Phospholamban Protein Research Articles

Effects of adiponectin on calcium-handling proteins in heart failure with preserved ejection fraction.

Despite the increasing prevalence of heart failure with preserved ejection fraction (HFpEF) in humans, there remains no therapeutic options for HFpEF. Adiponectin, an adipocyte-derived cytokine, exerts cardioprotective actions, and its deficiency is implicated in the development of hypertension and HF with reduced ejection fraction. Similarly, adiponectin deficiency in HFpEF exacerbates left ventricular hypertrophy, diastolic dysfunction, and HF. However, the therapeutic effects of adiponectin in HFpEF remain unknown. We sought to test the hypothesis that chronic adiponectin overexpression protects against the progression of HF in a murine model of HFpEF. Adiponectin transgenic and wild-type mice underwent uninephrectomy, a continuous saline or d-aldosterone infusion and given 1.0% sodium chloride drinking water for 4 weeks. Aldosterone-infused wild-type mice developed HFpEF with hypertension, left ventricular hypertrophy, and diastolic dysfunction. Aldosterone infusion increased myocardial oxidative stress and decreased sarcoplasmic reticulum Ca(2+)-ATPase protein expression in HFpEF. Although total phospholamban protein expression was unchanged, there was a decreased expression of protein kinase A-dependent phospholamban phosphorylation at Ser16 and CaMKII (Ca(2+)/calmodulin-dependent protein kinase II)-dependent phospholamban phosphorylation at Thr17. Adiponectin overexpression in aldosterone-infused mice ameliorated left ventricular hypertrophy, diastolic dysfunction, lung congestion, and myocardial oxidative stress without affecting blood pressure and left ventricular EF. This improvement in diastolic dysfunction parameters in aldosterone-infused adiponectin transgenic mice was accompanied by the preserved protein expression of protein kinase A-dependent phosphorylation of phospholamban at Ser16. Adiponectin replacement prevented the progression of aldosterone-induced HFpEF, independent of blood pressure, by improving diastolic dysfunction and by modulating cardiac hypertrophy. These findings suggest that adiponectin may have therapeutic effects in patients with HFpEF.

Differential regulation of the fiber type-specific gene expression of the sarcoplasmic reticulum calcium-ATPase isoforms induced by exercise training.

The regulatory role of adenosine monophosphate-activated protein kinase (AMPK)-α2 on sarcoplasmic reticulum calcium-ATPase (SERCA) 1a and SERCA2a in different skeletal muscle fiber types has yet to be elucidated. Sedentary (Sed) or exercise-trained (Ex) wild-type (WT) and AMPKα2-kinase dead (KD) transgenic mice, which overexpress a mutated and inactivated AMPKα2 subunit, were utilized to characterize how genotype or exercise training influenced the regulation of SERCA isoforms in gastrocnemius. As expected, both Sed and Ex KD mice had >40% lower AMPK phosphorylation and 30% lower SERCA1a protein than WT mice (P < 0.05). In contrast, SERCA2a protein was not different among KD and WT mice. Exercise increased SERCA1a and SERCA2a protein content among WT and KD mice, compared with their Sed counterparts. Maximal SERCA activity was lower in KD mice, compared with WT. Total phospholamban protein was higher in KD mice than in WT and lower in Ex compared with Sed mice. Exercise training increased phospholamban Ser(16) phosphorylation in WT mice. Laser capture microdissection and quantitative PCR indicated that SERCA1a mRNA expression among type I fibers was not altered by genotype or exercise, but SERCA2a mRNA was increased 30-fold in WT+Ex, compared with WT+Sed. In contrast, the exercise-stimulated increase for SERCA2a mRNA was blunted in KD mice. Exercise upregulated SERCA1a and SERCA2a mRNA among type II fibers, but was not altered by genotype. Collectively, these data suggest that exercise differentially influences SERCA isoform expression in type I and type II fibers. Additionally, AMPKα2 influences the regulation of SERCA2a mRNA in type I skeletal muscle fibers following exercise training.

Phosphorylation Alters the Transmembrane Binding Interface Between Phospholamban and SERCA

Cardiac muscle contraction is regulated through the release and reuptake of Ca2+ from the sarcoplasmic reticulum (SR), with the SR Ca2+-ATPase (SERCA) inducing relaxation by restoring Ca2+ to the SR lumen from the cytosol. SERCA is regulated by the single-pass membrane protein phospholamban (PLB), which acts as inhibitor unless phosphorylated at Ser16. The mechanism of SERCA-PLB regulation remains poorly understood, though our recent work demonstrated that PLB remains bound to SERCA following Ser16 phosphorylation, indicating that a structural rearrangement mediates inhibition relief. To further elucidate this mechanism, we have used site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study the interaction between the transmembrane (TM) domains of SERCA and PLB, where regulation is mediated. Spin labels were attached along PLB's TM helix for accessibility EPR measurements as well as double electron-electron resonance (DEER) distance measurements to spin-labeled SERCA. Our results suggest that Ser16 phosphorylation induces a change in the topology of PLB's TM helix that results in disruption of SERCA inhibition without dissociating the two proteins. Acknowledgments: spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, and funded by NIH grants to DDT (R01 GM27906, P30 AR507220).

