KarimZhang, David D. Thomas and Christine B.Zachary M. James, Qiang Xiong, Jianyi Naa-Adjeley D. Ablorh, Xiaoqiong Dong,  Phospholamban in Cardiac Muscleof the Four Phosphorylation States ofQuantitation of the Content and Function Synthetic Phosphopeptides EnableEnzymology:

doi: 10.1074/jbc.M114.556621 originally published online September 4, 20142014, 289:29397-29405.J. Biol. Chem. 

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Synthetic Phosphopeptides Enable Quantitation of theContent and Function of the Four Phosphorylation Statesof Phospholamban in Cardiac Muscle*

Received for publication, March 17, 2014, and in revised form, August 19, 2014 Published, JBC Papers in Press, September 4, 2014, DOI 10.1074/jbc.M114.556621

Naa-Adjeley D. Ablorh‡1, Xiaoqiong Dong‡, Zachary M. James‡2, Qiang Xiong§, Jianyi Zhang§, David D. Thomas‡3,and Christine B. Karim‡3,4

From the Departments of ‡Biochemistry, Molecular Biology and Biophysics and §Medicine, University of Minnesota,Minneapolis, Minnesota 55455

Background: Phosphorylation of phospholamban regulates cardiac calcium transport, but the content and function of thefour phosphorylation states of phospholamban are unknown.Results: Synthetic phosphopeptides solved both problems.Conclusion: The phosphorylation states were quantified in normal and hypertrophic pig hearts, and each has a distinct effect oncalcium transport.Significance: This information is needed for improved diagnosis and treatment of heart failure.

We have studied the differential effects of phospholamban (PLB)phosphorylation states on the activity of the sarcoplasmic reticu-lum Ca-ATPase (SERCA). It has been shown that unphosphory-lated PLB (U-PLB) inhibits SERCA and that phosphorylation ofPLB at Ser-16 or Thr-17 relieves this inhibition in cardiac sarco-plasmic reticulum. However, the levels of the four phosphorylationstates of PLB (U-PLB, P16-PLB, P17-PLB, and doubly phosphory-lated 2P-PLB) have not been measured quantitatively in cardiactissue, and their functional effects on SERCA have not been deter-mined directly. We have solved both problems through the chem-ical synthesis of all four PLB species. We first used the syntheticPLB as standards for a quantitative immunoblot assay, to deter-mine the concentrations of all four PLB phosphorylation states inpig cardiac tissue, with and without left ventricular hypertrophy(LVH) induced by aortic banding. In both LVH and sham hearts, allphosphorylation states were significantly populated, but LVHhearts showed a significant decrease in U-PLB, with a correspond-ing increase in the ratio of total phosphorylated PLB to U-PLB. Todetermine directly the functional effects of each PLB species, weco-reconstituted each of the synthetic peptides in phospholipidmembranes with SERCA and measured calcium-dependentATPase activity. SERCA inhibition was maximally relieved byP16-PLB (the most highly populated PLB state in cardiac tissuehomogenates), followed by 2P-PLB, then P17-PLB. Theseresults show that each PLB phosphorylation state uniquelyalters Ca2� homeostasis, with important implications for car-diac health, disease, and therapy.

To develop improved therapies for heart failure, the majorhealth problem for aging Americans, it is essential to develop

analytical procedures to assess cardiac physiology quantita-tively. During muscle relaxation, the sarcoplasmic reticulumcalcium ATPase (SERCA)5 actively transports Ca2� into thesarcoplasmic reticulum. The resulting electrochemical gradi-ent supplies most of the driving force for passive Ca2� effluxinto the cytosol during contraction (1). In cardiac sarcoplasmicreticulum, SERCA forms a complex with its 52-amino acid pep-tide inhibitor, phospholamban (PLB) (1– 4). It has been shownthat an increase in the ratio of unphosphorylated phospholam-ban (U-PLB) to SERCA (by overexpression of PLB, decreasedPLB phosphorylation, or decreased SERCA expression)decreases the apparent Ca2� affinity of SERCA (i.e. increasesKCa, the free Ca2� concentration corresponding to half-maxi-mal SERCA activation) and contributes to contractile dysfunc-tion (5, 6). Phosphorylation at Ser-16 and/or Thr-17 partiallyreverses this inhibition by decreasing KCa (5, 7–10). Spectro-scopic studies show that PLB phosphorylation induces struc-tural rearrangement within the PLB-SERCA complex withoutdissociating PLB, which is essentially a subunit of SERCA underphysiological conditions (11–14). Thus, PLB phosphorylationis a molecular switch that regulates SERCA activity. However,this is a complex switch because there are four distinct PLBphosphorylation states: U-PLB (no phosphorylation, dephos-phorylated by protein phosphatase-1) (15–17)), P16-PLB(phosphorylated by PKA (18)), P17-PLB (phosphorylated bycalmodulin-dependent kinase II, CaMKII), and 2P-PLB (phos-phorylated by both kinases). Their regulation of SERCAdepends on two factors: (a) the concentration of each state and(b) the potency of each state as a SERCA-bound inhibitor.

