Pentameric assembly of phospholamban facilitates inhibition of cardiac function in vivo.

Phospholamban has been proposed to coexist as pentamers and monomers in native sarcoplasmic reticulum membranes. To determine its functional unit in vivo, we reintroduced wild-type (pentameric) or monomeric mutant (C41F) phospholamban in the hearts of phospholamban knockout mice. Transgenic lines, expressing similar levels of mutant or wild-type phospholamban, were identified, and their cardiac phenotypes were characterized in parallel. Sarcoplasmic reticulum Ca2+ transport assays indicated similar decreases in SERCA2 Ca2+ affinity by mutant or wild-type phospholamban. However, the time constants of relaxation and Ca2+ transient decline in isolated cardiomyocytes were diminished to a greater extent by wild-type than mutant phospholamban, even without significant differences in the amplitudes of myocyte contraction and Ca2+ transients between the two groups. Langendorff perfusion also indicated that mutant phospholamban was not capable of depressing the enhanced relaxation parameters of the phospholamban knockout hearts to the same extent as wild-type phospholamban. Moreover, in vivo assessment of mouse hemodynamics revealed a greater depression of cardiac function in wild-type than mutant phospholamban hearts. Thus, the mutant or monomeric form of phospholamban was not as effective in slowing Ca2+ decline or relaxation in cardiomyocytes, hearts, or intact animals as wild-type or pentameric phospholamban. These findings suggest that pentameric assembly of phospholamban is necessary for optimal regulation of myocardial contractility in vivo.

Phospholamban (PLB) 1 is a transmembrane phosphoprotein that interacts with and reversibly inhibits the cardiac sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase. Phosphorylation of PLB by ␤-adrenergic agonists removes its inhibition and therefore facilitates Ca 2ϩ transport into the SR lumen, enhancing cardiac relaxation. The physiological importance of PLB has re-cently been elucidated using gene targeting (1) and transgenic methodology (2). These studies showed that ablation of PLB was associated with augmentation of the SR Ca 2ϩ -ATPase affinity for Ca 2ϩ and basal cardiac contractility as well as attenuation of the cardiac responses to ␤-adrenergic agonists. By contrast, the SR Ca 2ϩ -ATPase Ca 2ϩ affinity and contractile parameters were greatly diminished in cardiomyocytes overexpressing PLB. Thus, biochemical and functional characterization of these animal models indicates that PLB is a major regulator of cardiac contractility in vivo (3).
The functional unit of PLB in native SR membranes is presently unknown. Phospholamban was originally proposed to form homopentamers through interactions with its transmembrane domain in the SR, based on its migration pattern on SDS-polyacrylamide gels (4,5). Pentameric assembly has been further supported by site-directed mutagenesis studies in expression systems (6), computational modeling (7,8), gel filtration chromatography (9), and EPR spectroscopy studies (10). Recently, alanine-scanning mutagenesis studies in expression systems showed that a monomeric form of PLB, in which Leu 37 was changed to Ala, suppressed SR Ca 2ϩ -ATPase Ca 2ϩ affinity more effectively than wild-type or pentameric PLB (11,12). It was thus postulated that there might be an equilibrium between pentameric and monomeric PLB in SR membranes. Pentameric PLB may represent a less active reservoir, which dissociates to provide active monomeric PLB subunits. However, coexpression of other monomeric forms of PLB, associated with mutation of Cys 41 to Phe or Ser, with SR Ca 2ϩ -ATPase in HEK-293 cells failed to demonstrate superinhibition (or gain of function) by PLB monomers (13). Furthermore, recent studies indicated that a pentameric form of PLB, resulting from mutation of Asn 27 to Ala, was more potent than a monomeric mutant (L37A) PLB in inhibiting the SR Ca 2ϩ -ATPase Ca 2ϩ affinity (14,15) and suggested that the oligomeric structure of PLB may be important in SR Ca 2ϩ -ATPase regulation.
