A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.     

Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo-phosphorylated states

Human phospholamban (PLN), a 30 kDa homopentamer in the sarcoplasmic reticulum (SR) membrane, controls the magnitude of heart muscle contraction and relaxation by regulating the calcium pumping activity of the SR Ca(2+)-ATPase (SERCA). When PLN is not phosphorylated, it binds and inhibits SERCA. Phosphorylation of PLN at S16 or T17 releases such inhibitory effect. It remains a matter of debate whether phosphorylation perturbs the structure of PLN, which in turn affects its interaction with SERCA. Here we examine by NMR spectroscopy the structure and dynamics of PLN pentamer with a physiologically relevant, phosphorylation-mimicking mutation, S16E. Based on extensive NMR data, including NOEs, dipolar couplings, and solvent exchange of backbone amides, we conclude that the phosphorylation-mimicking mutation does not perturb the pentamer structure. However, (15)N R(1) and R(2) relaxation rates and (15)N((1)H) NOEs suggest subtle differences in the dynamics of the extramembrane portion of the protein.     

Sarcoplasmic reticulum Ca-ATPase-phospholamban interactions and dilated cardiomyopathy

Dilated cardiomyopathy is a disease of the heart muscle resulting from a diverse array of conditions that damages the heart and impairs myocardial function. Heart failure occurs when the heart is unable to pump blood at a rate which can accommodate the heart muscle's metabolic requirements. Several signaling pathways have been shown to be involved in the induction of cardiac disease and heart failure. Many of these pathways are linked to cardiac sarcoplasmic reticulum (SR) Ca cycling directly or indirectly. A large body of evidence points to the central role of abnormal Ca handling by SR proteins, Ca-ATPase pump (SERCA2a) and phospholamban (PLN), in pathophysiological heart conditions, compromising the contractile state of the cardiomyocytes. This review summarizes studies which highlight the key role of these two SR proteins in the regulation of cardiac function, the significance of SERCA2a-PLN interactions using transgenic approaches, and the recent discoveries of human PLN mutations leading to disease states. Finally, we will discuss extrapolation of experimental paradigms generated in animal models to the human condition.     

SERCA2a, phospholamban, sarcolipin, and ryanodine receptors gene expression in children with congenital heart defects

In animal models of conotruncal heart defects, an abnormal calcium sensitivity of the contractile apparatus and a depressed L-type calcium current have been described. Sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) is a membrane protein that catalyzes the ATP-dependent transport of Ca(2+) from the cytosol to the SR. The activity of SERCA is inhibited by phospholamban (PLN) and sarcolipin (SLN), and all these proteins participate in maintaining the normal intracellular calcium handling. Ryanodine receptors (RyRs) are the major SR calcium-release channels required for excitation-contraction coupling in skeletal and cardiac muscle. Our objective was to evaluate SERCA2a (i.e., the SERCA cardiac isoform), PLN, SLN, and RyR2 (i.e., the RyR isoform enriched in the heart) gene expression in myocardial tissue of patients affected by tetralogy of Fallot (TOF), a conotruncal heart defect. The gene expression of target genes was assessed semiquantitatively by RT-PCR using the calsequestrin (CASQ, a housekeeping gene) RNA as internal standard in the atrial myocardium of 23 pediatric patients undergoing surgical correction of TOF, in 10 age-matched patients with ventricular septal defect (VSD) and in 13 age-matched children with atrial septal defect (ASD). We observed a significantly lower expression of PLN and SLN in TOF patients, while there was no difference between the expression of SERCA2a and RyR2 in TOF and VSD. These data suggest a complex mechanism aimed to enhance the intracellular Ca(2+) reserve in children affected by tetralogy of Fallot.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.     

cAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban

Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.     

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.     

Maximal inhibition of SERCA2 Ca(2+) affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca(2+) affinity of the SR Ca(2+)-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca(2+) uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca(2+) with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (>/=2.6:1) exhibited increases in beta-myosin heavy chain expression, associated with cardiac hypertrophy. These findings demonstrate that overexpression of a non-phosphorylatable form of phospholamban in transgenic mouse hearts resulted in saturation of the functional phospholamban:SERCA2 ratio at 2.6:1 and suggest that approximately 40% of the SR Ca(2+) pumps are functionally regulated by phospholamban in vivo.     

Ca2+/calmodulin-dependent phosphorylation of the Ca2+-ATPase, uncoupled from phospholamban, stimulates Ca2+-pumping in native cardiac sarcoplasmic reticulum

