Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca²⁺ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca²⁺]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca²⁺ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca²⁺ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.     

Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function

The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) regulates the active transport of Ca²⁺ into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca²⁺ uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties.     

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

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.     

The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca²⁺-ATPase), pumps contractile-dependent Ca²⁺ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca²⁺ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca²⁺-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca²⁺ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca²⁺ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca²⁺ affinity and depresses cardiac contractility.     

Early administration of nifedipine protects against angiotensin II‐induced cardiomyocyte hypertrophy through regulating CaMKII–SERCA2a pathway and apoptosis in rat cardiomyocytes

The calcium channel blocker (CCB), nifedipine, is a more effective treatment for early‐ than late‐stage cardiac hypertrophy. We investigated the effects of early‐ and late‐stage nifedipine administration on calcium homeostasis, CaMKII (Ca²⁺/calmodulin‐dependent protein kinase II) activity and apoptosis of cardiomyocytes under hypertrophic stimulation with angiotensin II (AngII). Primary rat cardiomyocytes were divided into five treatment groups: AK, AngII plus the CaMKII inhibitor, KN‐93; AN‐1 (early‐stage), AngII plus nifedipine × 48 h; AN‐2 (late‐stage), AngII × 48 h, then AngII plus nifedipine × 48 h; C, untreated; and A, AngII × 48 h. The t1/2β [time required for intracellular Ca²⁺ concentration ([Ca²⁺]i) to decline to one half of the peak value] decreased; however, CaMKII and SERCA2a (sarcoplasmic reticulum Ca²⁺‐ATPase 2a) activities increased in the AN‐1 group compared with the AK group. In the AN‐2 group compared with the AN‐1 group, CaMKII activity, t1/2α [time required for [Ca²⁺]i to increase from the bottom to one half of peak value], t1/2β, and apoptosis increased. These results indicate that the timing of CCB administration affects the calcium concentration and apoptosis of hypertrophic cardiomyocytes through the CaMKII–SERCA2a signalling pathway, thereby influencing the drug's protective activity against cardiomyocyte hypertrophy. Copyright © 2016 John Wiley & Sons, Ltd.     

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca²⁺ affinity of SERCA2a by competing for Ca²⁺ binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca²⁺-ATPase activity and E1∼P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca²⁺ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca²⁺ for binding to SERCA2a. This was confirmed with the Ca²⁺ pump mutant, D351A, which is catalytically inactive but retains strong Ca²⁺ binding. Increasingly higher Ca²⁺ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB antagonizes Ca²⁺ binding. Finally, the specific conformation of E2 (Ca²⁺-free state of SERCA2a) that binds PLB was investigated using the Ca²⁺-pump inhibitors thapsigargin and vanadate. Cross-linking assays conducted in the absence of Ca²⁺ showed that PLB bound preferentially to E2 with bound nucleotide, forming a remarkably stable complex that is highly resistant to both thapsigargin and vanadate. In the presence of ATP, N30C-PLB had an affinity for SERCA2a approaching that of vanadate (micromolar), whereas PLB3 and PLB4 had much higher affinities, severalfold greater than even thapsigargin (nanomolar or higher). We conclude that PLB decreases Ca²⁺ binding to SERCA2a by stabilizing a unique E2·ATP state that is unable to bind thapsigargin or vanadate.     

Synthetic Phosphopeptides Enable Quantitation of the Content and Function of the Four Phosphorylation States of Phospholamban in Cardiac Muscle

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum Ca-ATPase (SERCA). It has been shown that unphosphorylated PLB (U-PLB) inhibits SERCA and that phosphorylation of PLB at Ser-16 or Thr-17 relieves this inhibition in cardiac sarcoplasmic reticulum. However, the levels of the four phosphorylation states of PLB (U-PLB, P16-PLB, P17-PLB, and doubly phosphorylated 2P-PLB) have not been measured quantitatively in cardiac tissue, and their functional effects on SERCA have not been determined directly. We have solved both problems through the chemical synthesis of all four PLB species. We first used the synthetic PLB as standards for a quantitative immunoblot assay, to determine the concentrations of all four PLB phosphorylation states in pig cardiac tissue, with and without left ventricular hypertrophy (LVH) induced by aortic banding. In both LVH and sham hearts, all phosphorylation states were significantly populated, but LVH hearts showed a significant decrease in U-PLB, with a corresponding increase in the ratio of total phosphorylated PLB to U-PLB. To determine directly the functional effects of each PLB species, we co-reconstituted each of the synthetic peptides in phospholipid membranes with SERCA and measured calcium-dependent ATPase activity. SERCA inhibition was maximally relieved by P16-PLB (the most highly populated PLB state in cardiac tissue homogenates), followed by 2P-PLB, then P17-PLB. These results show that each PLB phosphorylation state uniquely alters Ca²⁺ homeostasis, with important implications for cardiac health, disease, and therapy.     

