes probed with PLB, SR Ca2+-ATPase (SERCA) or calsequestrin (CSQ) antibodies. 0.2, 0.4, and 0.8 µg protein were loaded to generate linear regression lines, and the slopes were used to compare the protein levels between wild type (WT; n=5) and PLB/N27A (n=5) hearts. PLBp and PLBm, pentameric and monomeric PLB. (B) The initial rates of oxalate-supported SR Ca2+ uptake in the PLB/N27A (bullet) and WT ({circ}) hearts. Data are expressed as % of maximal uptake rates in each group. Values are mean±S.E.M. of five hearts, assayed in triplicate.

 
To determine whether the mutant PLB could functionally interact with the SR Ca2+ ATPase, the initial rates of oxalate-facilitated SR Ca2+ uptake were assessed in cardiac homogenates (Fig. 2B). Expression of mutant PLB resulted in a rightward supershift of the EC50 of SR Ca2+ uptake (0.52±0.04 µM) compared to wild-types (0.21±0.01 µM). However, the maximal rates of Ca2+-uptake in transgenic (49±7 nmol/mg/min) were not different from wild-type (48±3 nmol/mg/min) hearts.

3.2 Structural NMR analysis
The observed superinhibitory effects of PLB/N27A prompted us to examine its structure at the molecular level. Thus, wild-type and PLB/N27A peptides (AA 1–36) were synthesized, solubilized, and NMR spectra obtained. The N-terminal residues from V4 to I18 and the C-terminal residues from Q22 to F35 displayed an {alpha}-helical configuration (Fig. 3A). Analysis of the spectra of wild-type or mutated peptide did not reveal the presence of multiple conformations in slow exchange. However, a clear difference was observed in the region adjacent to the mutation site, where the C-terminal helix of the mutant PLB/N27A adopted a different course compared to the wild-type (AA 20–36: Fig. 3A). This is of particular importance, since the mobility of this region has been suggested to play an important role in transmitting the effect of phosphorylation in domain Ia resulting in dissociation of the PLB/SERCA heterodimer. The structural statistics confirmed that the number of nOe cross peaks and the quality of the spectra were comparable for the two peptide families (Table 1). Fig. 3B and C display expansions of the NOESY spectra from wild-type and PLB/N27A peptides. Changes in the chemical shifts (P21–A24) were restricted to AA around the mutation, while the rest of the structure remained unaffected.


Figure 3
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  		Fig. 3 NMR studies. (A) Main chain atoms of the C-terminal helices from residues M20 to S36 are shown from families of 40 structures of mouse WT (green) and mutant PLB (yellow). (B and C) The figures are expansions of the proton nOe NMR spectra and represent only 2% of the whole spectral area. These expansions show the chemical shift changes of AA 21–24 in PLB/N27A (C) compared to WT PLB (B), from 1 mM samples dissolved in 30% TFE at pH 3.0. The low field ends of the spectra display correlations from the N-terminal residues of peptides and from the residues at the beginning of the C-terminal helix (Q22 to A24) adjacent to the loop region between the two helices, comprising residues from I18 to P21.

 

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  		Table 1 Structural statistics

 
3.3 Functional measurements ex vivo
The decreased SERCA Ca2+-affinity, observed at the subcellular level, was associated with functional alterations at the cellular level: rates of shortening and relengthening were significantly depressed to 67% and 54%, respectively, in PLB/N27A compared to wild-type (100%) myocytes. The Ca2+ transient kinetics also demonstrated a significant prolongation in the times for 50% (2.2-fold) and 80% (1.6-fold) decay of the Ca2+ signal in transgenic myocytes compared to wild-types (1.0-fold), reflecting impaired SR Ca2+ re-uptake. Since PLB has been shown to act as a key mediator of the cardiac response to β-agonists, myocytes were maximally stimulated with 300 nM isoproterenol. Interestingly, isoproterenol could not fully relieve the superinhibitory effects of PLB/N27A, and the maximally stimulated mechanical and Ca2+ kinetic parameters remained significantly depressed compared to wild-types (data not shown).

Similar to observations at the myocyte level, Langendorff perfusions indicated that the depressed contractile parameters in transgenic hearts under basal conditions (Fig. 4A and B), remained decreased under maximal β-agonist stimulation (+dP/dt 52%; –dP/dt 70%; time to half-relaxation, RT1/2 134%) compared to wild-types (100%). Phosphorylation of PLB, assessed by PLB phosphoserine or phosphothreonine site-specific antibodies, indicated that the phosphothreonine signal was significantly lower in transgenics, while the phosphoserine signal was similar to wild-types (Fig. 4C). These results suggested that the physiologically relevant site (Ser16) for the isoproterenol stimulatory effects [19] was phosphorylated to the same extent in transgenic and wild-type hearts.