These in vitro structure-function studies of PLB were recently extended to in vivo systems, and a transgenic mouse model overexpressing a monomeric form of PLB, associated with the mutation C41F, was developed in our laboratory (16). Cardiac-specific overexpression of monomeric PLB was associated with less inhibition of the SR Ca 2ϩ -ATPase Ca 2ϩ affinity, cardiomyocyte mechanics, and Ca 2ϩ kinetics than overexpression of wild-type PLB. However, such studies are difficult to interpret since they are done in the presence of endogenous wild-type or pentameric PLB. The expressed monomers could either coexist with the endogenous pentamers or disrupt pentameric assembly, resulting in the observed phenotype. Thus, it was important to extend these findings to the PLB-deficient background and to reintroduce wild-type or monomeric (C41F) PLB in the cardiac compartment of the knockout mouse to * This work was supported by National Institutes of Health Grants HL-26057, HL-52318, and 1P40RR12358 (to E. G. K.), Grant HL-52318 (to B. D. H.), and Grant HL-30077 (to D. M. B.) and by Fellowship SW-97-17-F from the American Heart Association (to V. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom correspondence should be addressed: better clarify the physiological effects of monomeric PLB. Our results indicate that although PLB monomers (C41F) inhibit the apparent affinity of the SR Ca 2ϩ uptake system to the same extent as PLB pentamers, they are not as effective in inhibiting the rate of myocardial relaxation assessed in isolated cardiomyocytes, perfused hearts, or intact animals.

EXPERIMENTAL PROCEDURES
Generation of Transgenic Mice Expressing Mutant PLB in the Knockout Background-The site-specific mutation C41F was introduced into the PLB cDNA by polymerase chain reaction methodology as described previously (17). The entire expression construct was composed of the cardiac-specific ␣-myosin heavy chain (␣-MHC) promoter (5.5 kb), the PLB coding region with the C41F mutation (0.65 kb), and the SV40 poly(A) signal sequence (0.25 kb) (Fig. 1A). The KpnI-HindIII fragment of the vector pIBI31 containing the entire expression construct was released and purified for pronuclear microinjection of fertilized eggs derived from the intercrossing of male PLB knockout (KO) and female FVB/N mice. The founder mice, who were PLB heterozygous, were mated back with PLB knockout mice to generate transgenic mice expressing mutant PLB in the PLB knockout background (KOϩMU). Transgenic mice with the desired mutation in the knockout background were identified using polymerase chain reaction methodology and Southern blot analysis of genomic DNA isolated from tail biopsies. Transgenic mice with wild-type PLB reintroduced into the knockout mouse hearts (KOϩWT) were also generated in a similar manner as described previously (17), and these KOϩWT mice were used as controls for the KOϩMU mice in this study.
Western Blot Analysis-Quantitative immunoblotting was performed to determine the protein levels of PLB and SR Ca 2ϩ ATPase in the heart as described previously (16). The cardiac homogenates were incubated with equal volumes of loading buffer (20% glycerol, 2% ␤-mercaptoethanol, 4% SDS, 0.001% bromphenol blue, and 130 mM Tris-Cl, pH 6.8), subjected to 13% SDS-polyacrylamide gel electrophoresis (PAGE), and blotted onto nitrocellulose membranes (Schleicher & Schuell). The membranes were then reacted with a mouse monoclonal antibody to PLB or SR Ca 2ϩ -ATPase (Affinity Bioreagents Inc.) at a dilution of 1:1000. After washing out the unbound antibody with Tris-buffered saline (10 mM Tris-HCl and 150 mM NaCl, pH 7.8), the blots were incubated with an alkaline phosphatase-conjugated anti-mouse secondary antibody (1:1000; Cappel Division of Organon Teknika). The PLB and SR Ca 2ϩ -ATPase protein bands were visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates for the alkaline phosphatase reaction.