It has been proposed that the concentration of each PLBphosphorylation state differs with the etiology of heart disease.Qualitative Western blots have suggested that P16-PLB andP17-PLB concentrations decrease in dilated cardiomyopathy* This work was supported in part by National Institutes of Health Grants R37

AG26160, R21 AG42996, and R01 GM27906 (to D. D. T.), and HL114120 (to J. Z.).1 Supported by National Institutes of Health Training Grant T32 HL069764.2 Supported by National Institutes of Health Training Grant T32 AR07612.3 Co-senior authors.4 To whom correspondence should be addressed. Tel: 612-626-2304; Fax:

612-624-0632; E-mail: karim002@umn.edu.

5 The abbreviations used are: SERCA, sarcoplasmic reticulum calcium ATPase;PLB, phospholamban; CamKII, calcium/calmodulin-dependent kinase; U-PLB,unphosphorylated PLB; XPLB, mole fraction of PLB; LVH, left ventricular hyper-trophy; Ab, antibody; T-PLB, total PLB; ANOVA, analysis of variance.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 42, pp. 29397–29405, October 17, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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(19) and that P17-PLB concentration increases in ischemia (20)and acidosis (21). 2P-PLB has not been measured in eitherhealthy or failing hearts. Conflicting reports suggest that leftventricular hypertrophy (LVH) decreases, increases, or has noeffect on PLB phosphorylation (22). Moreover, these trends canvary with age (23) and gender (24). A complete phosphorylationprofile for each age group, gender, and etiology of heart failurecould aid diagnosis and guide rational design of disease-specificdrug or gene therapies (25–27), targeted toward specific PLBphosphorylation states. Such a phosphorylation profile couldalso evaluate protein phosphatase-1, CaMKII, and PKA as tar-gets for small molecules (28, 29). Assessing the role of PLBphosphorylation states in cardiac pathology and treatmentrequires quantification of their concentrations and functions.These goals require purified standards for each of the four spe-cies and antibodies that are at least partially specific for each ofthem (30). Antibodies to 2P-PLB have become available onlyrecently, and none of the PLB antibodies that are selective forother phosphorylation states are completely specific (30). Thus,the concentrations of the four species have not been deter-mined previously.

Previously, protein phosphatase-1, PKA, and CaMKII havebeen used in efforts to phosphorylate PLB selectively at Ser-16or Pro-17, but complete and exclusive phosphorylation at onesite has not been achieved, so the only clear conclusion was thatboth P16-PLB and P17-PLB decrease SERCA inhibition, com-pared with U-PLB (8, 31–37). The effect of double phosphory-lation (2P-PLB) is controversial. In one study, addition of asecond phosphate increased pKCa (38), whereas in two otherstudies, it had no effect (8, 39). Thus, the inhibitory potencies ofthe four PLB phosphorylation states are yet to be determined.

In the present study, we have used solid-phase peptide syn-thesis to produce pure samples of each of the four PLB phos-phorylation states, allowing the accurate quantification of boththe concentration and potency of each. To measure concentra-tions, the four synthetic peptides were used as standards toquantify antibody selectivity, enabling the accurate determina-tion of the mole fractions of all four species by immunoblot,despite the imperfect specificities of the four antibodies. Todemonstrate that the method can report site-specific changesin phosphorylation, we assayed pig hearts with and withoutLVH induced by aortic banding. To measure the potency ofeach PLB phosphorylation state, pKCa was measured after eachof the four synthetic peptides was co-reconstituted withSERCA, guaranteeing complete, exclusive, and site-specific phos-phorylation, as well as identical PLB/SERCA stoichiometry.

EXPERIMENTAL PROCEDURES

Synthesis of the Four Phosphorylation States of PLB—Thesequences of human U-PLB, P16-PLB, P17-PLB, and 2P-PLBwere prepared using Fmoc solid-phase peptide synthesis withor without incorporation of phosphorylated amino acids at spe-cific sites (13, 40, 41). Phosphorylation was accomplished byincorporation of Fmoc-Ser(PO(OBzl)OH)-OH for P16-PLBand Fmoc-Thr(PO(OBzl)OH)-OH for P17-PLB during peptidesynthesis. 2P-PLB was prepared by incorporation of both phos-phorylated amino acids at position Ser-16 and Thr-17 (13, 41).

