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Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump

Open AccessPublished:May 19, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102060
      The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA–micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB–SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB–SERCA regulatory interactions.

      Keywords

      Abbreviations:

      DWORF (dwarf open reading frame), ER (endoplasmic reticulum), PLB (phospholamban), SERCA (sarco/endoplasmic reticulum Ca2+-ATPase), SR (sarcoplasmic reticulum), TG (thapsigargin)
      The type 2a sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA2a) is a P-type ion transporter responsible for sequestering cytoplasmic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac muscle cells. The rate of Ca2+ transport by SERCA2a determines how quickly the heart muscle relaxes during the diastolic phase of the cardiac cycle as the ventricle is filling with blood. SERCA2a function also sets the amplitude of SR Ca2+ release, which determines the heart’s contractile strength during the systolic phase when blood is being ejected from the heart. Pathological decreases in SERCA expression, function, and regulation are associated with heart failure (
      • Currie S.
      • Smith G.
      Enhanced phosphorylation of phospholamban and downregulation of sarco/endoplasmic reticulum Ca ATPase type 2 (SERCA 2) in cardiac sarcoplasmic reticulum from rabbits with heart failure.
      ,
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      • Studer R.
      • Meyer M.
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      Relation between myocardial function and expression of sarcoplasmic reticulum Ca2+-ATPase in failing and nonfailing human myocardium.
      ), focusing attention on SERCA as a possible therapeutic target (
      • Hayward C.
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      The current and future landscape of SERCA gene therapy for heart failure: a clinical perspective.
      ,
      • Kranias E.G.
      • Hajjar R.J.
      Modulation of cardiac contractility by the phopholamban/SERCA2a regulatome.
      ). So far, attempts to enhance transport function in patients by increasing expression of SERCA2a with gene delivery have been unsuccessful (
      • Greenberg B.
      • Butler J.
      • Felker G.M.
      • Ponikowski P.
      • Voors A.A.
      • Desai A.S.
      • et al.
      Calcium upregulation by percutaneous administration of gene therapy in patients with cardiac disease (CUPID 2): a randomised, multinational, double-blind, placebo-controlled, phase 2b trial.
      ). Therefore, there is great interest in understanding physiological SERCA regulatory mechanisms to create a path toward rationally designed therapies that improve the function of the endogenous SERCA in patients with heart failure.
      The principal regulator of SERCA function in the heart is phospholamban (PLB), a single-pass transmembrane micropeptide (Fig. 1, blue). PLB physically interacts with SERCA and reduces its apparent affinity for Ca2+, decreasing Ca2+ uptake (
      • Cantilina T.
      • Sagara Y.
      • Inesi G.
      • Jones L.R.
      Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.
      ). This inhibition is relieved at high concentrations of cytoplasmic Ca2+ and after phosphorylation of PLB by cAMP-dependent PKA (
      • Tada M.
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      Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.
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      Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle.
      ), providing a mechanism to increase Ca2+ transport in response to exercise and other physiologic stress. Initially, the relief of SERCA inhibition was thought to require dissociation of the PLB–SERCA complex after PLB phosphorylation or Ca2+ binding to SERCA (
      • Asahi M.
      • McKenna E.
      • Kurzydlowski K.
      • Tada M.
      • MacLennan D.H.
      Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP.
      ,
      • James P.
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      Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum.
      ,
      • Toyoshima C.
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      • MacLennan D.H.
      Modeling of the inhibitory interaction of phospholamban with the Ca2+ ATPase.
      ,
      • Chen Z.
      • Akin B.L.
      • Jones L.R.
      Ca2+ binding to site I of the cardiac Ca2+ pump is sufficient to dissociate phospholamban.
      ,
      • Jones L.R.
      • Cornea R.L.
      • Chen Z.
      Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking.
      ). However, subsequent studies showed that the PLB–SERCA interaction can still occur after PLB phosphorylation or in high Ca2+ (
      • Karim C.B.
      • Zhang Z.
      • Howard E.C.
      • Torgersen K.D.
      • Thomas D.D.
      Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA.
      • Gustavsson M.
      • Verardi R.
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      • Mote K.R.
      • Traaseth N.J.
      • Gopinath T.
      • et al.
      Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban.
      ), suggesting that PLB acts more like a subunit of a persistent SERCA regulatory complex. These seemingly alternative models may be reconciled by observations that SERCA binds PLB with modestly reduced affinity in elevated Ca2+ (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ,
      • Alford R.F.
      • Smolin N.
      • Young H.S.
      • Gray J.J.
      • Robia S.L.
      Protein docking and steered molecular dynamics suggest alternative phospholamban-binding sites on the SERCA calcium transporter.
      ,
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ). PLB must bind more tightly to SERCA conformations that predominate at low Ca2+ and bind less avidly to SERCA states that prevail at elevated Ca2+. However, the energetics of PLB binding to specific SERCA conformers has not been measured. In the present study, we quantified PLB–SERCA interactions in intact cell membranes using FRET. Our goal was to compare the affinity of PLB for different intermediate states in the Ca2+ transport cycle.
      Figure thumbnail gr1
      Figure 1Regulatory interactions of SERCA (gray) with PLB (blue) and DWORF (red). Structures: PLB5, PDB:2KYV (
      • Verardi R.
      • Shi L.
      • Traaseth N.J.
      • Walsh N.
      • Veglia G.
      Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method.
      ); PLB1, PDB:1FJP, (
      • Lamberth S.
      • Schmid H.
      • Muenchbach M.
      • Vorherr T.
      • Krebs J.
      • Carafoli E.
      • et al.
      NMR solution structure of phospholamban.
      ); PLB-SERCA, DWORF–SERCA (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ,
      • Fernandez-de Gortari E.
      • Espinoza-Fonseca L.M.
      Structural basis for relief of phospholamban-mediated inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase at saturating Ca(2+) conditions.
      ); DWORF-7MPA, (
      • Reddy U.V.
      • Weber D.K.
      • Wang S.
      • Larsen E.K.
      • Gopinath T.
      • De Simone A.
      • Robia S.
      • Veglia G.
      A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca(2+)-ATPase.
      ). DWORF, dwarf ORF; PDB, Protein Data Bank; PLB, phospholamban; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase.
      In addition to direct regulation by phosphorylation, PLB inhibitory potency may be modulated indirectly by oligomerization of PLB into pentamers (Fig. 1, “PLB5”). PLB pentamers represent an inactive, reserve pool (
      • Kimura Y.
      • Kurzydlowski K.
      • Tada M.
      • MacLennan D.H.
      Phospholamban inhibitory function is activated by depolymerization.
      ) that serves as a buffer, reducing and stabilizing the concentration of active monomers. This buffering effect may be enhanced by adrenergic stimulation, since PLB pentamers are further stabilized by PLB phosphorylation (
      • Hou Z.
      • Kelly E.M.
      • Robia S.L.
      Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.
      ,
      • Cornea R.L.
      • Jones L.R.
      • Autry J.M.
      • Thomas D.D.
      Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers.
      ,
      • Song Q.
      • Pallikkuth S.
      • Bossuyt J.
      • Bers D.M.
      • Robia S.L.
      Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.
      ). The rates at which PLB exchanges between SERCA- and pentamer-bound pools must govern how quickly these complexes can redistribute in vivo. We (
      • Robia S.L.
      • Campbell K.S.
      • Kelly E.M.
      • Hou Z.
      • Winters D.L.
      • Thomas D.D.
      Forster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex.
      ) and others (
      • Koch D.
      • Alexandrovich A.
      • Funk F.
      • Kho A.L.
      • Schmitt J.P.
      • Gautel M.
      Molecular noise filtering in the beta-adrenergic signaling network by phospholamban pentamers.
      ) have provided evidence that the exchange of PLB monomers from pentamers occurs slowly relative to rapid exchange from the SERCA regulatory complex, but the underlying kinetics of these binding events have not been definitively measured. Therefore, the degree to which these regulatory complexes may dynamically redistribute in the cardiac SR remains unclear.
      PLB competes for SERCA binding with another membrane micropeptide expressed in the heart, dwarf open reading frame (DWORF) (
      • Reddy U.V.
      • Weber D.K.
      • Wang S.
      • Larsen E.K.
      • Gopinath T.
      • De Simone A.
      • Robia S.
      • Veglia G.
      A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca(2+)-ATPase.
      ), shown in red in Figure 1. In contrast to PLB, DWORF directly stimulates SERCA activity (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ,
      • Li A.
      • Yuen S.L.
      • Stroik D.R.
      • Kleinboehl E.
      • Cornea R.L.
      • Thomas D.D.
      The transmembrane peptide DWORF activates SERCA2a via dual mechanisms.
      ) and displaces the inhibitory PLB (
      • Makarewich C.A.
      • Munir A.Z.
      • Schiattarella G.G.
      • Bezprozvannaya S.
      • Raguimova O.N.
      • Cho E.E.
      • et al.
      The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.
      ,
      • Singh D.R.
      • Dalton M.P.
      • Cho E.E.
      • Pribadi M.P.
      • Zak T.J.
      • Seflova J.
      • et al.
      Newly discovered micropeptide regulators of SERCA form oligomers but bind to the pump as monomers.
      ,
      • Nelson B.R.
      • Makarewich C.A.
      • Anderson D.M.
      • Winders B.R.
      • Troupes C.D.
      • Wu F.
      • et al.
      A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.
      ,
      • Makarewich C.A.
      • Bezprozvannaya S.
      • Gibson A.M.
      • Bassel-Duby R.
      • Olson E.N.
      Gene therapy with the DWORF micropeptide attenuates cardiomyopathy in mice.
      ). Interestingly, our recent work showed that DWORF–SERCA affinity shows an opposite Ca2+-dependence compared to PLB–SERCA. That is, DWORF affinity for SERCA increases with elevated Ca2+ (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ). This implies dynamic competition of stimulatory and inhibitory micropeptides: Ca2+ elevations favor DWORF binding to SERCA, but PLB becomes more competitive as Ca2+ levels fall. Here, we measured the dynamic binding of DWORF and PLB to SERCA during cellular Ca2+ oscillations to understand how the exchange of micropeptides may be important for dynamically responsive regulation. We have previously used FRET to examine shifts in the PLB–SERCA binding equilibrium in paced cardiac myocytes (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ). However, motion artifacts and competing, non-FRET interactions with endogenous PLB/SERCA prevented a detailed analysis of exchange and equilibration rates in that study. To circumvent those experimental barriers we exploited a more well-controlled model system that mimics cardiac calcium handling. We interpreted these observations with a computational model that incorporates experimentally measured rate constants. This reductionist strategy yielded mechanistic insight into how regulatory equilibria shift during the cardiac cycle, and, on a longer timescale, how Ca2+ handling may adapt between rest and exercise. The results may inform future efforts to develop therapeutic strategies based on gene delivery of micropeptides (
      • Makarewich C.A.
      • Munir A.Z.
      • Schiattarella G.G.
      • Bezprozvannaya S.
      • Raguimova O.N.
      • Cho E.E.
      • et al.
      The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.
      ,
      • Makarewich C.A.
      • Bezprozvannaya S.
      • Gibson A.M.
      • Bassel-Duby R.
      • Olson E.N.
      Gene therapy with the DWORF micropeptide attenuates cardiomyopathy in mice.
      ,
      • Mbikou P.
      • Rademaker M.T.
      • Charles C.J.
      • Richards M.A.
      • Pemberton C.J.
      Cardiovascular effects of DWORF (dwarf open reading frame) peptide in normal and ischaemia/reperfused isolated rat hearts.
      ).