Recent studies have demonstrated phosphorylation of the cardiac and slow-twitch muscle isoform (SERCA2a) of the sarcoplasmic reticulum (SR) Ca2+-ATPase (at Ser38) by a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase). Analysis of the functional consequence of Ca2+-ATPase phosphorylation in the native SR membranes, however, is complicated by the concurrent phosphorylation of the SR proteins phospholamban (PLN) which stimulates Ca2+ sequestration by the Ca2+-ATPase, and the ryanodine receptor-Ca2+ release channel (RYR-CRC) which likely augments Ca2+ release from the SR. In the present study, we achieved selective phosphorylation of the Ca2+-ATPase by endogenous CaM kinase in isolated rabbit cardiac SR vesicles utilizing a PLN monoclonal antibody (PLN AB) which inhibits PLN phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which inhibits phosphorylation of RYR-CRC. Analysis of the Ca2+ concentration-dependence of ATP-energized Ca2+ uptake by SR showed that endogenous CaM kinase mediated phosphorylation of the Ca2+-ATPase, in the absence of PLN and/or RYR-CRC phosphorylation, results in a significant increase (approximately 50-70%) in the Vmax of Ca2+ sequestration without any change in the k0.5 for Ca2+ activation of the Ca2+ transport rate. On the other hand, treatment of SR with PLN AB (which mimics the effect of PLN phosphorylation by uncoupling Ca2+-ATPase from PLN) resulted in approximately 2-fold decrease in k0.5 for Ca2+ without any change in Vmax of Ca2+ sequestration. These findings suggest that, besides PLN phosphorylation, direct phosphorylation of the Ca2+-ATPase by SR-associated CaM kinase serves to enhance the speed of cardiac muscle relaxation.     

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

Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.     

Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs)

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) by about -1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.     

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.     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Thyroid hormone downregulates the expression and function of sarcoplasmic reticulum-associated CaM kinase II in the rabbit heart

Phosphorylation of sarcoplasmic reticulum (SR) Ca2+-cycling proteins by a membrane-associated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a well-documented physiological mechanism for regulation of transmembrane Ca2+ fluxes and the cardiomyocyte contraction-relaxation cycle. The present study investigated the effects of L-thyroxine-induced hyperthyroidism on protein expression of SR CaM kinase II and its substrates, endogenous CaM kinase II-mediated SR protein phosphorylation, and SR Ca2+ pump function in the rabbit heart. Membrane vesicles enriched in junctional SR (JSR) or longitudinal SR (LSR) isolated from euthyroid and hyperthyroid rabbit hearts were utilized. Endogenous CaM kinase II-mediated phosphorylation of ryanodine receptor-Ca2+ release channel (RyR-CRC), Ca2+-ATPase, and phospholamban (PLN) was significantly lower (30-70%) in JSR and LSR vesicles from hyperthyroid than from euthyroid rabbit heart. Western immunoblotting analysis revealed significantly higher (approximately 40%) levels of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) in JSR, but not in LSR, from hyperthyroid than from euthyroid rabbit heart. Maximal velocity of Ca2+ uptake was significantly increased in JSR (130%) and LSR (50%) from hyperthyroid compared with euthyroid rabbit hearts. Apparent affinity of the Ca2+-ATPase for Ca2+ did not differ between the two groups. Protein levels of PLN and CaM kinase II were significantly lower (30-40%) in JSR, LSR, and ventricular tissue homogenates from hyperthyroid rabbit heart. These findings demonstrate selective downregulation of expression and function of CaM kinase II in hyperthyroid rabbit heart in the face of upregulated expression and function of SERCA2 predominantly in the JSR compartment.     

Controlling the inhibition of the sarcoplasmic Ca2+-ATPase by tuning phospholamban structural dynamics

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.     

Possible role of acylphosphatase,Bcl-2 and Fas/Fas-L system in the early changes of cardiac remodeling induced by volume overload

To identify early adaptive processes of cardiac remodeling (CR) in response to volume overload, we investigated the molecular events that may link intracellular Ca(2+) homeostasis alterations and cardiomyocyte apoptosis. In swine heart subjected to aorto-cava shunt for 6, 12, 24, 48 and 96 h sarcoplasmic reticulum (SR) Ca(2+) pump activity was reduced until 48 h (-30%), but a recovery of control values was found at 96 h. The decrease in SR Ca(2+)-ATPase (SERCA2a) expression at 48 h, was more marked (-60%) and not relieved by a subsequent recovery, while phospholamban (PLB) concentration and phosphorylation were unchanged at all the considered times. Conversely, acylphosphatase activity and expression significantly increased from 48 to 96 h (+40%). Bcl-2 expression increased significantly from 6 to 24 h, but at 48 h, returned to control values. At 48 h, microscopic observations showed that overloaded myocardium underwent substantial damage and apoptotic cell death in concomitance with an enhanced Fas/Fas-L expression. At 96 h, apoptosis appeared attenuated, while Fas/Fas-L expression was still higher than control values and cardiomyocyte hypertrophy became to develop. These data suggest that in our experimental model, acylphosphatase could be involved in the recovery of SERCA2a activity, while cardiomyocyte apoptosis might be triggered by a decline in Bcl-2 expression and a concomitant activation of Fas.     