Phospholamban was characterized by amino acid analysis andmass spectrometry (41).

SERCA1a was purified from rabbit skeletal muscle usingreactive red in 0.1% octaethylene glycol monododecyl ether(C12E8) (42, 43), and the protein concentration was measuredwith the Pierce BCA assay (13, 41). SERCA1a, the fast-twitchskeletal muscle isoform, substitutes quantitatively for SERCA2a,the cardiac isoform, with respect to regulation by PLB (44).

Production of LVH—All experiments were performed inaccordance with the animal use guidelines of the University ofMinnesota, and the experimental protocol was approved by theUniversity of Minnesota Research Animal Resources Commit-tee. The investigation conformed to the “Guide for the Care andUse of Laboratory Animals” published by the National Insti-tutes of Health (NIH publication no. 85-23, revised 1985). Pro-cedures for producing and analyzing the porcine model of LVHsecondary to aortic banding have been described previously(45, 46). Briefly, Yorkshire pigs at �45 days of age were anes-thetized with inhaled isoflurane (2% v/v), intubated, and venti-lated with a respirator. A left thoracotomy was performed in thethird intercostal space, and the ascending aorta, �1.5 cm abovethe aortic valve, was mobilized and encircled with a polyethyl-ene band 2.5 mm in width. Whereas left ventricle (LV) anddistal aortic pressures were simultaneously measured, the bandwas tightened until a 40-mm Hg peak systolic pressure gradientwas achieved across the narrowing. The chest was then closedin layers, the pneumothorax was evacuated, and the animalswere allowed to recover (45). Left ventricular hypertrophyoccurred progressively as the area of aortic constrictionremained fixed in the face of normal body growth. Two monthsafter banding, animals were returned to the laboratory forstudy. Five size-matched normal pigs were studied as controls.

Myocardial Tissue Sample Preparation—After the terminalphysiological study (�8 weeks after aortic banding), each heartwas cross-sectioned into five short-axis rings. Odd-numberedrings were used for histological studies, which revealed clearevidence for LVH, and even-numbered rings were frozen formolecular biological analysis. For analysis, every LV short axisring was divided into 12 across the LV wall according to thecoronary anatomy. Tissues were embedded in Tissue-Tek OCTcompound (Fisher Scientific) and frozen in liquid nitrogen-cooled isopentane (45, 46). Frozen tissue sections were stored at�80 °C. Frozen tissue was homogenized in 10 mM NaHCO3, 10mM Tris-HCl (pH to 7.2 with KOH), 0.8 M benzamidine, 1mg/liter aprotinin, 1 mg/liter leupeptin, 1 M PMSF, and 1 mg/li-ter pepstatin A, using the Navy bead kit from Midwest Scien-tific. The homogenate was centrifuged at 4 °C for 5 min at speed12 with a bullet blender (Midwest Scientific). The pellet wasdiscarded, and the supernatant was frozen at �80 °C. Proteinconcentration was determined by the Pierce BCA assay.

Electrophoresis and Immunoblot—Samples were dissolved inLaemmli buffer (Bio-Rad) with 5% �-mercaptoethanol, loadedonto a 10 –20% Tris-Tricine gel (Bio-Rad), and separated bySDS-PAGE at constant voltage (120 V) at 25 °C for 90 min.Proteins were transferred to 0.45-micron Immobilon-FL PVDFmembranes (Millipore) in Towbin transfer buffer (47) for 50min at constant current (300 mA), blocked overnight in pureOdyssey blocking buffer (LI-COR Biosciences), and rinsed for 1