      Results

      SERCA affinity for PLB and DWORF is dependent on Ca2+-pump conformation

      We previously demonstrated that PLB–SERCA binding affinity is reduced in response to a sustained elevation of intracellular Ca2+, whereas the DWORF–SERCA interaction is slightly more stable with elevated [Ca2+] (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ,
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ). This reciprocal Ca2+ dependence prompted the hypothesis that PLB and DWORF may preferentially bind to different intermediate conformations of the SERCA enzymatic cycle. The SERCA enzymatic cycle (
      • Albers R.W.
      Biochemical aspects of active transport.
      ) is represented in Figure 2A. At basal Ca2+ (low nM), SERCA predominantly resides in the ATP-bound state, E1-ATP (
      • Autry J.M.
      • Rubin J.E.
      • Svensson B.
      • Li J.
      • Thomas D.D.
      Nucleotide activation of the Ca-ATPase.
      ) (Fig. 2A, blue box), waiting for Ca2+ to bind. During intracellular Ca2+ elevations, SERCA samples all the intermediate states of the Ca2+ transport cycle, but these states are not all equally populated during systole. Rather, there is relative accumulation in the autophosphorylated E1P/E2P intermediates that precede rate-limiting steps in its enzymatic cycle (
      • Clausen J.D.
      • Vandecaetsbeek I.
      • Wuytack F.
      • Vangheluwe P.
      • Andersen J.P.
      Distinct roles of the C-terminal 11th transmembrane helix and luminal extension in the partial reactions determining the high Ca2+ affinity of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b).
      ,
      • Dode L.
      • Andersen J.P.
      • Leslie N.
      • Dhitavat J.
      • Vilsen B.
      • Hovnanian A.
      Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses.
      ). Those states are outlined with red boxes in Figure 2A.
      Figure thumbnail gr2
      Figure 2Dynamics of PLB and DWORF binding to SERCA during elevations in intracellular Ca2+. A, a simplified Post-Albers scheme of the SERCA enzymatic cycle, highlighting states that predominate at low (blue) and high (red) intracellular [Ca2+]. B, FRET-based binding curves displaying a shifted dissociation constant (KD) of PLB–SERCA binding between the ATP-bound (blue) and TG-bound (black) states of SERCA. C and D, apparent KDs of PLB or DWORF binding to different SERCA enzymatic states of the catalytic cycle as in panel (A) with lines representing mean ± SEM (n = 6). Ligands used to stabilize each state are shown in parentheses. Differences in micropeptide KDs between SERCA states were analyzed by one-way ANOVA with Tukey’s post hoc (∗p < 0.05). E, apparent KDs of PLB and DWORF for SERCA in ATP-containing solutions with low (blue) and high (red) concentrations of intracellular Ca2+ with lines representing mean ± SEM (n = 5). Differences in KD evaluated by Student’s t test. F, confocal microscopy quantification of intracellular Ca2+ measured by X-rhod-1 fluorescence (gray raw data, with black smoothed trendline) with simultaneous measurement of changes in PLB–SERCA FRET (YFP/Cer ratio) (gray raw data, with blue smoothed trendline). G, quantification of ER luminal Ca2+ measured by R-CEPIA1er fluorescence with simultaneous measurement of PLB–SERCA FRET (YFP/Cer ratio). H, quantification of intracellular Ca2+ measured by X-rhod-1 fluorescence with simultaneous measurement of DWORF–SERCA FRET (YFP/Cer ratio) I, quantification of ER luminal Ca2+ measured by R-CEPIA1er fluorescence with simultaneous measurement of DWORF–SERCA FRET (YFP/Cer ratio). DWORF, dwarf ORF; ER, endoplasmic reticulum; PLB, phospholamban; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase.
      To determine the relative affinity of both micropeptides for these and other key SERCA enzymatic states, we transfected HEK-293 cells with Cerulean (Cer)-labeled SERCA2a (FRET donor) and YFP-labeled PLB (FRET acceptor) and quantified the interaction of these proteins using acceptor sensitization FRET microscopy. Protein expression levels in membrane fractions prepared from HEK cells were comparable to protein concentrations in membranes prepared from human heart tissues (Fig. S1). To control the conformational poise of SERCA, cells were permeabilized with 0.05 mg/ml saponin in bath solutions appropriate for stabilization of the transporter in various conformations (see Experimental procedures and Fig. S2). The affinity of the PLB–SERCA interaction was quantified by measuring FRET with automated fluorescence microscopy, as previously described (
      • Hou Z.
      • Kelly E.M.
      • Robia S.L.
      Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.
      ,
      • Singh D.R.
      • Dalton M.P.
      • Cho E.E.
      • Pribadi M.P.
      • Zak T.J.
      • Seflova J.
      • et al.
      Newly discovered micropeptide regulators of SERCA form oligomers but bind to the pump as monomers.
      ). FRET was low in cells with low fluorescence (low protein expression), increasing to a maximum in the brightest cells (high protein expression), yielding a FRET based “binding curve” (Fig. 2B). A hyperbolic fit to the data yielded the maximal FRET efficiency at high protein concentration or FRETmax (representing the intrinsic FRET of the bound PLB–SERCA complex) and the apparent dissociation constant (KD), the protein concentration that yields half-maximal FRET efficiency. The KD value is inversely related to the affinity of the PLB–SERCA complex. Figure 2B shows the conditions that yielded the greatest difference in relative KD for the PLB regulatory complex. PLB bound to the E1-ATP state of SERCA is obtained with a solution containing 3 mM ATP and low Ca2+ and represents the prevailing conformation in resting (diastolic) conditions. Addition of thapsigargin (TG) resulted in a significant right-shift of the binding curve (p = 8 × 10−3) (Table S1), indicating a decrease in PLB–SERCA affinity. Reduced affinity of PLB for TG-inhibited SERCA is consistent with previous observations from our lab (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ) and others (
      • Jones L.R.
      • Cornea R.L.
      • Chen Z.
      Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking.
      ).
      The summary of fitting of binding curves obtained from six independent transfections for each of eight conditions is shown in Figure 2, C and D and Table 1 (Figs. S3 and S4). We observed especially avid binding of PLB to the E1-ATP state, which may indicate stabilization of that conformation of SERCA. This is the state that most SERCA pumps are in during the diastolic phase of the cardiac cycle when cytoplasmic Ca2+ is low, the muscle is relaxed, and the heart is filling with blood. The affinity of PLB for this state was not altered over a range of pH from 6 to 8 (Fig. S5), indicating no preference for “E2” or “E1” transmembrane domain conformations stabilized in acidic or alkaline pH, respectively (
      • Hughes G.
      • Starling A.P.
      • Sharma R.P.
      • East J.M.
      • Lee A.G.
      An investigation of the mechanism of inhibition of the Ca(2+)-ATPase by phospholamban.
      • Mintz E.
      • Mata A.M.
      • Forge V.
      • Passafiume M.
      • Guillain F.
      The modulation of Ca2+ binding to sarcoplasmic reticulum ATPase by ATP analogues is pH-dependent.
      ).
      Table 1Acceptor-sensitization FRET-binding curve parameters
      Enzymatic stateStabilizing ligand(s)PLB-SERCADWORF–SERCA
      KD (AU)FRETmax (%)KD (AU)FRETmax (%)
      E2TG4.4 ± 0.226 ± 0.82.7 ± 0.128 ± 0.5
      Eapo3.3 ± 0.225 ± 0.92.5 ± 0.229 ± 1.1
      E1-ATPATP1.8 ± 0.2∗22 ± 0.32.5 ± 0.229 ± 1.4
      E1Ca2+3.5 ± 0.226 ± 0.92.6 ± 0.129 ± 0.7
      E1Ca2+-AMPPCP3.6 ± 0.226 ± 0.82.6 ± 0.229 ± 1.6
      E1PCa2+-ADP-AlF43.0 ± 0.122 ± 0.92.1 ± 0.225 ± 0.8
      E2PAlF43.4 ± 0.224 ± 0.92.2 ± 0.327 ± 1.0
      E2PVi3.5 ± 0.326 ± 0.82.7 ± 0.331 ± 2.1
      Data are reported as mean ± SEM (n = 6). Differences were determined by one-way ANOVA with Dunn’s post hoc (∗p < 0.05, See Table S1 for complete statistical analysis).
      In contrast to PLB, DWORF showed a much flatter affinity profile across SERCA enzymatic states (Fig. 2D). We noted slightly lower KD values for E1PCa and E2P states. The population of SERCA pumps increasingly accumulates in these states during the systolic phase of the cardiac cycle when cytoplasmic Ca2+ is elevated and the heart is contracting to eject blood. DWORF may have a preference for those conformations, though the difference was not statistically significant by one-way ANOVA (Tables 1 and S1). We did resolve statistically significant differences in PLB–SERCA and DWORF–SERCA affinities in a simpler comparison of affinities in ATP-containing solutions: low Ca2+ “relaxing solution” versus a high Ca2+ solution that induced enzymatic cycling of SERCA (see Experimental procedures). In these physiological buffers mimicking diastole versus systole, respectively, we observed reciprocal Ca2+-dependent binding affinity for PLB–SERCA versus DWORF–SERCA (Figs. 2E and S6), consistent with our previous study (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ).
      Taken together, these data indicate that PLB and DWORF prefer to bind different SERCA conformations corresponding to different enzymatic intermediate states of the transport cycle. To determine how this differential Ca2+-dependent affinity may cause dynamic shifts in PLB and DWORF binding equilibria during intracellular Ca2+ elevations, we exploited a cardiomimetic model system described previously (
      • Bovo E.
      • Martin J.L.
      • Tyryfter J.
      • de Tombe P.P.
      • Zima A.V.
      R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes.
      ). HEK-293 cells expressing RyR and SERCA2a show spontaneous ER Ca2+ release events that give rise to large, prolonged increases in cytoplasmic Ca2+. Ca2+ transients were detected by confocal microscopy as an increase in X-rhod-1 cytoplasmic Ca2+ indicator fluorescence or a decrease in R-CEPIA1er ER Ca2+ indicator fluorescence (Fig. 2, FI, black). In some cells, these Ca2+ elevations coincided with small decreases in PLB–SERCA FRET, shown in Figure 2, F and G as gray data overlaid with a smoothed trendline (blue, see Experimental procedures) corresponding to a loss of PLB–SERCA affinity at elevated Ca2+. These were very small changes in FRET, as expected from the small differences in affinity quantified in equilibrium experiments (Fig. 2, C and E). Indeed, we were previously unable to detect a PLB–SERCA FRET change over a single Ca2+ transient in electrically paced cardiac myocytes, only observing a modest decrease in average FRET over a period of repeated Ca2+ transients during rapid pacing (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ). However, in HEK cells the Ca2+ elevations were more prolonged (increasing time for quantification), expression of endogenous SERCA/PLB was very low (reducing competing non-FRET interactions), and the cells are noncontractile (eliminating confounding cell motions). These advantages enabled detection of small shifts in the binding equilibria in the present study.
      Next, we tested whether there was a change in DWORF–SERCA FRET in response to Ca2+ elevations. These FRET changes were even smaller than those observed for PLB–SERCA binding (Fig. 2H), consistent with DWORF’s relatively flat affinity profile across SERCA enzymatic states (Fig. 2D). The DWORF–SERCA FRET change was easier to appreciate in experiments where a low affinity Ca2+ indicator localized to the ER lumen (R-CEPIA1er) was used instead of a cytoplasmic Ca2+ indicator dye (Fig. 2I), perhaps because ER Ca2+ buffering by the indicator resulted in larger amplitude Ca2+ release events. Interestingly, the direction of the DWORF–SERCA FRET change was the opposite of that observed for PLB–SERCA; we observed modestly increased FRET during Ca2+ transients (Fig. 2, H and I), consistent with enhanced DWORF–SERCA binding affinity at elevated cytoplasmic Ca2+ (Fig. 2E) (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ).