Reduced sarcoplasmic reticulum Ca2+-ATPase activity and dephosphorylated phospholamban contribute to contractile dysfunction in human hibernating myocardium

Human hibernating myocardium (HHM) is characterized by reversible contractile dysfunction during chronic ischemia. A disturbed calcium-homeostasis is a decisive factor for reduced functional capacity in heart diseases. We therefore investigated calcium-handling proteins in HHM. In 12 patients suffering from multi-vessel coronary artery disease and contractile dysfunction with indication for bypass surgery, HHM was detected preoperatively by thallium scintigraphy, radionuclide ventriculography and dobutamine echocardiography. Transmural biopsies of these regions were taken and analyzed by immunohistochemistry and electron microscopy. Furthermore, SR-calcium ATPase (SERCA2a), phospholamban (PLN), the phosphorylated forms of PLN (PLN-Ser16, PLN-Thr17) as well as sodium-calcium exchanger (NCX) and ryanodine receptor (RyR2) were investigated by RT-PCR and Western-blotting. Additionally, SERCA2a activity was measured by an enzyme-coupled assay. In all patients complete functional recovery could be documented 3 months after revascularization by repeating all preoperative investigations. In HHM maximal SERCA2a activity was significantly reduced (HHM: 424.5± 33.9, control: 609.0± 48.5 nmol ATP mg protein⁻¹ min⁻¹, p≤ 0.05), whereas SERCA2a protein levels were unchanged. mRNA levels (HHM: 1.36± 0.08 vs. control: 0.78± 0.04, p≤ 0.05) and protein amount (HHM:1.67± 0.14 vs. control: 1.00± 0.04, p≤ 0.05) of PLN (A1) were increased resulting in an increased PLN:SERCA2a-ratio. PLN-Ser16 (HHM: 0.60± 0.08 vs. control: 1.00± 0.11, p≤ 0.05) and PLN-Thr17 (HHM: 0.63± 0.11 vs. control: 1.00± 0.06, p≤ 0.05) phosphorylation was significantly decreased. RyR2 and NCX showed no significant alteration. In HHM a decreased activity of SERCA2a due to an impaired phosphorylation of PLN contributes to contractile dysfunction. The increase in the relative ratio of PLN/SERCA2a leads to a decreased calcium affinity of SERCA2a.     

Co-Expression of SERCA Isoforms,Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca²⁺-ATPases (SERCAs) by reducing their apparent affinity for Ca²⁺. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca²⁺ affinity of SERCA (K _Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.     

Sarco(endo)plasmic Reticulum Calcium ATPase (SERCA) Inhibition by Sarcolipin Is Encoded in Its Luminal Tail

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is regulated in a tissue-dependent manner via interaction with the short integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). Although defects in SERCA activity are known to cause heart failure, the regulatory mechanisms imposed by PLN and SLN could have clinical implications for both heart and skeletal muscle diseases. PLN and SLN have significant sequence homology in their transmembrane regions, suggesting a similar mode of binding to SERCA. However, unlike PLN, SLN has a conserved C-terminal luminal tail composed of five amino acids (²⁷RSYQY), which may contribute to a distinct SERCA regulatory mechanism. We have functionally characterized alanine mutants of the C-terminal tail of SLN using co-reconstituted proteoliposomes of SERCA and SLN. We found that Arg²⁷ and Tyr³¹ are essential for SLN function. We also tested the effect of a truncated variant of SLN (Arg²⁷stop) and extended chimeras of PLN with the five luminal residues of SLN added to its C terminus. The Arg²⁷stop form of SLN resulted in loss of function, whereas the PLN chimeras resulted in superinhibition with characteristics of both PLN and SLN. Based on our results, we propose that the C-terminal tail of SLN is a distinct, essential domain in the regulation of SERCA and that the functional properties of the SLN tail can be transferred to PLN.     

Regulation of the calcium ion pump of sarcoplasmic reticulum: reversible inhibition by phospholamban and by the calmodulin binding domain of the plasma membrane calcium ion pump.

A 45 amino acid peptide (A45) corresponding to the phospholamban (PLN) binding domain of the sarcoplasmic reticulum (SR) ATPase was synthesized. Circular dichroism experiments have shown that the peptide had a predominantly random-coil conformation but adopted a higher proportion of secondary structure in the presence of a synthetic 32 amino acid peptide corresponding to the hydrophilic portion of PLN. A similar conformational change was induced by the synthetic calmodulin binding domain of the plasma membrane Ca2+ pump (peptide C28W), which acts as an endogenous inhibitor of the pump and is homologous to PLN. Cross-linking experiments have shown that peptide C28W interacted with peptide A45. The Ca(2+)-pumping activity of cardiac SR, which contains endogenous PLN, was stimulated about 30% by peptide A45. The stimulation was maximal at submicromolar Ca2+ levels and tended to disappear at higher Ca2+ concentrations. By contrast, the Ca(2+)-pumping activity of skeletal muscle SR, which lacks endogenous PLN, was unaffected. Peptide C28W strongly inhibited the pumping activity of skeletal muscle SR, and peptide A45 reversed the inhibition. The results suggest that peptide A45 competed with the ATPase for phospholamban or for peptide C28W, removing the inhibition of the pump. Thus, the exogenous inhibitor of the SR Ca(2+)-ATPase, PLN, and the internal inhibitor of the plasma membrane Ca(2+)-ATPase, peptide C28W, are functionally analogous.