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min in double-distilled H2O. Each membrane was incubatedwith one primary antibody (Ab) per the manufacturer’s instruc-tions, washed three times for 15 min with TBST (Tris-bufferedsaline with 0.1% Tween), and then incubated with a secondprimary antibody per the manufacturer’s instructions andwashed three times for 15 min with TBST before simultane-ously adding two secondary antibodies, LI-COR IR-680LT andLI-COR 800CW, directed against the appropriate animal, eachat a dilution of 1:15,000 for 25 min. The membrane was thenwashed three times for 15 min with TBST, and the blots werestored in TBS. Proteins were detected and analyzed using LI-COR Odyssey. The 700-nm channel detected the goat anti-rabbit IR-680LT. The 800 nm channel detected the goat-anti-mouse IR-800CW and donkey-anti-goat IR-800CW (LI-CORBiosciences). All incubation buffers consisted of 50% Odysseyblocking buffer (LI-COR Biosciences) and 50% TBST exceptthe incubation of the antibody for 2P-PLB. This was incubatedin 5% w/v BSA, 1� TBS, 0.1% Tween 20 at 4 °C, per the manufac-turer’s instructions. Analysis by densitometry was accom-plished with Odyssey software. The resolution was 169 �m, andthe focus offset was 0.0 mm. Boxes were drawn around an areathat encompassed all PLB bands (monomeric and oligomeric).The background was local, determined as the average ormedian of the intensities at the top and the bottom of the box.The greatest linearity was obtained at a border width of 1 pixel.

Quantification of Four Phosphorylation States of PLB—Quantification of the four phosphorylation states of PLB in theco-reconstituted system or in pig cardiac homogenates wasaccomplished by an extension of our previous method forquantification of two states (30), using the antibodies given inTable 2. Antibodies entirely specific for each of these four spe-cies are not available, so it is necessary to start with four anti-bodies having partial selectivity, determine this selectivityquantitatively using purified synthetic standards, run theunknown on the same four blots, and then solve a system of fourequations with four unknowns to determine the composition ofthe unknown sample (Equation 1),

I i � ��ijcj, i � 1, . . . 4; j � 1, . . . 4 (Eq. 1)

where i refers to the primary antibody, and j refers to the phos-phorylation state of the standard, as defined in Table 2. Thus, �ijis the slope of the standard curve from phosphorylation state j(obtained using purified synthetic standards) from a blot usingprimary antibody i, and cj is the concentration of the phosphor-ylation state j of PLB in the sample. The result is a 4 � 4 matrixof simultaneous equations, which was solved in MATLAB todetermine the unknown concentration cj. The mole fraction ofeach PLB phosphorylation state (Xj) was calculated from Equa-tion 2.

X j � cj ��cj (j � 1, . . . 4) (Eq. 2)

To validate the method, it was applied to mixtures of syntheticstandards containing known concentrations of each phosphor-ylation state (Table 1). The PLB primary antibodies were allbased on epitopes that exclude residues 2 and 27 (the only dif-ferences between human and pig sequences), so the method isequally valid for analysis of both species. Secondary antibodies

Ab800CW (goat anti-mouse), Ab800CW (donkey anti-goat),and Ab680LT (goat anti-rabbit), were obtained from Li-CORBiosciences. Quantification of SERCA for co-reconstitutionswas accomplished by SDS-page, stained with Coomassie Bril-liant Blue, using purified SERCA1a.

ATPase Assay—Ca-ATPase activity was measured after co-reconstitution of SERCA with each of the four synthetic PLBstandards (13, 41, 48 –50). PLB and SERCA were co-reconsti-tuted in dioleoyl-phosphatidylcholine and dioleoyl-phosphati-dylethanolamine, at a molar ratio of 4:1 (51) and at a molar ratioof PLB/SERCA/lipid of 5/1/700 and 10/1/700. Both ratiosyielded indistinguishable functional results, indicating thatSERCA was saturated by PLB in each case. Ca-ATPase activitywas measured as a function of [Ca2�] using an NADH-coupled,enzyme-linked ATPase assay (42, 52) at a temperature of 25 °C.Each data set was fitted by the Hill equation,

V � Vmax�(1 � 10�nH(pKCa � pCa)) (Eq. 3)

where Vmax is the maximum ATPase rate (at saturating [Ca2�]),pCa is the negative log of [Ca2�], pKCa is the pCa value corre-sponding to half-maximal SERCA activation (where V �0.5Vmax), and nH is the Hill coefficient. Inhibitory potency ofeach PLB species is defined as ��pKCa, the decrease in pKCacompared with that measured for SERCA in the absence ofPLB.

Statistical Analysis—For validation of the method, the accu-racies of XPLB values, performed on known mixtures of stan-dards, were calculated as �XPLB � XPLB (apparent) � XPLB(known). Precision is expressed as S.E./mean. XPLB in pighomogenates and pKCa in the co-reconstitutions were com-pared using one-way analysis of variance (ANOVA). The Bon-ferroni method was used for comparisons between individualpairs. A two sample t test was used to compare sham and LVHgroups. A p value of �0.05 was considered significant. Pear-son’s r value of �0.95 was required for all slopes in Fig. 1.