Figure 4
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  		Fig. 4 Altered response to β-adrenergic stimulation. Langendorff-hearts were perfused in the presence of isoproterenol (1.0 µM) and frozen at the peak of the inotropic response. Basal (–) and isoproterenol-stimulated (+) values for (A) maximal rates of pressure development (+dP/dt) and (B) pressure decline (–dP/dt). (C) Phosphorylation of PLB, using PLB pSer and pThr site-specific antibodies. Quantification of pSer and pThr levels in PLB/N27A relative to WTs. (D) Isoproterenol (iso) effects on SR Ca2+ uptake in WT (EC50 0.26±0.01 to 0.19±0.01 µM) and PLB/N27A (EC50 1.00±0.10 to 0.52±0.01 µM) perfused hearts. Values are mean±S.E.M. (n=5). *P<0.05 vs. WT.

 
To examine the effects of mutant PLB-phosphorylation on SR function, ATP-dependent, oxalate-supported SR Ca2+ uptake was measured, using transgenic and wild-type hearts perfused in the absence or presence of isoproterenol (Fig. 4D). There were no significant differences in the maximal rates of Ca2+ uptake (Vmax) among the four groups. Isoproterenol stimulation shifted the EC50 to the left of both wild-type and transgenic hearts, but the values in transgenics remained higher, suggesting that phosphorylation of mutant PLB could not completely relieve its superinhibitory effects on the SR Ca2+ ATPase.

3.4 Invasive hemodynamics and contractile reserve in intact animals
To determine whether compensatory mechanisms may overcome the PLB inhibitory effects in vivo, hemodynamic parameters of mice were assessed using LV-catheterization (Fig. 5). At intrinsic heart rates (WT 402.4±11.0 vs. PLB/N27A 338.5±5.5; P<0.05), LV systolic pressure was preserved in PLB/N27A (Fig. 5A), but +dP/dt was moderately decreased (Fig. 5B). Interestingly, diastolic parameters were significantly altered in the PLB/N27A hearts, as indicated by a doubling in LV end-diastolic pressure (Fig. 5C), and a significantly prolonged time constant for isovolumic left ventricular relaxation, {tau} (Fig. 5D). The response to β-adrenergic stimulation was also significantly attenuated (data not shown). To test whether PLB-superinhibition by PLB/N27A alters cardiac reserve, we examined the effect of heart rate on hemodynamic parameters. Incremental pacing resulted in a significant increase in the rate of contraction in hearts expressing wild-type PLB, but this frequency response was blunted in PLB/N27A hearts, as indicated by a leftward and downward shift of the force–frequency relation (Fig. 5E).


Figure 5
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  		Fig. 5 Invasive hemodynamics in closed chest animals. Left ventricular indices are: (A) systolic pressure (LVSP), (B) rate of pressure development (+dP/dt), (C) left ventricular end diastolic pressure (LVEDP), and (D) time constant ({tau}) for relaxation. (E) Group data for force–frequency relationships from PLB/N27A and WT. For each group, data points denote maximal rates of contraction (+dP/dt max) with increasing heart rates. Data are means±S.E.M. (n=5). bpm, beats per min. *P<0.05 versus WT.

 
3.5 Effects of age
To assess the long-term effects of increased SERCA inhibition by PLB/N27A, non-invasive M-mode and Doppler echocardiography was employed (Table 2). In 3-month-old transgenic mice, heart rate was significantly lower, the ratio of early to late diastolic transmitral velocity was depressed, and the isovolumic relaxation time was significantly prolonged (187%), indicating the presence of impaired left ventricular relaxation. However, fractional shortening and velocity of circumferential fiber shortening, corrected for differences in heart rate, were not different in transgenic mice, suggesting that systolic function was compensated at a young age. Increases in end-diastolic posterior wall thickness to cavity ratio, and calculated LV/body mass indicated the presence of mild LV concentric hypertrophy in transgenic mice (Table 2). Cardiac hypertrophy (24%) was also confirmed by gravimetric analysis.