Sarcoplasmic Reticulum Ca 2ϩ Uptake Assays-Mouse hearts were excised, frozen in liquid nitrogen, and stored at Ϫ80°C until processed for SR Ca 2ϩ uptake experiments. The frozen hearts were powdered in liquid nitrogen and homogenized in 50 mM KH 2 PO 4 , pH 7.0, 10 mM NaF, 1 mM EDTA, 0.3 mM sucrose, 0.3 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. The initial rates of calcium uptake in cardiac homogenates were obtained as described previously (1).
Cardiomyocyte Preparations and Contraction Measurements-Isolation of mouse left ventricular myocytes was carried out as described previously (18). Briefly, mouse hearts were perfused in a Langendorff mode with nominally Ca 2ϩ -free Tyrode's solution for 6 min at 37°C. Perfusion was then switched to the same solution containing 0.8 mg/ml collagenase (type B; Boehringer Mannheim) and 0.03 mg/ml Pronase, with perfusion continuing until the heart became flaccid (ϳ7-12 min). The isolated cells were perfused with normal Tyrode's solution at room temperature (22-23°C) and field-stimulated at 0.5 Hz. Cell shortening was measured using a video-edge detection system (Crescent Electronics).
Measurements of Ca 2ϩ Transients-Cells were incubated with the acetoxymethyl ester form of indo-1 (10 M; Molecular Probes, Inc.) for 20 min at room temperature. After loading, the cells were superfused with normal Tyrode's solution for 30 min to wash out excess indicator and to allow de-esterification (19). Fluorescence was measured as described by Li et al. (18), with excitation at 355 Ϯ 5 nm restricted to a circular spot of 100 m in diameter and with emission measured at 405 and 485 nm (F 405 and F 485 ) (40-nm bandwidth; Chroma Technology Corp.) by two photomultiplier tubes (Hamamatsu Phototonic Systems Corp.).
Langendorff Perfusions-Mouse hearts were rapidly excised and cannulated for retrograde aortic perfusion with modified Krebs-Henseleit buffer as described previously (20). The left ventricular pressure was monitored through a PE-50 polyethylene catheter, which was connected to a pressure transducer (041-500-803, COBE Cardiovascular, Inc.). The heart rate, left ventricular systolic pressure (LVSP) and its first derivatives (ϮdP/dt), end diastolic pressure, time-to-peak pressure, and time for half-relaxation were continuously obtained on-line with a heart performance analyzer (HPA-, Micro-Med, Inc.).
In Vivo Phosphorylation-Mouse hearts were perfused in a Langendorff mode as described above. After 30 min of perfusion to stabilize the contraction, the perfusion system was switched to a recirculating mode with modified Krebs-Henseleit buffer containing 2 mCi of [ 32 P]orthophosphate for 30 min at 37°C. At the end of this labeling period, isoproterenol (0.1 M) was administered into the perfusion system (20). After a 2-min stimulation, the hearts were freeze-clamped, stored at Ϫ80°C, and processed for cardiac SR membrane and myofibrillar preparations (21), which were subsequently subjected to SDS-PAGE and autoradiography.
Closed-chest Preparations-Mouse hemodynamics were obtained using closed-chest preparations as described previously (22). Briefly, mice were anesthetized with intraperitoneal injection of a mixture of ketamine (100 mg/kg), xylazine (5 mg/kg), and morphine (2.5 mg/kg). The trachea was intubated with a 24-gauge Jelco intravenous catheter. A custom-made 1-French bipolar pacemaker (Numed) was positioned in the right atrium via the right internal jugular vein. A 1.4-French Millar high fidelity catheter was introduced into the right carotid artery and advanced into the left ventricle. Analog signals for left ventricular systolic pressure and rates of left ventricular pressure increase and decline as well as electrocardiogram were obtained on-line, recorded on a Gould WindowGraf four-channel recorder, and digitized via an A-D board at 1000 Hz.
Statistical Analysis-Data are expressed as means Ϯ S.E. Statistical analysis was performed using analysis of variance for multiple comparisons and Student's t test for unpaired observations. Values of p Ͻ 0.05 were considered statistically significant. For the SR Ca 2ϩ uptake assays, the data were analyzed by nonlinear regression analysis using MicroCal Origin software. Statistical analysis was performed using one-way analysis of variance followed by Dunnett's test for multiple comparisons and the one-tailed Student's t test for comparison of means.