RESULTS

Validation of Immunoblot Method—The assay was applied tofive mixtures of synthetic standards containing known concen-trations of each of the four PLB phosphorylation states (Table1). Two identical gels, with the same concentrations of eachpure synthetic PLB standard and equal volumes of the same fivemixtures (Table 1) were run and blotted with four differentantibodies (two antibodies on each gel). The mixtures were runin duplicate. The use of fluorescent secondary antibodies madeit possible to visualize two blots on the same membrane (Fig.1A), provided that the primary antibodies were produced indifferent animals. For the top blot in Fig. 1A, mouse primaryantibody (AbU) was paired with rabbit primary antibody(Ab17). For the bottom blot in Fig. 1A, goat primary antibody

TABLE 1Known Xj values (Eq. 2) in mixtures of PLB standards

Mixture U P16 P17 2P n

a 0.25 0.25 0.25 0.25 6b 0.10 0.20 0.30 0.40 6c 0.20 0.30 0.40 0.10 6d 0.30 0.40 0.10 0.20 6e 0.40 0.10 0.20 0.30 6

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(Ab16) was paired with rabbit primary antibody (Ab2P). Visu-alizing two different epitopes on one membrane requiredthat the secondary antibodies emitted at resolved wave-lengths. LI-COR-800CW is conjugated to a dye detected at800 nm, using a green pseudo-color for display. In contrast,LI-COR680LT is conjugated to a dye detected at 680 nm, usinga red pseudo-color for display. In Fig. 1A (top), the green signalcorresponds to goat anti-mouse LI-COR-800CW secondaryantibody bound to primary mouse antibody AbU, and the redsignal corresponds to goat anti-rabbit LI-COR-680LT second-ary antibody bound to primary rabbit antibody Ab17. In Fig. 1A(bottom), the green signal corresponds to donkey anti-goatLI-COR-800CW secondary antibody bound to primary goatantibody Ab16, and the red signal corresponds to goat anti-rabbit LI-COR-680LT secondary antibody bound to primaryrabbit antibody Ab2P. Fig. 1B shows the red and green signalsresolved in separate images. The same accuracy and precision

were obtained when four blots, as in Fig. 1B, each stained withone antibody (1, 2, 3, or 4), were performed and analyzed (datanot shown).

Fig. 1C shows standard curves obtained from the 2.5, 5.0, and11-ng PLB standards in Fig. 1B. In C, “Ii” indicates the primaryantibody that was used to stain the corresponding blot in B(Table 2), and the slopes of these standard curves are the �ijvalues in Equation 1. All slopes had a Pearson’s r value of �0.95.These �ij values were used to calculate the mole fraction of eachspecies in the five mixtures, and the results are compared withthe known values, in Fig. 1D and Table 1. �XPLB is the differ-ence between the measured and known mole fractions in thePLB mixtures. The results show that the assay is quite accurate,with all 20 measurements coming within 0.1 of the correctvalue. Total PLB (T-PLB) was also measured for the mixtures inTable 1. The average T-PLB(app)/T-PLB(actual), the ratio ofthe measured (apparent) value of T-PLB to the known value,

FIGURE 1. Validation of the method for calculating mole fractions (XPLB, Equation 2) of all four PLB phosphorylation states. A, two-color immunoblots,each stained with two primary antibodies. B, individual images from A. Numbers in parentheses indicate i and j values in Equation 1 and Table 2. C, standardcurves produced from intensity values Ii (i indicates the antibody as in B), obtained from densitometry of standards in B. Slopes are the �ij values in Equation 1.D, the error �XPLB � S.E., obtained from known mixtures (Table 1), plotted against the actual XPLB for all four PLB phosphorylation states. E, ratio of apparentT-PLB to actual, � S.E.

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was calculated for each mixture from n � 5 immunoblot exper-iments. This was also quite accurate (close to 1), ranging from0.88 to 1.09.

Application to a Model of LVH—Left ventricular tissue washarvested from five LVH pigs and five sham controls, asdescribed under “Experimental Procedures.” Left ventricularhypertrophy was quite evident, based on an increase of left ven-tricular mass by 65% 4% (S.E.). There was no significant effectof LVH on left ventricular ejection fraction, indicating that thehypertrophy was compensatory and that end-stage heart failurewas not present.