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  		Table 2 M-mode and Doppler echocardiographic measurements in PLB/N27A and wild-type mice

 
Despite this compensated LV dysfunction at 3 months of age, Kaplan–Meier analysis demonstrated that mutant mice died prematurely, between 10 and 15 months (Fig. 6A), reaching 50% of the cumulative survival rate at 13 months. Therefore, we examined transgenic animals at 10 months, the beginning of this period. There were further increases (90.3%) in calculated LV/body mass and LV geometry became eccentric by 10 months (Fig. 6B, Table 2). The increase in wall stress resulted in significant depression of systolic ejection phase indices (fractional shortening and velocity of circumferential fiber shortening), whereas there was no alteration in cardiac function of wild-type mice (Fig. 6C, Table 2). Besides deterioration of LV function in PLB mutants, a significant increase in lung/body weight ratios in PLB/N27A mice compared to wild-type was observed, indicative of lung congestion and decompensation of left heart failure (Fig. 6D). These structural and functional alterations were associated with reactivation of a fetal gene program, such as β-myosin heavy chain, skeletal {alpha}-actin, and ANF (Fig. 6E). Histopathological and gross examination revealed substantial interstitial fibrosis and hypertrophic myocytes in PLB/N27A hearts compared with no alterations in aging WT hearts (Fig. 7).


Figure 6
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  		Fig. 6 Progressive remodeling and early mortality in aging N27A mice. (A) Kaplan–Meier survival curves comparing WT (n=18) and transgenic N27A (n=13) mice over a 14-month period. (B and D) Gravimetric analysis of biventricular and lung weights, respectively, of PLB/N27A (grey) and WT (black) mice. (C) Representative M-mode echocardiograms from 10-month-old WT and transgenic N27A hearts. (E) Quantitative grouped dot-blot analysis of ventricular RNA expression normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in wild-type control (C) or 3-month (3) and 10-month (10) PLB/N27A hearts. Values are mean±S.E.M. (n=6). *P<0.05 versus WT.

 

Figure 7
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  		Fig. 7 Histopathology of WT and N27A hearts: representative Masson’s trichrome staining for fibrosis (blue) in sectioned hearts from 10-month-old WT and mutant PLB/N27A mice (original magnification x200). Upon aging, N27A hearts revealed interstitial fibrosis and myocyte hypertrophy.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
To critically address the question of the role of PLB activity in the onset and progression of cardiomyopathy, the current study introduced a PLB mutant, which is a potent inhibitor of SERCA, in the null background. A single point-mutation of N27A in the hinge region of PLB caused a defined alteration in the 3-D structure of this regulatory protein, which was associated with increased inhibition of cytosolic Ca2+ sequestration by the SR Ca2+ ATPase. This translated into predominantly impaired relaxation in isolated myocytes, perfused hearts, and intact mice that eventually progressed to cardiac remodeling and congestive heart failure.

Since neither the native stoichiometry of PLB/SERCA nor the pentamer/monomer ratio was altered in this model, we hypothesized that a change in the structure of the mutant PLB/N27A might be the underlying mechanism of increased SERCA inhibition. Using NMR methodology, we demonstrated that mutant and wild-type PLB-peptides form N-terminal (V4 to I18) and C-terminal (Q22 to F35) {alpha}-helices connected by a turn, which is in agreement with previously published data by Pollesello et al. [20]. However, in the mutant peptide, the C-terminal helix adjacent to the site of the mutation was found to adopt a different course compared to wild-type. This structural alteration in PLB was likely associated with enhancement of its interaction with SERCA, possibly involving a long-range transmission among the PLB cytoplasmic and transmembrane domains [15]. Such enhanced association would be consistent with the increases (twofold) in the amount of SERCA co-immunoprecipitated with PLB/N27A [15] and the inability of phosphorylation, induced by β-agonists, to fully relieve the superinhibitory effects of PLB/N27A in vivo. Indeed, a change in mobility of the hinge domain has been proposed to be essential in transmitting the effects of phosphorylation in the cytoplasmic domain to the hydrophobic domain, resulting in dissociation of the PLB/SERCA heterodimer [20]. Furthermore, recent data suggest that a potential SERCA1-PLB interaction involves the cytoplasmic loop connecting M6 and M7 of SERCA1 (Asn810 to Asp813) and PLB domain IB (Asn27 and Asn30) [26]. Alternatively, pentameric PLB has also been proposed to form an ion pore in the SR membranes [27]. Nuclear magnetic resonance structure of PLB residues 1–36 revealed a clustering of glutamine and asparagine (Gln22, Gln23, Gln26 and Asn30) lining the inner side of the cytoplasmic portion of pentameric PLB, and it was suggested that the polar residues in the cytoplasmic portion of PLB might modulate Ca2+ leakage from the SR thereby contributing to the inhibitory effect of PLB on SR Ca2+ sequestration [20]. It is therefore conceivable that pentameric PLB/N27A may exert some of its inhibitory effects by facilitating SR Ca2+ leakage. Interestingly, Asn27 in PLB is the only amino acid, which is replaced by Lys in human (N27K), and this is associated with increased inhibitory function [15], suggesting that AA 27 in PLB may also account for functional differences among species.