      PLB reassociation with SERCA after Ca2+ transients is delayed by the PLB pentamer

      To examine how the kinetics of cellular Ca2+ signaling determines SERCA–micropeptide binding dynamics, we quantified the rates of change of FRET and [Ca2+]. Specifically, exponential decay fitting of these changes revealed characteristic time constants (τ) for each process. Analysis of the Ca2+ release process (Fig. 3A) showed that DWORF–SERCA binding occurred with a time course that was similar to the rate of rise of Ca2+ (τ = 0.22 ± 0.03 s and 0.21 ± 0.03 s, respectively) (Fig. 3B). In contrast, PLB–SERCA unbinding was slower (τ = 0.74 ± 0.14 s), lagging behind the Ca2+ upstroke (p = 4 × 10−8) (Fig. 3C). In addition, we evaluated how Ca2+-dependent changes in PLB–SERCA binding affects the PLB monomer–pentamer equilibrium, measuring intrapentameric FRET between Cer-PLB and YFP-PLB. Interestingly, PLB–PLB FRET increased rapidly during Ca2+ elevations (Fig. S6). The inverse changes in PLB–SERCA FRET and PLB–PLB FRET show that as PLB is displaced from SERCA, it is rapidly incorporated into pentamers. The time course of PLB–PLB binding (τ = 0.59 ± 0.04 s) also lagged slightly behind Ca2+ release (p = 2 × 10−4) (Fig. 3D). This delay was similar to that of PLB–SERCA unbinding (Fig. 3E), suggesting that PLB–SERCA unbinding may be rate limiting for subsequent PLB oligomerization during Ca2+ elevations. The kinetics of SERCA–micropeptide binding dynamics during Ca2+ release are summarized in Fig. S8 and Tables S2–S4.
      Figure thumbnail gr3
      Figure 3PLB reassociation with SERCA after Ca2+ transients are delayed by slow dissociation from the PLB pentamer. A, schematic diagram of shifts in PLB and DWORF binding equilibria during Ca2+ release. B, representative single exponential decay fit of the kinetics of DWORF–SERCA binding during Ca2+ release. C, kinetics of PLB–SERCA unbinding during Ca2+ release. D, kinetics of PLB–PLB binding during Ca2+ release. E, the latency of FRET ratio changes compared to Ca2+ release with lines representing mean ± SEM. Differences determined by one-way ANOVA with Dunn’s post hoc test (∗p < 0.05, see for complete statistical analysis). F, schematic diagram of shifts PLB and DWORF binding equilibria during Ca2+ uptake. G, representative single exponential decay fit of the kinetics of DWORF–SERCA unbinding during Ca2+ uptake. H, kinetics of PLB–SERCA rebinding during Ca2+ uptake. I, kinetics of PLB–PLB binding during Ca2+ release. J, the latency of FRET ratio changes compared to Ca2+ release with lines representing mean ± SEM. Differences determined by one-way ANOVA with Dunn’s post hoc test (∗p < 0.05, see for complete statistical analysis). DWORF, dwarf ORF; PLB, phospholamban; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase.
      Next, we examined changes in SERCA–micropeptide binding as Ca2+ declined to baseline during the Ca2+-reuptake phase of the Ca2+ transient (Fig. 3F). DWORF dissociated rapidly from SERCA, as measured by the decrease in the DWORF–SERCA FRET ratio back to baseline (Fig. 3G). This unbinding process occurred with a τ of 1.2 ± 0.4 s, similar to the Ca2+ transient relaxation time. However, PLB–SERCA reassociation displayed a significant lag compared to the Ca2+ transient relaxation, with FRET continuing to increase back toward maximum for several seconds after Ca2+ had already stabilized at a basal level (Fig. 3H). PLB–SERCA reassociation occurred with a τ of 4.9 ± 0.6 s, much slower than the Ca2+ relaxation time (τ = 1.6 ± 0.2 s, p = 2 × 10−8).
      To understand this unexpectedly slow rate for PLB–SERCA rebinding, we considered whether the rate of PLB–SERCA recovery could be limited by slow dissociation of PLB monomers from the PLB pentamer (
      • Robia S.L.
      • Campbell K.S.
      • Kelly E.M.
      • Hou Z.
      • Winters D.L.
      • Thomas D.D.
      Forster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex.
      ,
      • Koch D.
      • Alexandrovich A.
      • Funk F.
      • Kho A.L.
      • Schmitt J.P.
      • Gautel M.
      Molecular noise filtering in the beta-adrenergic signaling network by phospholamban pentamers.
      ). Indeed, PLB–PLB FRET relaxation (unbinding of monomers from the pentamer) displayed a slow time course (τ = 5.2 ± 0.8 s) (Fig. 3I) that closely matched the slow PLB–SERCA reassociation (4.9 ± 0.6 s) (Fig. 3J). The data suggest PLB pentamer dissociation is rate limiting for PLB rebinding SERCA after Ca2+ elevations. This interpretation was further supported by the observation that destabilization of the PLB pentamer by mutagenesis (PLB-AFA) (
      • Ha K.N.
      • Masterson L.R.
      • Hou Z.
      • Verardi R.
      • Walsh N.
      • Veglia G.
      • et al.
      Lethal Arg9Cys phospholamban mutation hinders Ca2+-ATPase regulation and phosphorylation by protein kinase A.
      ) accelerated the PLB–SERCA reassociation rate (τ = 2.5 ± 0.5 s, p = 8 × 10−3), such that the FRET change no longer lagged behind the Ca2+ transient decay (Figs. 3J and S9).
      We also examined how pentamer dissociation kinetics could be physiologically tuned to control the PLB–SERCA reassociation rate. Equilibrium measurements of PLB oligomerization have shown that PLB pentamers are stabilized by PKA phosphorylation of serine 16 of PLB (
      • Cornea R.L.
      • Jones L.R.
      • Autry J.M.
      • Thomas D.D.
      Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers.
      ), which occurs as an outcome of adrenergic signaling during physiological stress. Here, we found that stabilization of pentameric interactions by a phosphomimetic S16E mutation further slowed the rate of PLB–SERCA reassociation (τ = 8.6 ± 0.7 s, p = 2 × 10−6) (Figs. 3J and S10). The kinetics of SERCA–micropeptide binding dynamics during Ca2+ uptake are summarized in Fig. S11 and Table S5–S7. Overall, the results support the hypothesis that PLB pentamers delay reassociation of the dynamic fraction of PLB with SERCA after Ca2+ elevations, and this physiological mechanism is tuned under the control of adrenergic signaling.

      Modeling the redistribution of PLB and DWORF regulatory complexes

      To interpret our FRET-based measurements of SERCA–micropeptide binding dynamics, we developed a computational model to integrate the measured rates of the dynamic interactions of PLB and DWORF with SERCA in the context of the human cardiac cycle. Using this model, we simulated the dynamic redistribution of these regulatory complexes between systole and diastole, or on a longer timescale, between rest and exercise. The model describes the kinetics of these regulatory interactions with a set of numerically solved ordinary differential equations (see Supplemental Methods). A genetic algorithm was used to fit mean rate constants for the forward and reverse reactions in the model from experimentally measured FRET data as previously described (
      • Stewart B.D.
      • Scott C.E.
      • McCoy T.P.
      • Yin G.
      • Despa F.
      • Despa S.
      • et al.
      Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes.
      ). For simplicity, we considered the population of SERCA pumps to be distributed between two ensembles of pump enzymatic states, representing the “diastolic” condition (Ca-free) and the “systolic” condition (Ca-bound), as diagrammed in Figure 4A. The relative population of these two ensemble states is dependent on the relative concentration of Ca2+, as quantified by confocal microscopy experiments. Monomeric PLB (PLB1) can bind either the Ca-free or Ca-bound ensemble of SERCA (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ) but with higher affinity for the Ca-free population (Fig. 2E). The relative affinity of PLB for SERCA was constrained to be twofold higher for Ca-free versus Ca-bound ensembles, as determined experimentally from FRET-binding curves (Fig. 2C) (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ). Monomeric PLB (PLB1) is also in equilibrium with the pentameric population (PLB5). The relative affinity of DWORF for SERCA was constrained to increase by 25% between Ca-free and Ca-bound ensembles, consistent with FRET measurements (Fig. 2D) (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ). The computed kinetic parameters are provided in the Supplemental data (Fig. S12).
      Figure thumbnail gr4
      Figure 4A computational model simulated the dynamics of PLB and DWORF interactions with SERCA. A, simplified diagram of modeled regulatory interactions. B, a fit of the model to a representative FRET change measured by confocal microscopy. C, simulation of changes in the populations of regulatory species during a Ca2+ elevation, where relative amounts of SERCA and PLB are equal. Black circles represent the results of the model of the normal, nonfailing heart. Red triangles represent adjustment of the model to simulate heart failure. D, simulation of the effect of cardiac pacing on PLB–SERCA at three heart rates (beats per min, BPM), where the ratio of SERCA:PLB was 1:3. E, increasing pacing frequency modestly decreased PLB–SERCA binding. F, increasing pacing frequency increased PLB oligomerization. G, PLB–SERCA oscillation amplitude decreased with faster pacing. H, simulation of the effect of increasing DWORF expression relative to SERCA on PLB–SERCA binding. I, increasing DWORF resulted in a decrease in the equilibrium level of PLB–SERCA. J, increasing DWORF increased PLB oligomerization. K, increasing DWORF relative to SERCA resulted in larger oscillations in PLB–SERCA binding. For HK, the ratio of SERCA:PLB was 1:3 and pacing rate was 180 BPM. DWORF, dwarf ORF; PLB, phospholamban; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase.