Cardiac-specific Expression of Mutant PLB in the Knockout
Background-Cardiac specific expression of wild-type or mutant PLB was driven using the ␣-MHC promoter (Fig. 1A) as described previously (2). The codon TGT (Cys 41 ) in the coding region of PLB was mutated to TTT (Phe) by site-directed polymerase chain reaction mutagenesis. The expression construct sequence and mutation were confirmed by restriction digestion and DNA sequencing and subsequently used for pronuclear microinjection to generate transgenic mice. Eleven founder mice harboring the mutated PLB transgene in the knockout background were identified using polymerase chain reaction analysis of genomic DNA isolated from tail biopsies. Furthermore, the transgene and its expression were detected using Southern and Northern blot analyses in the transgenic lines (data not shown) as described previously (16).
To examine the PLB protein expression levels, cardiac homogenates from transgenic mice expressing either wild-type or mutant PLB in the knockout background were processed in parallel with nontransgenic wild-type mice for Western blot analysis. Quantitative immunoblotting indicated that the reintroduced mutant PLB migrated as monomers, whereas the reintroduced wild-type PLB migrated mainly as pentamers, similar to endogenous PLB in wild-type hearts (Fig. 1B). In 11 transgenic lines, the cardiac PLB protein expression levels varied between 20 and 70% of those present in nontransgenic wild-type hearts. Transgenic mice with 70% PLB expression levels ( Fig. 1C) were selected to breed and propagate for further biochemical and physiological studies. Transgenic mice expressing similar levels of wild-type PLB in the knockout hearts (17) were used as controls. Assessment of the SR Ca 2ϩ -ATPase protein levels showed no significant alterations upon expression of either wild-type or mutant PLB in the knockout mouse hearts (data not shown).
Sarcoplasmic Reticulum Ca 2ϩ Uptake Assays-To examine the effects of mutant PLB expression on SR Ca 2ϩ pump func-tion, cardiac homogenates from knockout mice and mice expressing either wild-type or mutant PLB in the knockout background were processed in parallel for SR Ca 2ϩ uptake studies. The initial rates of SR Ca 2ϩ uptake were assessed over a wide range of [Ca 2ϩ ] (Fig. 2). Reintroduction of wild-type PLB was associated with a significant increase (0.22 Ϯ 0.02 M) in the EC 50 value compared with knockout hearts (0.11 Ϯ 0.004 M). Expression of mutant PLB also resulted in a significantly elevated EC 50 value (0.27 Ϯ 0.02 M) of the SR Ca 2ϩ uptake system, and this EC 50 value was similar (p Ͼ 0.05) to that in transgenic hearts expressing wild-type PLB. The maximal velocities of SR Ca 2ϩ uptake were similar among all three groups of hearts (Fig. 2).
Cardiomyocyte Mechanics and Ca 2ϩ Transients-To determine whether the observations at the subcellular level were associated with similar findings at the cellular level, left ventricular myocytes were isolated, paced at 0.5 Hz, and used for measurements of contractile parameters and Ca 2ϩ transients. Reintroduction of wild-type PLB in the knockout background was associated with significant depression of the cell shortening fraction (7.7 Ϯ 0.7%, n ϭ 28) and prolongation of the time constant () of relaxation (83.3 Ϯ 5.2 ms, n ϭ 28) compared with PLB knockout cardiomyocytes (cell shortening: 10.6 Ϯ 0.8%, n ϭ 33; ϭ 34.0 Ϯ 2.1 ms, n ϭ 33). Fig. 3 shows representative traces of steady-state cell shortening and Ca 2ϩ transients of transgenic myocytes with wild-type PLB or mutant PLB reintroduced in the knockout background. Myocytes expressing wild-type PLB relaxed more slowly than those expressing mutant PLB (Fig. 3, A and B). The time constant for cell relaxation during the steady-state twitch was significantly faster in the mutant PLB myocytes ( ϭ 45.1 Ϯ 2.5 ms in mutant versus 83.3 Ϯ 5.2 ms in wild-type). The amplitude of cell shortening was slightly but not significantly higher in mutant PLB myocytes (Fig. 3B). Consistent with cell mechanics, the decline of the Ca 2ϩ transient was faster in myocytes with mutant PLB compared with wild-type PLB ( ϭ 162 Ϯ 7.5 ms in mutant versus 196 Ϯ 11 ms in wild-type). Peak [Ca 2ϩ ] i tended to be higher with mutant PLB, but again this effect was not significant (Fig. 3, C and D).