Concentrations of the four PLB phosphorylation states weredetermined from immunoblots obtained from tissue homoge-nates (Fig. 2A), using the method described in Fig. 1. Asdescribed in Fig. 1, the accuracy and precision of the assay wasverified by including known mixtures of synthetic standards ineach immunoblot (left side of Fig. 2A). Two controls containingknown values of X (X � 0.25 for each phosphorylation state asin mixture A in Table 1) gave apparent X values of XU � 0.31

0.01, XP16 � 0.24 0.01, XP17 � 0.20 0.01, and X2P � 0.25 0.01. The apparent/actual total PLB value of the control was1.05 0.03.

Mole fractions of the four PLB species, determined fromthe right half of Fig. 2A, are plotted in Fig. 2B. In sham (con-trol) hearts (open bars in Fig. 2B), the greatest mole fractionwas observed for P16-PLB (XP16 � 0.47 0.006), followed byU-PLB (XU � 0.30 0.014), doubly phosphorylated 2P-PLB(X2P � 0.16 0.007), and P17-PLB (XP17 � 0.07 0.007).ANOVA showed significant differences among all the molefractions of all four PLB phosphorylation states, and theBonferroni test for multiple comparisons showed significantdifferences between all pairs (p � 0.05). In summary, all fourstates are significantly populated, and the order of mole fractions isP16-PLB U-PLB 2P-PLB P17-PLB. The mole fraction ofTP-PLB, the combined mole fraction of all phosphorylated PLB(P16-PLB � P17-PLB � 2P-PLB) was 0.70 0.005.

A similar pattern was seen in LVH hearts (closed bars in Fig.2B). Again, ANOVA showed significant differences among all

FIGURE 2. Mole fractions (XPLB, Equation 2) of all four PLB phosphorylation states in sham and LVH hearts. A, immunoblots stained with primaryantibodies AbU, Ab16, Ab17, and 2P, each with 2.5, 5.0, and 11 ng of U-PLB, P16-PLB, P17-PLB, and 2P-PLB synthetic standards, and 25 �g total protein from fivesham hearts or five LVH hearts. B, mole fractions (XPLB, Equation 2) of all four PLB phosphorylation states in sham (open) and LVH (solid) hearts (**, p � 0.005).C, the ratios of P16-PLB, P17-PLB, and 2P-PLB, and total phosphorylated PLB (TP-PLB) to U-PLB for sham (open) and LVH (solid) (*, p � 0.05). D, T-PLB for sham(open) and LVH (solid) hearts.

TABLE 2Primary antibodiesIndices (i) and (j) correspond to antibodies and PLB species, respectively, in Eq. 1. Either AbA1 or Ab2D12 were used in both Figs. 1 and 2 to emphasize the robustness ofthe assay. Cell Signaling Technology reports that the Ab8496 epitope has 100% sequence homology with pig and human, thus excluding E2D and K27N.

Antibody Epitope Ab (i) PLB selectivity (j) Reactivity Dilution Company

AbA1 7–16 AbU (1) U-PLB (1) Mouse 1:1000 MilliporeAb2D12 9–17 AbU (1) U-PLB (1) Mouse 1:600 AbcamAb12963 11–21 Ab16 (2) P16-PLB and 2P-PLB (2) Goat 1:1000 Santa Cruz BiotechnologyAb17024-R 12–22 Ab17 (3) P17-PLB and 2P-PLB (3) Rabbit 1:2000 Santa Cruz BiotechnologyAb8496 proprietary Ab2P (4) 2P-PLB (4) Rabbit 1:2000 Cell Signaling Technology

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the mole fractions of all four PLB phosphorylation states, andthe Bonferroni test showed significant differences between allpairs (p � 0.05). All four states were significantly populated,and the order of mole fractions is P16-PLB U-PLB 2P-PLB P17-PLB. The mole fraction TP-PLB was 0. 74 �0.008.

LVH caused a significant decrease in the mole fraction ofU-PLB (Fig. 2B, p � 0.005), and a corresponding increase inboth P16-PLB and TP-PLB, when normalized to the U-PLBmole fraction (Fig. 2C, p � 0.05). LVH did not cause a signifi-cant change in total PLB for the two groups but did cause asignificant increase in the variability (S.E.) of this measure (Fig.2D).

Effects of Specific Phosphorylation States of PLB on SERCAActivity—Synthetic human U-PLB, P16-PLB, P17-PLB, and2P-PLB were evaluated for their potency to inhibit SERCA (Fig.3). By fitting the data with Equation 3, the inhibition of Ca-ATPase activity was quantified by the decrease in pKCa (the pCavalue corresponding to 50% activation) in Fig. 3A. Each PLBphosphorylation state displayed a significantly different valueof pKCa (Fig. 3B), according to both ANOVA and the Bonfer-roni comparisons, showing that they are functionally distinct.Inhibitory potency, expressed as ��pKCa, the decrease in pKCacompared with SERCA alone, is plotted in Fig. 3C. As expected,U-PLB was the most inhibitory species. P16-PLB was the leastinhibitory, followed by 2P-PLB and P17-PLB (Fig. 3C). Thus,phosphorylation at Ser-16 provides the greatest reversal ofSERCA-PLB inhibition, followed by phosphorylation at bothSer-16 and Thr-17, followed by phosphorylation at Thr-17alone.