      Dynamic responses to Ca2+ transients

      The computational model provided good descriptions of data from physical experiments showing time-dependent changes in PLB–SERCA FRET and cytoplasmic Ca2+ (measured simultaneously). Figure 4B provides a best-fit model prediction of the FRET compared to a representative experimental measurement. The model captured the rapid reduction of FRET with the rise of the Ca2+ transient. The decrease in PLB–SERCA is followed by a slower restoration of the FRET as Ca2+ returns to baseline. The similarity of the predicted and experimentally measured FRET data suggests that the model appropriately represents key aspects of PLB–SERCA binding dynamics.
      Using these fit parameter values, we simulated the time-dependent changes in PLB–SERCA and PLB–PLB binding dynamics that we observed in HEK cells. Importantly, we observed that a majority of the PLB–SERCA regulatory complexes remained intact (Fig. 4C, purple) during a Ca2+ elevation (Fig. 4C, gray shaded region), consistent with the “subunit model” (
      • Mueller B.
      • Karim C.B.
      • Negrasshov I.V.
      • Kutchai H.
      • Thomas D.D.
      Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer.
      ). Thus, PLB can remain bound to SERCA when SERCA is bound to Ca2+; this Ca2+-bound PLB–SERCA fraction is highlighted in Figure 4C (red). However, a fraction of the population of PLB–SERCA dissociated during a Ca2+ transient (Fig. 4C, purple). This fraction comprised ∼30% of PLB–SERCA complexes under these simulation conditions. This shift impacted the PLB monomer/pentamer equilibrium (Fig. 4C, blue/green). Expectedly, there was very little observed change in the small population of PLB monomers, as the dynamic fraction was quickly incorporated into pentamers. These reciprocal shifts are the cause of the transient decrease in SERCA–PLB FRET and increase in PLB–PLB FRET observed by confocal microscopy (Fig. 3, C and D).
      Next, we simulated the shorter, faster Ca2+ transients observed in the heart, pacing at a range of frequencies from 60 to 180 beats per minute, to investigate regulatory dynamics under conditions of rest, moderate exercise, and intense exercise. Increasing pacing frequency progressively decreased PLB–SERCA binding (Fig. 4, D and E, black circles) and increased accumulation of PLB in pentamers (Fig. 4F, black circles). The systole–diastole difference in PLB–SERCA binding was small. This oscillation became even smaller with increasing pacing frequency (Fig. 4, D and G, black circles), as the shifts in binding equilibria began to lag behind the rapid Ca2+ changes. These results suggest that the exchange dynamics of PLB may impact adaptive SERCA regulation between resting and exercising heart rates.
      To assess how PLB–SERCA binding dynamics may be altered in disease, we modified the model conditions to reflect changes observed in heart failure. Specifically, the SERCA population was reduced by 40% (
      • Hasenfuss G.
      • Reinecke H.
      • Studer R.
      • Meyer M.
      • Pieske B.
      • Holtz J.
      • et al.
      Relation between myocardial function and expression of sarcoplasmic reticulum Ca2+-ATPase in failing and nonfailing human myocardium.
      ) and diastolic Ca2+ was increased by 50% (
      • Stewart B.D.
      • Scott C.E.
      • McCoy T.P.
      • Yin G.
      • Despa F.
      • Despa S.
      • et al.
      Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes.
      ). The heart failure condition is shown in Figure 4 as red triangles. As expected, the heart failure condition decreased the population of PLB bound to SERCA at equilibrium (Fig. 4E), decreased PLB–SERCA oscillation amplitude (Fig. 4G), and blunted the responsiveness of these parameters to increasing heart rates (Fig. 4, EG). This suggests frequency-dependent changes in PLB inhibition may be reduced in heart failure.
      Introduction of DWORF–SERCA interactions to the model yielded the expected effect of competition of DWORF and PLB for SERCA binding (Fig. 4A). Increasing the amount of DWORF relative to PLB decreased PLB–SERCA binding (Fig. 4, H and I, black circles) and increased PLB oligomerization (Fig. 4J, black circles). DWORF competes potently with PLB even at low stoichiometry because oligomerization of PLB reduces the effective concentration of the active monomeric species. Interestingly, the Ca2+-dependent increase in DWORF affinity for SERCA exaggerated the oscillations in PLB–SERCA binding during pacing (Fig. 4K, black circles). That is, during Ca2+ elevations DWORF (which binds SERCA better at high Ca2+) increasingly displaced PLB (which binds SERCA better at low Ca2+).
      Under conditions representing heart failure (decreased SERCA expression, increased diastolic Ca2+) (Fig. 4, IK, red triangles), there was decreased formation of the PLB–SERCA complex (Fig. 4E), and therefore, the effect of DWORF competition was blunted (Fig. 4I). Moreover, since changes in PLB–SERCA binding between systole and diastole were smaller in heart failure, the ability of DWORF to exaggerate this change through calcium-dependent competition was also decreased (Fig. 4I). These results suggest that the impact of DWORF on PLB–SERCA binding dynamics may be reduced in heart failure.

      Discussion

      Differential Ca2+-dependence of micropeptides for SERCA

      The principal finding of the present study is that PLB and DWORF preferentially bind to different conformations of SERCA (Fig. 2, C and D), and this is the underlying cause of the reciprocal Ca2+-dependent affinities of these micropeptides (Fig. 2E) (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ). The FRET-based binding assay showed that PLB has the highest affinity for the E1-ATP state (Fig. 2C), which is the predominant state at resting Ca2+ (Fig. 2A, blue box). In contrast, DWORF shows a much flatter SERCA-binding profile, with more avid binding to E1P and E2P states (Fig. 2D). Much of the SERCA population accumulates in these states when cytoplasmic Ca2+ is elevated. This accumulation occurs because of rate-limiting, slow steps in the SERCA enzymatic cycle (
      • Clausen J.D.
      • Vandecaetsbeek I.
      • Wuytack F.
      • Vangheluwe P.
      • Andersen J.P.
      Distinct roles of the C-terminal 11th transmembrane helix and luminal extension in the partial reactions determining the high Ca2+ affinity of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b).
      ,
      • Dode L.
      • Andersen J.P.
      • Leslie N.
      • Dhitavat J.
      • Vilsen B.
      • Hovnanian A.
      Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses.