Contractile Parameters in Isolated Heart Preparations-Previous studies in our laboratory have shown that the rates of cardiac contraction and relaxation were significantly increased upon ablation of PLB compared with wild-type hearts (1,23). However, reintroduction of wild-type PLB in the knockout hearts reversed their hyperdynamic function (17). To determine whether reintroduction of monomeric PLB had similar regulatory effects as wild-type or pentameric PLB, the hearts from transgenic mice with wild-type or mutant PLB reintroduced into the knockout background were perfused in parallel with knockout hearts in a retrograde mode. Langendorff perfusion indicated that there was no difference in the ϩdP/dt values between the two transgenic groups, but the ϪdP/dt values were depressed to a greater extent by reintroduction of wild-type PLB compared with mutant PLB in the knockout hearts (Table I). This observation was also consistent with the time parameters in the same cardiac preparations. There was no difference in the time-to-peak pressure, whereas the time to half-relaxation was prolonged significantly more in wild-type PLB heart than in mutant PLB hearts (Table I). Since PLB has been implicated to be a major regulator of the ␤-agonist stimulatory responses, the effects of isoproterenol on cardiac contractile parameters were also examined. Isoproterenol increased the rates of contraction and relaxation (ϮdP/dt) in wild-type and mutant PLB hearts in a dose-dependent manner, eventually reaching similar maximal values (Fig. 4, A and B). Assessment of the time-to-peak pressure and time to halfrelaxation values also indicated decreases in an isoproterenol dose-dependent manner, even though the basal value for time to half-relaxation was much lower in mutant PLB hearts than in wild-type PLB transgenic hearts (Fig. 4, C and D). In Vivo Phosphorylation of Mutant PLB-The stimulatory effects of isoproterenol suggested that both mutant and wildtype PLB can become phosphorylated and that this relieves their inhibitory effects. Thus, it was of special interest to determine the degree of mutant PLB phosphorylation and to compare it to that of wild-type PLB. Transgenic mouse hearts were perfused with buffer containing [ 32 P]orthophosphate in the presence of optimal isoproterenol concentration (0.1 M), and at the peak of the inotropic response, the hearts were freeze-clamped. Cardiac SR membranes and myofibrillar fractions were then prepared and subjected to SDS-PAGE. Autoradiography indicated that wild-type PLB migrated mainly as pentamers, whereas mutant PLB migrated as monomers (Fig.  5A), consistent with Western blotting analysis (Fig. 1B). Furthermore, the degree of phosphorylation ( 32 P incorporation) was similar between mutant and wild-type PLB, suggesting that mutant PLB was phosphorylated to the same extent as wild-type PLB. There was no alteration in the degree of phosphorylation of troponin I, which served as an internal control in these experiments, between the same wild-type and mutant PLB hearts (Fig. 5B). In addition, pentameric wild-type PLB could be dissociated to monomers upon boiling, prior to loading onto SDS-polyacrylamide gel (data not shown).