DISCUSSION

This study demonstrates the potential of synthetic peptidechemistry for solving important problems in cardiac patho-physiology. The availability of accurate synthetic standards ofall four phosphorylation states of PLB, we were able to providenew accuracy and precision in determining (a) the concentra-tions of these proteins in normal and diseased cardiac tissueand (b) their functional properties.

Quantification of PLB Phosphorylation States in Cardiac Tis-sue Homogenates—We have extended our previous quantita-tive immunoblot assays (30), which measured mole fractions of

two PLB phosphorylation states, to measure mole fractions ofall four PLB phosphorylation states (Fig. 1). Our assay allowsaccurate quantification even though AbU, Ab16, and Ab17 arenot completely specific (Fig. 1C). The use of the synthetic pep-tides and two-color immunoblots provides accurate concentra-tions of all four species in known mixtures (Fig. 1D). We usedthis assay to determine the unknown concentrations of thesephosphorylation states of PLB in tissue homogenates fromsham and LVH hearts, showing that all four states are present inboth groups (Fig. 2B), with the concentration of P16-PLB nearlytwice that of U-PLB, four times that of 2P-PLB, and six timesthat of P17-PLB. 2P-PLB is significantly populated even thoughit requires phosphorylation at adjacent sites. Including 2P-PLBincreases the mole fraction of Thr-17 phosphorylated PLB (2P-PLB � P17-PLB) from 0.07 to 0.23, which suggests that CamKIIhas substantial activity in the resting heart, even in the absenceof �-adrenergic stimulation. LVH causes a small but significantdecrease in U-PLB (Fig. 2B), a corresponding increase in theratios of P16-PLB and total P-PLB to U-PLB (Fig. 2C), and anincrease in the variability of total PLB (Fig. 2D).

The effect of cardiac hypertrophy on PLB phosphorylation iscontroversial. Some authors have reported a decrease in phos-phorylation at Ser-16 (53, 54), Thr-17 (55, 56), or both (57),whereas other reports show unaltered PLB phosphorylation(56) or an increase in total PLB phosphorylation (58) (59). Someof this variability may be due to different models of cardiachypertrophy being used, but the present method offers unprec-edented accuracy and precision, so its future application todiverse forms of heart disease should provide invaluableinsight into cardiac pathology. For example, the presentstudy suggests that measuring a ratio of mole fractions (e.g.X16/XU) provides the most reliable biomarkers for LVH (Fig.2C). The observed increase in PLB phosphorylation withLVH may be due to the need to compensate for the decreasedin SERCA activity that occurs in heart failure (60). Indeed,the hypertrophic pig hearts used in this study did not exhibitsignificant loss of cardiac function; future studies on moreadvanced hypertrophy, including end-stage heart failure,will be informative.

In the future, this assay can be used to further investigatecorrelations between the mole fractions of PLB phosphoryla-

FIGURE 3. SERCA activity due to PLB phosphorylation states. A, Ca-ATPase data, showing best fits to Equation 3, with data normalized to the Vmax valueobtained in the fit. B, pKCa values from fits in A. C, inhibitory potency, expressed as the decrease in pKCa relative to no PLB. Error bars are S.E. for n � 4. All valuesin B and C are significantly different (p � 0.01).

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tion states and cardiac physiology and pathology, as affected byCaMKII, PKA, and protein phosphatase-1. This assay requires asmall sample such that precious cardiac tissues from biopsies ordonors can be assayed. Biopsy is routinely performed for diag-nosis of the presence and/or etiology of cardiomyopathy (61–63). It has a �1% complication rate (62). When this assay isapplied to biopsy tissue, the effects of age, gender, the etiologyof heart failure, and the stage of heart failure on PLB phosphor-ylation can be determined quantitatively.