      ), which cause rapidly cycling pumps to build up in “traffic jams” in E1P and E2P (Fig. 2A, red boxes). Figure 3, A and F show how differential binding of micropeptides leads to exchange of a dynamic fraction of PLB and DWORF from the binding site on SERCA during intracellular Ca2+ elevations. In high Ca2+ (Fig. 3A), DWORF increasingly outcompetes PLB, displacing a fraction of monomeric PLB (“PLB1”), which is increasingly incorporated into PLB pentamers (“PLB5”). Conversely, in low Ca2+ (Fig. 3F), DWORF binding is decreased, and PLB monomers increasingly bind to SERCA because the pump accumulates in the E1-ATP state that PLB prefers. This recovery process is rate limited by the slow dissociation of monomeric PLB from pentamers (Fig. 3I).
      We propose that differential Ca2+ dependence is the key determinant of whether a micropeptide is inhibitory or stimulatory for SERCA function. In particular, micropeptide–SERCA binding affinity may be taken as an index of the relative energetics of the regulatory complex, much as melting temperature may be quantified as a proxy for the stability of a protein–protein complex (
      • Fukuzawa A.
      • Koch D.
      • Grover S.
      • Rees M.
      • Gautel M.
      When is an obscurin variant pathogenic? The impact of Arg4344Gln and Arg4444Trp variants on protein-protein interactions and protein stability.
      ). Thus, avid PLB binding to the E1-ATP structure (Fig. 2A, blue box) is expected to stabilize that complex and favor the population of that state of SERCA. The functional consequence of stabilizing this state is to slow the subsequent Ca2+-binding step in the enzymatic cycle. This may account for the principal inhibitory effects of PLB, reducing the apparent Ca2+ affinity of the pump and slowing pump turnover (
      • Cantilina T.
      • Sagara Y.
      • Inesi G.
      • Jones L.R.
      Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.
      ). Moreover, we propose that while PLB binding to E1-ATP deepens a depression in the energy landscape, DWORF reduces the height of a peak, lowering an energy barrier by stabilizing E1P/E2P states. Increasing pump flux through these rate-limiting steps enhances enzyme turnover when Ca2+ is high (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ).
      In addition, the dynamics of PLB and DWORF binding and unbinding from SERCA described here have important functional implications. Reciprocal binding may be important for reacting to transient Ca2+ changes during the cardiac cycle or, on a longer timescale, responding to adrenergic signaling and changes in pacing frequency during exercise. The present data provide insight into Ca2+ transport regulation on both timescales, as discussed in the following sections.

      Functional implications of micropeptide exchange during the cardiac cycle

      The change in PLB–SERCA binding with Ca2+ is only approximately twofold, so PLB–SERCA complexes do not completely dissociate during systole (Fig. 4D) (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ,
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ). However, cardiac Ca2+ elevations simultaneously reduce PLB-binding affinity and favor DWORF binding, so a fraction of the population of these micropeptides must exchange from SERCA during each Ca2+ transient. Our computational model showed that the expected functional impact of this exchange is to exaggerate the intrinsic response of SERCA to changing Ca2+. That is, during diastole (cardiac relaxation), SERCA activity is already low because Ca2+ is low. SERCA is further inhibited in diastole by an increase in the fraction of pumps that bind PLB (Fig. 2, F and G). Then, during systole (cardiac contraction), Ca2+ is high, supporting high SERCA activity. This high SERCA activity is further enhanced by increased DWORF binding (Fig. 2, H and I), which increasingly displaces PLB and directly stimulates SERCA maximal activity (
      • Fisher M.E.
      • Bovo E.
      • Aguayo-Ortiz R.
      • Cho E.E.
      • Pribadi M.P.
      • Dalton M.P.
      • et al.
      Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
      ,
      • Reddy U.V.
      • Weber D.K.
      • Wang S.
      • Larsen E.K.
      • Gopinath T.
      • De Simone A.
      • Robia S.
      • Veglia G.
      A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca(2+)-ATPase.
      ,
      • Li A.
      • Yuen S.L.
      • Stroik D.R.
      • Kleinboehl E.
      • Cornea R.L.
      • Thomas D.D.
      The transmembrane peptide DWORF activates SERCA2a via dual mechanisms.
      ,
      • Makarewich C.A.
      • Munir A.Z.
      • Schiattarella G.G.
      • Bezprozvannaya S.
      • Raguimova O.N.
      • Cho E.E.
      • et al.
      The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.
      ,
      • Nelson B.R.
      • Makarewich C.A.
      • Anderson D.M.
      • Winders B.R.
      • Troupes C.D.
      • Wu F.
      • et al.
      A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.
      ,
      • Makarewich C.A.
      • Bezprozvannaya S.
      • Gibson A.M.
      • Bassel-Duby R.
      • Olson E.N.
      Gene therapy with the DWORF micropeptide attenuates cardiomyopathy in mice.
      ,
      • Mbikou P.
      • Rademaker M.T.
      • Charles C.J.
      • Richards M.A.
      • Pemberton C.J.
      Cardiovascular effects of DWORF (dwarf open reading frame) peptide in normal and ischaemia/reperfused isolated rat hearts.
      ). Together, low Ca2+ inhibition and high Ca2+ stimulation should enhance the apparent cooperativity of the Ca2+ response, which would benefit cardiac function by conserving ATP consumption until Ca2+ transport is most efficient and most needed. Thus, competitive binding of SERCA by DWORF decreases both the basal level of PLB–SERCA binding as previously proposed (
      • Nelson B.R.
      • Makarewich C.A.
      • Anderson D.M.
      • Winders B.R.
      • Troupes C.D.
      • Wu F.
      • et al.
      A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.
      ) and also increases the amplitude of the oscillations in the population of the PLB–SERCA complex (Fig. 4, IK). The dose dependence of these effects revealed in the computational model may provide guidance for future DWORF-based gene therapy approaches (
      • Makarewich C.A.
      • Munir A.Z.
      • Schiattarella G.G.
      • Bezprozvannaya S.
      • Raguimova O.N.
      • Cho E.E.
      • et al.
      The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.
      ,
      • Makarewich C.A.
      • Bezprozvannaya S.
      • Gibson A.M.
      • Bassel-Duby R.
      • Olson E.N.
      Gene therapy with the DWORF micropeptide attenuates cardiomyopathy in mice.
      ,
      • Mbikou P.
      • Rademaker M.T.
      • Charles C.J.
      • Richards M.A.
      • Pemberton C.J.
      Cardiovascular effects of DWORF (dwarf open reading frame) peptide in normal and ischaemia/reperfused isolated rat hearts.
      ).