Sarcolipin Protein Interaction with Sarco(endo)plasmic Reticulum Ca2+ATPase (SERCA) Is Distinct from Phospholamban Protein,and Only Sarcolipin Can Promote Uncoupling of the SERCA Pump

Sarco(endo)plasmic reticulum Ca²⁺ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547–554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575–1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the V_max of SERCA-mediated Ca²⁺ uptake but not the pump affinity for Ca²⁺; 2) SLN can bind to SERCA in the presence of high Ca²⁺, but PLB can only interact to the ATP-bound Ca²⁺-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca²⁺ causes uncoupling of the SERCA pump and increased heat production.     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys²⁷ of PLB was cross-linked to the Ca²⁺ pump at the cytoplasmic extension of M4 (at or near Lys³²⁸) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca²⁺ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca²⁺ affinity conformation of the Ca²⁺-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca²⁺-ATPase activity and Ca²⁺ transport. The results suggest that the inhibitory effect of PLB on Ca²⁺-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca²⁺ to the Ca²⁺ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca²⁺ pump are largely unchanged in failing myocardium.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca²⁺) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg^–1 i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca²⁺-uptake activity in diabetic animals. The reduction in SR Ca²⁺-uptake was consistent with a significant decrease in the level of SR Ca²⁺-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca²⁺-uptake was also due to a reduction in the phosphorylation of PLB by the Ca²⁺-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca²⁺-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart. (Mol Cell Biochem 261: 245–249, 2004)     

CaM kinase II and phospholamban contribute to caffeine-induced relaxation of murine gastric fundus smooth muscle

Caffeine has been shown to increase the Ca²⁺ release frequency (Ca²⁺ sparks) from the sarcoplasmic reticulum (SR) through ryanodine-sensitive stores and relax gastric fundus smooth muscle. Increased Ca²⁺ store refilling increases the frequency of Ca²⁺ release events and store refilling is enhanced by CaM kinase II (CaMKII) phosphorylation of phospholamban (PLB). These findings suggest that transient, localized Ca²⁺ release events from the SR may activate CaMKII and contribute to relaxation by enhancing store refilling due to PLB Thr17 phosphorylation. To investigate this possibility, we examined the effects of caffeine on CaMKII, muscle tone, and PLB phosphorylation in murine gastric fundus smooth muscle. Caffeine (1 mM) hyperpolarized and relaxed murine gastric fundus smooth muscle and activated CaMKII. Ryanodine, tetracaine, or cyclopiazonic acid each prevented CaMKII activation and significantly inhibited caffeine-induced relaxation. The large-conductance Ca²⁺-activated K⁺ channel blocker iberiotoxin, but not apamin, partially inhibited caffeine-induced relaxation. Caffeine-induced CaMKII activation increased PLB Thr17, but not PLB Ser16 phosphorylation. 3-Isobutyl-1-methylxanthine increased PLB Ser16 phosphorylation, but not PLB Thr17 phosphorylation. The CaMKII inhibitor KN-93 inhibited caffeine-induced relaxation and PLB Thr17 phosphorylation. These results show that caffeine-induced CaMKII activation and PLB phosphorylation play a role in the relaxation of gastric fundus smooth muscles. Ca²⁺/CaM-dependent protein kinase II     

Exendin-4 inhibits structural remodeling and improves Ca2+ homeostasis in rats with heart failure via the GLP-1 receptor through the eNOS/cGMP/PKG pathway

The glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 is a long-acting analog of GLP-1, which stimulates insulin secretion and is clinically used in the treatment of type 2 diabetes. Previous studies have demonstrated that GLP-1 agonists and analogs serve as cardioprotective factors in various conditions. Disturbances in calcium cycling are characteristic of heart failure (HF); therefore, the aim of this study was to investigate the effect of exendin-4 (a GLP-1 mimetic) on the regulation of calcium handling and to identify the underlying mechanisms in an HF rat model after myocardial infarction (MI). Rats underwent surgical ligation of the left anterior descending coronary artery or sham surgery prior to infusion with vehicle, exendin-4, or exendin-4 and exendin9-39 for 4 weeks. Exendin-4 treatment decreased MI size, suppressed chamber dilation, myocyte hypertrophy, and fibrosis and improved in vivo heart function in the rats subjected to MI. Exendin-4 resulted in an increase in circulating GLP-1 and GLP-1R in ventricular tissues. Additionally, exendin-4 activated the eNOS/cGMP/PKG signaling pathway and inhibited the Ca²⁺/calmodulin-dependent kinase II (CaMKII) pathways. Myocytes isolated from exendin-4-treated hearts displayed higher Ca²⁺ transients, higher sarcoplasmic reticulum Ca²⁺ content, and higher l-type Ca²⁺ current densities than MI hearts. Exendin-4 treatment restored the protein expression of sarcoplasmic reticulum Ca²⁺ uptake ATPase (SERCA2a), phosphorylated phospholamban (PLB) and Cav1.2 and decreased the levels of phosphorylated ryanodine receptor (RyR). Moreover, the favorable effects of exendin-4 were significantly inhibited by exendin9-39 (a GLP-1 receptor antagonist). Exendin-4 treatment of an HF rat model after MI inhibited cardiac and cardiomyocytes progressive remodeling. In addition, Ca²⁺ handling and its molecular modulation were also improved by exendin-4 treatment. The beneficial effects of exendin-4 on cardiac remodeling may be mediated through activation of the eNOS/cGMP/PKG pathway.     