In Vivo Assessment of Cardiac Function-To determine whether these cardiac alterations in ex vivo preparations reflected similar findings in vivo, anesthetized closed-chest preparations of transgenic mice were atrially paced at 300 beats/ min and studied in parallel with PLB knockout mice. Reintroduction of wild-type PLB into the knockout background significantly repressed cardiac contractile parameters, whereas reintroduction of monomeric mutant PLB only slightly inhibited mouse hemodynamics (Table II). The maximal rates of left ventricular systolic pressure development (ϩdP/dt) and decline (ϪdP/dt) and were significantly different between wild-type and mutant transgenic mice (Table II). These data  corroborated our ex vivo findings and suggested that pentameric PLB was a stronger repressor of cardiac function compared with monomeric PLB, associated with the C41F mutation. DISCUSSION This study was designed to determine the functional unit of PLB in vivo by reintroducing wild-type (pentameric) or monomeric mutant (C41F) PLB into the cardiac compartment of PLB knockout mice. Although expression of either PLB form was capable of inhibiting the SR Ca 2ϩ -ATPase Ca 2ϩ affinity to the same extent, our findings indicate that monomeric PLB was less effective than the pentameric form in reversing the enhanced relaxation parameters of the PLB knockout hearts.
The availability of the PLB knockout mouse coupled with a strong cardiac-specific ␣-MHC promoter allowed us for the first time to assess the functional significance of pentameric or monomeric PLB in a background free of the endogenous protein in vivo. Transgenic lines expressing similar levels of mutant or wild-type PLB were chosen to assess the effects of each PLB form in a physiologically relevant manner. Calcium transport studies showed that wild-type or mutant PLB inhibited the apparent affinity of SR Ca 2ϩ -ATPase for Ca 2ϩ to the same extent. Furthermore, there were no alterations in the maximal velocity of the enzyme, consistent with the finding that the levels of SERCA2 were similar between these transgenic lines. However, the pentameric form of PLB depressed the relaxation rate during the steady-state twitch and prolonged the Ca 2ϩ transient decline to a greater extent than the mutant or mono-  meric PLB in isolated cardiomyocytes. Consistent with the findings at the cellular level, this differential effect was also observed in Langendorff perfused hearts. Furthermore, assessment of cardiac contractile parameters in the intact mouse revealed a greater depression of cardiac relaxation rates by wild-type PLB than by mutant PLB. These findings on cardiac contractile parameters were similar to previous results in models overexpressing similar levels of wild-type or mutant PLB in the presence of the endogenous protein (16). However, the findings on SR Ca 2ϩ transport measurements were different from those in the overexpression models (16). Overexpression of pentameric PLB was more effective than that of monomeric PLB in inhibiting the initial rates of SR Ca 2ϩ transport, whereas reintroduction of either form of PLB in the null background inhibited this SR parameter to the same extent. The reason for this apparent discrepancy could be due to the following: 1) the stoichiometric ratio of PLB to SERCA2 (0.7 in the transgenic mice in this study versus 2.0 in the overexpression models compared with 1.0 in the wild-type mice), which may affect the nature of interaction between these proteins; 2) the coexistence of PLB pentamers and monomers in the overexpression models, which may mask the maximal inhibitory effects by monomers; 3) compensatory mechanisms, associated with altered PLB expression levels, which may be different between the knockout and overexpression models; and 4) the genetic background of the two models (129/SvJ and CF-1 for the knockout models versus FVB/N for the overexpression models), which may contain different modifier alleles. The C41F mutation, chosen in this study, was based on previous in vitro (13) and in vivo (16) studies, which indicated that this mutation resulted in pentamer destabilization and formation of PLB monomers as revealed by SDS-PAGE. However, it is not entirely clear whether the migration pattern of C41F versus wild-type PLB on SDS-polyacrylamide gels reflects the in vivo situation. Future studies using chemical cross-linking (24), EPR spectroscopy (10), or low-angle