      The function of the PLB pentamer kinetic trap in rest and exercise

      The observations presented here also have implications for the enhancement of cardiac function during exercise. PLB dissociation from pentamers is slow (τ ∼5 s) (Fig. 3D), a noteworthy finding since this is significantly slower than the cardiac cycle. Even at a resting heart rate, a 1 s interval between beats would not be sufficient time for the equilibrium to fully relax between systole and diastole. Therefore, Ca2+ signals must also integrate over a longer timescale, such as between rest and exercise. This is visualized in our computational model, which showed increasing accumulation of PLB in pentamers at faster heart rates at the expense of the population of PLB–SERCA (Fig. 4D). Such “kinetic trapping” of PLB in pentamers at faster heart rates is further increased by phosphorylation of PLB by PKA or CaMKII, which stabilizes the pentamer complex (
      • Hou Z.
      • Kelly E.M.
      • Robia S.L.
      Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.
      ,
      • Cornea R.L.
      • Jones L.R.
      • Autry J.M.
      • Thomas D.D.
      Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers.
      ,
      • Song Q.
      • Pallikkuth S.
      • Bossuyt J.
      • Bers D.M.
      • Robia S.L.
      Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.
      ). These kinases are activated in exercise through adrenergic signaling and prolonged Ca2+ elevation, respectively. The functional consequence of decreased SERCA inhibition by PLB is increased SR Ca2+ load and enhanced cardiac contractility. Thus, one may speculate that frequency-dependent accumulation of PLB in pentamers contributes to the Bowditch effect, a positive force–frequency relationship in which a faster heart rate causes more forceful contractions of the heart. The mechanism we propose may explain previous studies that showed the Bowditch effect was absent in PLB KO mice (
      • Bowditch H.P.
      Über die Eigenthümlichkeiten der Reizbarkeit, welche die Muskelfasern des Herzens zeigen. Arbeiten aus der Physiologischen Anstalt zu Leipzig.
      ,
      • Bluhm W.F.
      • Kranias E.G.
      • Dillmann W.H.
      • Meyer M.
      Phospholamban: a major determinant of the cardiac force-frequency relationship.
      ,
      • Meyer M.
      • Bluhm W.F.
      • He H.
      • Post S.R.
      • Giordano F.J.
      • Lew W.Y.
      • et al.
      Phospholamban-to-SERCA2 ratio controls the force-frequency relationship.
      ,
      • Wu Y.
      • Luczak E.D.
      • Lee E.J.
      • Hidalgo C.
      • Yang J.
      • Gao Z.
      • et al.
      CaMKII effects on inotropic but not lusitropic force frequency responses require phospholamban.
      ). Moreover, simulations showed that under pathological conditions SERCA regulation is decreased and the frequency dependence of that regulation is diminished (Fig. 4, EG). This may explain why the Bowditch effect is blunted in patients with heart failure (
      • Mulieri L.A.
      • Hasenfuss G.
      • Leavitt B.
      • Allen P.D.
      • Alpert N.R.
      Altered myocardial force-frequency relation in human heart failure.
      ).

      Summary and future directions

      Dynamic changes in the competition of regulatory micropeptides on seconds and minutes timescales represent an important aspect of the responsiveness of SERCA regulation to Ca2+ transients. The present data clarify the role of the PLB pentamer as a phosphorylation-tunable kinetic trap that limits the rate of SERCA rebinding by a dynamic fraction of PLB. Future studies will test the functional predictions derived from the present results: DWORF enhances transitions through E1P, E2P states in the SERCA enzymatic cycle; DWORF increases the size of the fraction of PLB that binds/unbinds SERCA with each Ca2+ elevation, exaggerating the SERCA inhibition/stimulation cycle; DWORF and PLB act in concert to increase the cooperativity of Ca2+ transport. Overall, the data suggest that dynamic competition of PLB and DWORF is an important determinant of cardiac function. More generally, the results may provide insight into the interplay of other tissue-specific micropeptide regulators of ion transporters.

      Experimental procedures

      Plasmid constructs

      For all plasmid constructs, we used pEGFP-C1 as the expression vector in mammalian cells. All micropeptides (PLB, DWORF, PLB-AFA, and PLB-S16A) and SERCA constructs consisted of mCerulean3, enhanced YFP, or TagRFP fused via a five amino acid linker to the N terminus of the micropeptide or SERCA (
      • Robia S.L.
      • Campbell K.S.
      • Kelly E.M.
      • Hou Z.
      • Winters D.L.
      • Thomas D.D.
      Forster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex.
      ,
      • Makarewich C.A.
      • Munir A.Z.
      • Schiattarella G.G.
      • Bezprozvannaya S.
      • Raguimova O.N.
      • Cho E.E.
      • et al.
      The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.
      ,
      • Autry J.M.
      • Rubin J.E.
      • Pietrini S.D.
      • Winters D.L.
      • Robia S.L.
      • Thomas D.D.
      Oligomeric interactions of sarcolipin and the Ca-ATPase.
      ). We previously showed SERCA singly or doubly labeled with fluorescent protein still retains normal ATPase activity (
      • Hou Z.
      • Hu Z.
      • Blackwell D.J.
      • Miller T.D.
      • Thomas D.D.
      • Robia S.L.
      2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding.
      ,
      • Gruber S.J.
      • Cornea R.L.
      • Li J.
      • Peterson K.C.
      • Schaaf T.M.
      • Gillispie G.D.
      • et al.
      Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.
      ) and Ca2+ transport function (
      • Hou Z.
      • Hu Z.
      • Blackwell D.J.
      • Miller T.D.
      • Thomas D.D.
      • Robia S.L.
      2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding.
      ,
      • Raguimova O.N.
      • Smolin N.
      • Bovo E.
      • Bhayani S.
      • Autry J.M.
      • Zima A.V.
      • et al.
      Redistribution of SERCA calcium pump conformers during intracellular calcium signaling.
      ,
      • Bovo E.
      • Nikolaienko R.
      • Bhayani S.
      • Kahn D.
      • Cao Q.
      • Martin J.L.
      • et al.
      Novel approach for quantification of endoplasmic reticulum Ca(2+) transport.
      ). In addition, SERCA fused to two fluorescent proteins can be regulated by PLB fused to a third fluorescent protein (
      • Hou Z.
      • Hu Z.
      • Blackwell D.J.
      • Miller T.D.
      • Thomas D.D.
      • Robia S.L.
      2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding.
      ). The data suggest that the tags are benign for the pump and regulator.

      Cell culture and transfection

      AAV 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) cell culture medium supplemented with 10% fetal bovine serum (ThermoScientific). Following culture, the cells were transiently transfected using either MBS mammalian transfection kit (Agilent Technologies, Stratagene) or Lipofectamine 3000 transfection kit (Invitrogen) as per instructions provided with the respective kits. Twenty-four hours post-transfection, the cells were trypsinized (ThermoScientific) and replated onto poly-D-lysine–coated glass bottom chambers and allowed to settle down for 1 h before imaging. We have previously estimated that this approach achieves ER protein expression levels on the order of 2000:1 or 4000:1 lipids:protein ratio (
      • Blackwell D.J.
      • Zak T.J.
      • Robia S.L.
      Cardiac calcium ATPase Dimerization measured by cross-linking and fluorescence energy transfer.
      ), versus 750:1 lipid:protein ratio quantified for native SR (
      • Mueller B.
      • Karim C.B.
      • Negrasshov I.V.
      • Kutchai H.
      • Thomas D.D.
      Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer.
      ,
      • Ferrington D.A.
      • Yao Q.
      • Squier T.C.
      • Bigelow D.J.
      Comparable levels of Ca-ATPase inhibition by phospholamban in slow-twitch skeletal and cardiac sarcoplasmic reticulum.
      ). Thus, native protein concentrations are threefold to fivefold more concentrated, and we do not consider this heterologous model to represent “overexpression” of SR proteins.