Inhibition of CaMKII Does Not Attenuate Cardiac Hypertrophy in Mice with Dysfunctional Ryanodine Receptor

In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca²⁺/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2^ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2^ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2^ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2^ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca²⁺ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2^ADA/ADA mice.     

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport

The sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) is responsible for intracellular Ca²⁺ homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca²⁺-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca²⁺ transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues ²ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.     

Solid-state H and N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with ²H and ¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). ²H and ¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.     

Sarcoplasmic Phospholamban Protein Is Involved in the Mechanisms of Postresuscitation Myocardial Dysfunction and the Cardioprotective Effect of Nitrite during Resuscitation

Objectives

Sarcoplasmic reticulum (SR) Ca²⁺-handling proteins play an important role in myocardial dysfunction after acute ischemia/reperfusion injury. We hypothesized that nitrite would improve postresuscitation myocardial dysfunction by increasing nitric oxide (NO) generation and that the mechanism of this protection is related to the modulation of SR Ca²⁺-handling proteins.

Methods

We conducted a randomized prospective animal study using male Sprague-Dawley rats. Cardiac arrest was induced by intravenous bolus of potassium chloride (40 µg/g). Nitrite (1.2 nmol/g) or placebo was administered when chest compression was started. No cardiac arrest was induced in the sham group. Hemodynamic parameters were monitored invasively for 90 minutes after the return of spontaneous circulation (ROSC). Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after ROSC.

Results

Myocardial function was significantly impaired in the nitrite and placebo groups after resuscitation, whereas cardiac function (i.e., ejection fraction and fractional shortening) was significantly greater in the nitrite group than in the placebo group. Nitrite administration increased the level of nitric oxide in the myocardium 5 min after resuscitation compared to the other two groups. The levels of phosphorylated phospholamban (PLB) were decreased after resuscitation, and nitrite increased the phosphorylation of phospholamban compared to the placebo. No significant differences were found in the expression of sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) and ryanodine receptors (RyRs).

Conclusions

postresuscitation myocardial dysfunction is associated with the impairment of PLB phosphorylation. Nitrite administered during resuscitation improves postresuscitation myocardial dysfunction by preserving phosphorylated PLB protein during resuscitation.     

Ischemia induces phospholamban dephosphorylation via activation of calcineurin, PKC-α, and protein phosphatase 1, thereby inducing calcium overload in reperfusion

Cardiac sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a) promotes Ca²⁺ uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a activity. We found a distinct dephosphorylation of PLB at Thr¹⁷ and Ser¹⁶ after 20-30 min of ischemia produced by coronary artery occlusion in rats. The aim of the study was to investigate how PLB is dephosphorylated in ischemia and to determine whether PLB dephosphorylation causes myocardial hypercontraction and calpain activation through Ca²⁺ overload in reperfusion. Protein inhibitor-1 (I-1) specifically inhibits protein phosphatase 1 (PP1), the predominant PLB phosphatase in heart. A Ca²⁺-dependent phosphatase calcineurin may also induce PLB dephosphorylation. Ischemia for 30 min induced PKC-α translocation, resulting in inactivation of I-1 through PKC-α-dependent phosphorylation at Ser⁶⁷. The PP1 activation following I-1 inactivation was thought to induce PLB dephosphorylation in ischemia. Ischemia for 30 min activated calcineurin, and pre-treatment with a calcineurin inhibitor, cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at Ser⁶⁷, and PLB dephosphorylation in ischemia. Reperfusion for 5 min following 30 min of ischemia induced spreading of contraction bands (CBs) and proteolysis of fodrin by calpain. Both CsA and an anti-PLB antibody that inhibits binding of PLB to SERCA2a reduced the CB area and fodrin breakdown after reperfusion. These results reveal a novel pathway via which ischemia induces calcineurin-dependent activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation at Ser⁶⁷, and PP1-dependent PLB dephosphorylation. The pathway contributes to the spreading of CBs and calpain activation through Ca²⁺ overload in early reperfusion.