Direct Measurement of Inhibitory Potencies of All Four PLBPhosphorylation States—We used four phosphorylation state-specific co-reconstitutions of PLB with SERCA to determinethe inhibitory potencies of PLB phosphorylation states (Fig. 3).Our in vitro system has facilitated the simultaneous control ofPLB/SERCA stoichiometry and the purity of each PLB phos-phorylation state, which is not feasible in cardiac tissue samples(8). Although our assay utilizes SERCA1a from fast-twitch skel-etal muscle, our measured potencies apply as well to the heart,where SERCA2a predominates, as these two isoforms havebeen shown to have identical regulatory interactions with PLB(44, 64).

Each PLB phosphorylation state has a distinct effect onSERCA function (Fig. 3 and Table 3). These differences are notdue to differences in affinity for SERCA, as doubling the PLB/SERCA ratio had no effect on SERCA activity. This is consistentwith previous studies showing that PLB, phosphorylated PLB,and phosphomimetic PLB mutants saturate SERCA, both phys-ically and functionally, at protein/lipid concentrations compa-rable with those in this study, which are lower than physiolog-ical concentrations (11–14, 27). Thus, PLB is effectively asubunit of SERCA, and phosphorylation relieves SERCA inhi-bition by changing the structure of the SERCA-PLB complex(11–14, 27).

U-PLB decreased pKCa by 0.39, corresponding to an increaseof KCa by a factor of 2.4. P16-PLB was the least inhibitory PLBspecies, decreasing pKCa by only 0.14, corresponding to anincrease in KCa by a factor of 1.4. Thus phosphorylation at P16has the greatest effect in relieving SERCA inhibition. Interme-diate results were observed for P17-PLB (�pKCa � 0.33) and for2P-PLB (�pKCa � 0.22) (Fig. 3 and Table 3), showing that theeffects of phosphorylation at the two sites are not additivebecause double phosphorylation produces an effect intermedi-ate between the two singly phosphorylated states of Ser-16phosphorylation when both Ser-16 and Thr-17 are phosphory-lated on the same PLB (Fig. 3 and Table 3).

Determination of the contribution of any given phosphory-lation state to the activation of SERCA requires knowledge ofboth its mole fraction and its potency (pKCa shift). We nowknow that P16-PLB makes the largest contribution, both in

terms of its concentration and pKCa shift, followed by 2P-PLB,which has the next largest concentration and pKCa shift, andfinally P17-PLB, which has the smallest concentration and thesmallest pKCa shift from the reference point of the pKCa ofSERCA bound to U-PLB (Fig. 3C). Thus, increases in P16-PLBprovide coarse adjustments to SERCA inhibition, whereas2P-PLB and P17-PLB finely tune the PLB/SERCA regulatome.

Implications for Cardiac Physiology and Pathology—SERCAactivation is an established therapeutic goal in treatment ofheart failure (65). Therefore, the high concentration of P16-PLB in cardiac tissue homogenates (Fig. 2) and its low inhibi-tory potency (Fig. 3 and Table 3) establishes it (not P17-PLB or2P-PLB) as the best template for loss-of-inhibition PLBmutants in gene therapy (25, 27, 66, 67). Similarly, these resultsindicate that activation of PKA should be therapeutically moreeffective than activation of CaMKII. In fact, elevation ofCaMKII activity could reduce the effects of PKA activationbecause both P17-PLB and 2P-PLB are more inhibitory thanP16-PLB (Fig. 3 and Table 3). This may explain why P17-PLBelevation at the onset of ischemia fails to improve SERCA activ-ity and cardiac function (21). This small effect of Thr-17 phos-phorylation on SERCA activity could be therapeutically usefulin acidosis and ischemia, where elevated CaMKII leads toarrhythmia, necrosis and apoptosis (21), so treatment withsmall-molecule CaMKII inhibitors (68, 69) can occur withoutappreciable decreases in SERCA activity.

Conclusions—We have used solid-phase peptide synthesis toproduce pure samples of each of the four PLB phosphorylationstates, to permit the accurate quantification of both the concen-tration and potency of each PLB phosphorylation state. Thesefour states are all present in pig cardiac tissue in different con-centrations, with small but significant perturbations caused byinduced cardiac hypertrophy, and each has a distinct inhibitoryeffect on SERCA activity. This assay is applicable to extremelysmall tissue samples. Thus, it is now feasible to determine theseconcentrations in human tissue samples and to relate themquantitatively to cardiac physiology and pathology, providingessential information for future therapeutic developments.These tools are particularly important for the fight against heartfailure, the leading health problem for aging Americans.

Acknowledgments—This work used the facilities of the BiophysicalSpectroscopy Center and the Peptide Synthesis Facility (University ofMinnesota). We thank Florentin Nitu for expert advice and OctavianCornea for help in preparing the manuscript.

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