In the current study, some of the effects of PLB/N27A expression on SR Ca2+ uptake and cardiac function in young adult mice qualitatively confirm results previously obtained in a mouse model overexpressing the mutant form in wild-type background [17]. However, the presence of endogenous PLB made it impossible to distinguish between the superinhibitory effect of the mutation and the impact of relative protein abundance. It was therefore mandatory to repeat those experiments in the knock-out background and in order to establish a causal relationship between the induction of a hinge region mutant, prolonged Ca2+ transients, contractile dysfunction and the initiation of progressive remodeling due to chronic SERCA inhibition. An interesting feature of the impaired SR Ca2+ sequestration in PLB/N27A hearts was a blunted force–frequency relation, observed in intact mice. Previous reports have indicated that the level of PLB and therefore, the magnitude of the SR Ca2+ loading is an important determinant of the force–frequency relation in intact mice [24,28]. Importantly, this impairment was found to precede LV dilation and heart failure in our model, consistent with clinical findings, where it appears to serve as an early marker for the transition from physiological to pathophysiological hypertrophy [29]. In this context it is noteworthy, that the phosphothreonine signal was significantly lower in PLB/N27A, while the phosphoserine signal was phosphorylated to the same extent in transgenic and wild-type hearts. Recent studies reported a decrease in Thr17 PLB phosphorylation in failing human myocardium [30]. Furthermore, frequency-dependent Thr17 PLB phosphorylation has been suggested to provide an intrinsic mechanism for cardiac myocytes to adapt to a sudden change in heart rate [31]. Thus, it could be hypothesized that a reduced ability of PLB/N27A to become phosphorylated at the threonine17 site might contribute to the blunted frequency-dependent inotropic response in this model.

Several studies have proposed that impaired diastolic function may precede systolic dysfunction [2,32] and decreases in SERCA/PLB protein ratio or the degree of PLB phosphorylation are key characteristics in human and experimental heart failure [4,5,18]. Importantly, the current study documents that increased PLB inhibition of SERCA may be a critical early event for the initiation of compensated, predominantly LV diastolic dysfunction, which progresses to cardiac remodeling and congestive heart failure upon aging. It is thus reasonable to speculate, that if such a mutation were to occur in the human PLB gene, it would lead to a gain of PLB inhibitory function and cardiomyopathies, comparable to polymorphisms in structural or contractile proteins [9,33]. However, besides the diminished SR Ca2+ resequestration leading to inadequate filling of SR stores and Ca2+ available for subsequent contractions in PLB/N27A hearts, a number of secondary players may be activated and effect the transition from compensated to decompensated phase. One of these compensatory responses is the altered MHC-isoform expression, observed upon cardiac remodeling in the mutant hearts. Interestingly, cardiac overexpression of β-MHC induced neither hypertrophic remodeling nor heart failure in transgenic models [34], indicating that β-MHC alone is not a primary candidate for the PLB/N27A cardiac phenotype. Other secondary alterations may include depletion of energy-rich phosphates [2] and decreases in cyclic AMP levels [35]. The downstream molecular pathways that couple SR Ca defects to reprogramming of gene expression and initiation of the hypertrophic response in this and other models are currently unclear. Surprisingly, diastolic dysfunction preceded induction of fetal genes in PLB/N27A hearts, indicating that impaired SR Ca cycling may be a prime candidate for the activation of intrinsic hypertrophic signaling pathways, leading to cardiac remodeling. According to this view, improved SR Ca2+ sequestration by either downregulation [36], ablation of PLB [37] or overexpression of SERCA [38,39] was able to either prevent dilated cardiomyopathy [37] or reverse contractile dysfunction in myocytes from animal models or end-stage failing human hearts [36,38,39]. Longitudinal studies, employing gene expression profiling and proteomics in PLB/N27A hearts may delineate the cellular mechanisms involved in the transition from compensated diastolic dysfunction to combined diastolic and systolic heart failure.

In summary, the current study addresses the potential causative role of impaired SR Ca2+ cycling in diastolic dysfunction without major cardiac remodeling, and its progression to heart failure in vivo. The findings support the importance of the phospholamban/SERCA complex as a nodal point in the pathophysiology of dilated cardiomyopathy. Furthermore, it is conceivable that targeted disruption of the PLB hinge domain may be important in enhancing cardiac performance. Future studies on elucidating the precise mechanism of the hinge domain transmitting its regulatory effects on SERCA are warranted.

Time for primary review 26 days.


   Acknowledgements
 
We are grateful to Ms. M. Nieman for excellent technical assistance, and Dr. P.R. Rosevear for critical discussions. This work was supported by NIH grants HL-26057, HL-64018, HL-52318, and P40RR12358 (E.G.K.), 5 T32 HL-07382 (A.C.), and HL-52318 (B.D.H).


   Notes
 
1 Each author contributed equally to this manuscript. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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