laser light scattering measurements in conjunction with high-performance gel chromatography (25) may provide further insights into the oligomeric/monomeric state of PLB in vivo and its molecular dynamics upon mutagenesis or phosphorylation. Furthermore, it could be argued that replacement of cysteine with phenylalanine, which has a bulky side chain, may diminish the inhibitory effects of monomeric (C41F) PLB on cardiac relaxation parameters due to its inefficient interaction with the SR Ca 2ϩ -ATPase. However, biochemical reactivity studies of the three Cys residues (Cys 36 , Cys 41 , and Cys 46 ) in the transmembrane domain of wild-type (pentameric) PLB indicated that Cys 36 and Cys 46 reacted with 5,5Ј-dithiobis(2-nitrobenzoic acid), whereas Cys 41 remained unreactive (26). When PLB was denatured or a monomeric (L37A) form of PLB was used, all three Cys residues including Cys 41 reacted with 5,5Ј-dithiobis(2-nitrobenzoic acid) (26). These studies suggest that Cys 41 in wild-type PLB may be located within the pore of the PLB pentamer, which is protected and most insensitive to chemical modification. Furthermore, it has been hypothesized that the PLB transmembrane helix is oriented with Cys 36 and Cys 46 projecting into the lipid hydrocarbon, thus allowing interaction with the SR Ca 2ϩ -ATPase, and Cys 41 on the helix interface within the pentameric structure of PLB away from the binding region of SR Ca 2ϩ -ATPase. Thus, mutation of Cys 41 to Phe should not alter the interface of the inhibitory domain of PLB with the SR Ca 2ϩ -ATPase and their physical interaction. This assumption is also supported by previous in vitro expression studies of SR Ca 2ϩ -ATPase and PLB (13) and by this study, in which C41F mutant or monomeric PLB was capable of inhibiting the SR Ca 2ϩ -ATPase Ca 2ϩ affinity to the same extent as wild-type or pentameric PLB.
Langendorff perfusion in the presence of 32 P-containing buffer revealed that mutant PLB could be phosphorylated by isoproterenol. The degree of phosphorylation of monomeric PLB appeared to be similar to that of pentameric PLB, suggesting that the functional differences in cardiac preparations expressing wild-type or mutant PLB were not due to alterations in the phosphorylation capability of the mutant form of PLB. Under maximal stimulation by isoproterenol, the functional differences between the two forms of PLB, observed under basal conditions, were abolished, and the contractile parameters were similar between the two groups. It is interesting to note that both the non-phosphorylated and phosphorylated forms of mutant PLB migrated with similar apparent monomeric molecular weight, indicating that phosphorylation did not promote oligomerization of PLB in vivo. These findings using in vivo phosphorylation conditions and SDS-PAGE are different from previous results based on EPR studies, which suggested that in vitro phosphorylation of wild-type or monomeric (L37A) PLB resulted in oligomer formation in lipid bilayers (10).
In summary, our findings indicate that cardiac-specific expression of the monomeric form of PLB (C41F) is associated with inhibition of the SR Ca 2ϩ -ATPase Ca 2ϩ affinity, similar to wild-type or pentameric PLB, but the mutant form is not as effective as wild-type PLB in suppressing cardiac relaxation parameters, assessed at the cellular, organ, and intact animal levels. This apparent discrepancy between biochemical and physiological findings may be explained by the following possibilities. 1) The assay used to measure initial rates of SR Ca 2ϩ uptake is not sufficiently sensitive to detect small differences in Ca 2ϩ affinity. 2) The Ca 2ϩ uptake assay conditions in vitro do not adequately reflect the dynamic situation in vivo.
3) The pentameric form of PLB may induce a Ca 2ϩ leak in SR membranes via PLB pentamers (7,8,27), ryanodine receptors, or uncoupled Ca 2ϩ -ATPase, which slows down net Ca 2ϩ uptake in the intact cell, but not the apparent initial SR Ca 2ϩ uptake rates under in vitro conditions. 4) There could be an increased SR Ca 2ϩ load in the myocytes expressing pentameric PLB that slows down net SR Ca 2ϩ uptake during relaxation in vivo. These possibilities may be addressed in future studies designed to elucidate the mechanisms underlying the differences between the regulatory effects of the C41F PLB mutant in SR Ca 2ϩ transport assays in vitro and in functional measurements in vivo.