      Human heart tissue procurement

      Human left ventricular tissue was provided by Loyola Cardiovascular Research Institute Biorepository. The sample collection was approved by Loyola University Review Board (IRB number 210940821918) and written informed consent was obtained for collection of heart tissue according to Declaration of Helsinki.

      Human tissue membrane protein enrichment

      Membrane protein enriched fractions were prepared from human heart tissue as previously described (
      • Seflova J.
      • Habibi N.R.
      • Yap J.Q.
      • Cleary S.R.
      • Fang X.
      • Kekenes-Huskey P.M.
      • et al.
      Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.
      ). Briefly, frozen heart tissue was placed in 5 ml of a solution containing 100 mM KCl, 2.5 mM K2HPO4, 2.5 mM KH2PO4, 2 mM EDTA, and protease inhibitors. Samples were mechanically homogenized with an Omni International GLH-01 homogenizer and rotated for 1 h at 4 °C. Homogenates were centrifuged at 10,000g for 20 min at 4 °C. Supernatants were collected and centrifuged at 48,000g for 1 h at 4 °C. The pellet containing membrane fractions was resuspended in 100 μl of a solution containing 1 M sucrose and 50 mM KCl.

      HEK cell membrane protein enrichment

      Membrane protein enriched fractions were prepared from transfected HEK cells as previously described (
      • Seflova J.
      • Habibi N.R.
      • Yap J.Q.
      • Cleary S.R.
      • Fang X.
      • Kekenes-Huskey P.M.
      • et al.
      Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.
      ). Fourty-eight hours post-transfection, HEK cells were scraped in 5 ml of homogenizing solution containing 250 mM sucrose, 10 mM Tris, and 2 mM EDTA pH 7.4 with protease inhibitors and centrifuged at 4000g for 10 min at 4 °C. Cell pellets were resuspended in homogenizing solution and mechanically homogenized. Cell homogenates were centrifuged at 4000g for 20 min at 4 °C. Supernatants were collected and centrifuged at 55,000g for 30 min at 4 °C. The pellet containing membrane fractions was resuspended in 100 μl of a solution containing 1 M sucrose and 50 mM KCl.

      Western blotting

      Sample total protein concentrations were determined using a Pierce bicinchoninic acid protein assay kit. Thirty micrograms of sample were diluted in 4× Laemmli sample buffer with β-mercaptoethanol at 1:1 ratio, denatured at 90 °C for 5 min, run on a 4 to 15% polyacrylamide gradient gel, and transferred to a polyvinylidene difluoride membrane. The membrane was stained with Revert total protein stain (LI-COR Biosciences) for 5 min to obtain total protein in each lane and then blocked for 1 h at room temperature in Intercept blocking buffer (LI-COR Biosciences) diluted at a 1:1 ratio in PBS with Tween 20 (PBS-T). Blots were incubated overnight at 4 °C with primary antibody diluted 1:1000 in PBS-T: mouse anti-SERCA2 (abcam; ref# ab2817), mouse anti-PLB (Invitrogen; ref# MA3-922;2D12), or rabbit anti-DWORF (
      • Nelson B.R.
      • Makarewich C.A.
      • Anderson D.M.
      • Winders B.R.
      • Troupes C.D.
      • Wu F.
      • et al.
      A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.
      ) (a gift of Catherine Makarewich, Cincinnati Children’s Hospital). The anti-DWORF antibody was a custom polyclonal antibody derived against the N-terminal region of the mouse DWORF protein sequence (MAEKESTSPHLI) and did not react with human DWORF (Fig. S1). The blots were then incubated with antimouse (IRDye 680RD; LI-COR Biosciences) or anti-rabbit (IRDye 800CW; LI-COR Biosciences) secondary antibody diluted 1:10,000 in PBS-T. Blots were imaged using an Azure c600 gel imaging system and analyzed using the LI-COR Image Studio software.

      FRET acceptor sensitization in permeabilized HEK-293 cells

      Acceptor sensitization FRET was quantified as previously described (
      • Bidwell P.
      • Blackwell D.J.
      • Hou Z.
      • Zima A.V.
      • Robia S.L.
      Phospholamban binds with differential affinity to calcium pump conformers.
      ). Briefly, AAV 293 cells were transiently transfected with Cer-donor and YFP acceptor–labeled FRET-binding partners in a 1:5 M plasmid ratio. FRET acceptor sensitization was measured by automated fluorescence microscopy before and after permeabilization with 0.005% w/v saponin. For each condition, two sets of 72 images (∼500 total cells per condition) were collected from six independent experiments with a 20 × 0.75 numerical aperture objective with 50 ms exposure for each channel: Cer, YFP, and FRET (Cer excitation/YFP emission). Cells that expressed Cer had an area of 136 to 679 μm2 and were at least 40% circular, were automatically scored for Cer, YFP, and FRET fluorescence intensity with a rolling background subtraction using a plugin in Fiji (imagej.net/software/fiji/). FRET efficiency was calculated according to E=G/(G+2.782FCer), where G=FFRETaFYFPdFCer, where FFRET, FYFP, and FCer are the fluorescence intensities from FRET, YFP, and Cer images, respectively, and G represents FRET intensity corrected for the bleedthrough of the channels. The parameters a and d are bleedthrough constants calculated as a=FFRET/FYFP for a control sample transfected only with YFP-SERCA and d=FFRET/FCer for a control sample transfected only with Cer-SERCA. For our experimental setup, a and d were 0.185 and 0.405, respectively. FRET efficiency for each scored cell was plotted as a function of expressed protein concentration, as determined from the fluorescence intensity of the YFP channel (
      • Hou Z.
      • Kelly E.M.
      • Robia S.L.
      Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.
      ,
      • Singh D.R.
      • Dalton M.P.
      • Cho E.E.
      • Pribadi M.P.
      • Zak T.J.
      • Seflova J.
      • et al.
      Newly discovered micropeptide regulators of SERCA form oligomers but bind to the pump as monomers.
      ). FRET was low in cells with low fluorescence (low protein expression), increasing to a maximum in the brightest cells (high protein expression), yielding a FRET-based “binding curve.” The data were fit to a hyperbolic function of the form [FRET = FRETmax [YFP]/(KD+[YFP]), where FRETmax is the maximal FRET efficiency at high protein concentration (representing the intrinsic FRET of the bound complex), [YFP] is inferred from YFP fluorescence emission in each cell, and KD is the apparent dissociation constant (the protein concentration that yields half-maximal FRET efficiency).
      To control the conformational poise of SERCA, cells were permeabilized with 0.05 mg/ml saponin in bath solutions, appropriate for stabilization of the transporter in various conformations. Solutions for ligand stabilization of enzymatic intermediate states were prepared by addition of corresponding substrates to a calcium-free base solution, which includes 100 mM KCl, 5 mM MgCl2, 2 mM EGTA, and 10 mM imidazole, pH 7.0. The base solution was used to characterize SERCA in a ligand-free state, Eapo. The following ligands were used to prepare specific solutions corresponding to SERCA biochemical state: 100 μM TG for E2(TG); 3 mM ATP for E1-ATP; 2.1 mM CaCl2 for E1(Ca2) with free [Ca2+]i = 100 μM (
      • Schoenmakers T.J.
      • Visser G.J.
      • Flik G.
      • Theuvenet A.P.
      Chelator: an improved method for computing metal ion concentrations in physiological solutions.
      ); 2.1 mM CaCl2 and 500 μM nonhydrolyzable ATP analog (AMPPCP) for E1(Ca2-AMPPCP); 2.1 mM CaCl2, 500 μM ADP, 50 μM AlCl3, and 3 mM KF for E1(Ca2–ADP–AlF4); 0.1 mM orthovanadate for E2(Vi); and 50 μM AlCl3 and 3 mM KF for E2(AlF4). Concentrations of AMPPCP nucleotide analog and ADP nucleotide were reduced from previously published conditions (3 mM) used to stabilize SERCA in microsomal fractions (
      • Makarewich C.A.
      • Munir A.Z.
      • Schiattarella G.G.
      • Bezprozvannaya S.
      • Raguimova O.N.
      • Cho E.E.
      • et al.
      The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.
      ) in order to prevent altering fluorescent protein emission intensity. The data for each set of binding partners were analyzed for differences in KD between buffer conditions using a one-way ANOVA with Tukey’s post hoc test (significance = p < 0.05).
      For experiments measuring PLB-binding preference for SERCA enzymatic states E2-ATP and E1-ATP, which are in equilibrium under low Ca2+, high ATP conditions (e.g., cardiac diastole), a buffer containing 100 mM KCl, 5 mM MgCl2, 2 mM EGTA, 10 mM imidazole, and 3 mM ATP was prepared. The pH was then adjusted to the following concentrations: pH = 6.0, 6.5, 7.0, 7.5, and 8.0. For these experiments, a TagRFP-PLB acceptor construct (pKa = 3.8) was used in place of the YFP-PLB acceptor because YFP fluorescence is sensitive to pH changes in this range (pKa = 6.9) (
      • Lambert T.J.
      FPbase: a community-editable fluorescent protein database.
      ).
      For experiments with low and elevated intracellular [Ca2+], solutions were prepared containing potassium aspartate 120 mM, KCl 15 mM, KH2PO4 5 mM, MgCl2 0.75 mM, dextran 2%, ATP 5 mM, Hepes 20 mM, and EGTA 2 mM, pH 7.2. The elevated [Ca2+] buffer was prepared with CaCl2 1.7 mM for a free [Ca2+]i = 2 μM (
      • Schoenmakers T.J.
      • Visser G.J.
      • Flik G.
      • Theuvenet A.P.
      Chelator: an improved method for computing metal ion concentrations in physiological solutions.
      ). The data for each set of binding partners were analyzed for differences in KD between high and low Ca2+ conditions using a Student’s t test (significance = p < 0.05).

      Confocal fluorescence microscopy to measure intermolecular FRET and HEK-293 cytoplasmic Ca2+

      HEK-293 cells exhibiting spontaneous Ca2+ oscillations were generated by transient transfection with GFP-tagged RyR2 and either Cer or unlabeled SERCA2a and cotransfected with SERCA, PLB, PLB-AFA, PLB-S16E, or DWORF FRET pairs tagged with Cer and YFP fluorophores. Transfected cells were cultured for 24 h and seeded into poly-D-lysine–coated glass bottom chamber slides in DMEM plus 10% fetal bovine serum. Twenty-four hours after seeding, cell culture medium was changed with PBS (+Ca2+/+Mg2+), and experiments were conducted with a Leica SP5 laser scanning confocal microscope equipped with a 63× water objective. To observe transient changes in cytoplasmic calcium, cells were incubated with 10 μM X-Rhod-1/AM (X-Rhod) for 20 min in PBS (+Ca2+/+Mg2+). X-Rhod was excited with the 543 nm line of a He-Ne laser, and emitted fluorescence was measured at wavelength 580 nm. FRET pair fluorophores Cer and YFP were excited with the 430 and 514 nm lines of an argon laser, respectively, and emitted fluorescence was measured at wavelengths 485 ± 15 and 537 ± 15 nm, respectively. Images were acquired in line scan with averaging of four every 134 ms for ∼2 min.
      A select set of concurrent experiments were conducted with a Zeiss LSM 880 Airyscan confocal microscope using a 40× oil immersion objective. X-Rhod was excited with the 594 nm line of a He-Ne laser, and emitted fluorescence was measured at wavelength 580 nm. FRET pair fluorophores Cer and YFP were excited with the 458 nm line of an argon laser, and emitted fluorescence was measured at wavelengths 485 ± 15 and 537 ± 15 nm, respectively. Images were acquired in line scan every 24 ms for ∼2 min. This faster acquisition rate was used to resolve time-dependent changes in FRET signals during the fast upstroke of cellular Ca2+ elevations.
      FRET ratio was determined by dividing the acceptor fluorescence by the donor fluorescence and plotted as a function of time with X-Rhod to indicate concurrent changes in [Ca2+]. FRET ratio data was smoothed using a Savitzky–Golay binomial filter with a 4.08 s averaging window. Changes in FRET ratio and X-Rhod fluorescence associated with Ca2+ uptake were fit to the single-exponential decay function, y=A1x/τ+y0 in origin, to estimate the time constant or τ of the change, where A1 is the amplitude of change and y0 is the initial detected fluorescence. Changes in FRET ratio and X-Rhod fluorescence associated with Ca2+ release were fit using the single-exponential decay function, y=A1(xx1)/τ+y1, where x1 is the time in seconds of Ca2+ release, and y1 is the baseline fluorescence prior to x1, defined by the linear function y=A1+y1. Differences in the characteristic time constants (τ) for each process were analyzed using a one-way ANOVA with Dunn’s post hoc test (significance = p < 0.05).

      Kinetic modeling

      We implemented systems of ordinary differential equations according to the schematic provided in Figure 4A (
      • Sun B.
      • Stewart B.D.
      • Kucharski A.N.
      • Kekenes-Huskey P.M.
      Thermodynamics of cation binding to the sarcoendoplasmic reticulum calcium ATPase pump and impacts on enzyme function.
      ). This kinetic diagram describes the populations of the SERCA states under diastolic and systolic conditions. Model parameters were informed or constrained by experimental observations where appropriate, such as the PLB dissociation rates reported in this study. Mean rates were determined by averaging over several transients from multiple cells. Kinetic parameters for PLB–SERCA binding dynamics were fit to 15 independent FRET transients, while kinetic parameters for DWORF–SERCA interactions were fit to nine independent FRET measurements. All other parameters were assigned initial values that were subject to fitting against time-varying FRET data. The relative affinity of PLB for SERCA (here described by the rate fraction of kon/koff) was constrained to be twofold higher for Ca-free versus Ca-bound ensembles, as determined experimentally from FRET-binding curves. Likewise, the relative affinity of DWORF for SERCA (kon/koff) was constrained to increase by 25% between Ca-free and Ca-bound ensembles, consistent with FRET measurements. The ordinary differential equation system was numerically solved using the scipy (v1.5.0) SOLV_IVP function, using the experimentally measured intracellular Ca2+ transient as an input to the model. The resulting numerically estimated PLB–SERCA population was compared against the experimentally reported FRET data by assuming that the PLB–SERCA population was proportional to the FRET efficiency. Mean-squared error between the predicted PLB–SERCA population and the FRET data was computed to assess the fit. Fitting was optimized using a genetic algorithm we developed previously (
      • Stewart B.D.
      • Scott C.E.
      • McCoy T.P.
      • Yin G.
      • Despa F.
      • Despa S.
      • et al.
      Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes.
      ) that reduces the mean-squared error by randomizing model parameters and selecting those that reduce error. Forward simulations of the SERCA/PLB/DWORF equilibria for the cardiac pacing experiments used simulated Ca2+ transients with amplitudes and frequencies in place of those measured in HEK cells for fitting purposes. A parameter sensitivity analysis is provided in Fig. S13.

      Data availability

      All code written in support of this publication are publicly available at https://github.com/huskeypm/pkh-lab-analyses.

      Supporting information

      This article contains supporting information (
      • Hughes G.
      • Starling A.P.
      • Sharma R.P.
      • East J.M.
      • Lee A.G.
      An investigation of the mechanism of inhibition of the Ca(2+)-ATPase by phospholamban.
      ,
      • Raguimova O.N.
      • Smolin N.
      • Bovo E.
      • Bhayani S.
      • Autry J.M.
      • Zima A.V.
      • et al.
      Redistribution of SERCA calcium pump conformers during intracellular calcium signaling.
      ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Howard S. Young, Gianluigi Veglia, J. Michael Autry, Daniel Koch, Catherine Makarewich, and L. Michel Espinoza-Fonseca for helpful discussions. We thank Elisa Bovo and Aleksey Zima for technical assistance. We thank L. Michel Espinoza-Fonseca for providing the structural models of SERCA bound to PLB/DWORF in Figures 1 and 3. Anti-DWORF antibody was a gift of Catherine Makarewich.

      Author contributions

      S. R. C. and S. L. R. conceptualization; S. R. C., X. F., J. R. B., P. M. K.-H., and S. L. R. methodology; X. F. and P. M. K.-H., software; X. F. validation; S. R. C., X. F., E. E. C., and M. P. P. formal analysis; S. R. C., X. F., E. E. C., M. P. P., and J. S. investigation; J. R. B., P. M. K.-H., and S. L. R. resources; S. R. C., X. F., E. E. C., and M. P. P. data curation; S. R. C. and S. L. R. writing–original draft; S. R. C. and S. L. R. writing–review & editing; S. R. C., J. S., and S. L. R. visualization; J. S., P. M. K.-H., and S. L. R. supervision; P. M. K.-H. and S. L. R. project administration; J. B., P. M. K.-H., and S. L. R. funding acquisition.

      Funding and additional information

      This investigation was supported by the National Institutes of Health (NIH): Maximizing Investigators’ Research Awards (MIRA) R35GM124977 from the National Institute of General Medical Sciences (NIGMS) to P. M. K.-H.; MIRA R35GM138183 from NIGMS and K22HL133150 from the National Heart, Lung, and Blood Institute (NHLBI) to J. R. B.; R01HL092321 and R01HL143816 from the NHLBI to S. L. R. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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