Multifrequency EPR Detects Orientation of Calcium Transport Proteins in Lipid Bicelles

We have used electron paramagnetic resonance (EPR) at X and Q band to probe the structural dynamics of the integral membrane protein phospholamban (PLB), as a function of phosphorylation and addition of its regulatory target, the sarcoplasmic reticulum calcium ATPase (SERCA). We found previously that PLB remains bound to SERCA after phosphorylation, suggesting that a structural transition within the SERCA-PLB complex is responsible for relief of inhibition. Our current goal is to elucidate this mechanism through orientation and accessibility EPR, supporting rational design of therapies to improve calcium transport in muscle cells. We used the monomeric mutant AFA-PLB with the rigid spin label TOAC incorporated within the transmembrane domain. This protein was reconstituted with purified SERCA into lipid bicelles and magnetically aligned at both X and Q band field strength, allowing for a robust global fitting approach. We measured changes in PLB tilt upon phosphorylation, in the absence and presence of SERCA. Experiments were performed in the Biophysical Spectroscopy Center at the University of Minnesota. This work was funded by grants from NIH (R01 GM27906, P30 AR0507220, and T32 AR007612).

Distinct Functional Effects of Phospholamban Phosphorylation States

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). It is known that PLB inhibits SERCA and that PLB phosphorylation at S16 and/or T17 relieves this inhibition in cardiac SR. However, the functional effects of the two PLB phosphorylation sites have not been compared quantitatively. Therefore, we co-reconstituted SERCA1a with synthetic PLB, pS16-PLB, pT17-PLB, or pS16/pT17-PLB in phospholipid vesicles and measured Ca2+-dependent SERCA activity. Inhibition was maximally relieved by phosphorylation at S16, followed by doubly phosphorylated PLB (pS16/pT17-PLB) and pT17-PLB. Thus, T17 phosphorylation shows the least capacity for SERCA inhibition relief, and actually decreases the effect of S16 phosphorylation on the same PLB. Quantitative Western blots were used to demonstrate that all four PLB phosphorylation states are present in cardiac SR. Cardiac SR was treated with PLB antibodies and protein kinase A (PKA) to quantify the effects of PLB phosphorylation states on SERCA inhibition in a biological milieu. These results show that each PLB phosphorylation state uniquely alters Ca2+ homeostasis. These effects are relevant to cardiovascular health, disease and treatment.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Microwave Assisted Peptide Synthesis as a New Gold Standard in Solid Phase Peptide Synthesis: Phospholamban as an Example

In this study, we report the microwave assisted synthesis of Phospholamban protein (WT-PLB) as a new gold standard in solid phase peptide synthesis. Microwave energy offers benefits for both the coupling and deprotection reactions during peptide synthesis. The use of microwave energy for both the coupling and deprotection steps makes the microwave peptide synthesizers the most versatile and powerful systems available. It produces high yield and fast synthesis when compared to conventional peptide synthesizers.

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

ObjectivesSarcoplasmic 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.MethodsWe 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.ResultsMyocardial 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).Conclusionspostresuscitation 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.

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.

Increased Sarcolipin Expression and Adrenergic Drive in Humans With Preserved Left Ventricular Ejection Fraction and Chronic Isolated Mitral Regurgitation

There is currently no therapy proven to attenuate left ventricular (LV) dilatation and dysfunction in volume overload induced by isolated mitral regurgitation (MR). To better understand molecular signatures underlying isolated MR, we performed LV gene expression analyses and overlaid regulated genes into ingenuity pathway analysis in patients with isolated MR. Gene arrays from LV tissue of 35 patients, taken at the time of surgical repair for isolated MR, were compared with 13 normal controls. Cine-MRI was performed in 31 patients before surgery to measure LV function and volume from serial short-axis summation. LV end-diastolic volume was 2-fold (P=0.005) higher in MR patients than in normal controls, and LV ejection fraction was 64±7% (50%-79%) in MR patients. Ingenuity pathway analysis identified significant activation of pathways involved in β-adrenergic, cAMP, and G-protein-coupled signaling, whereas there was downregulation of pathways associated with complement activation and acute phase response. SERCA2a and phospholamban protein were unchanged in MR versus control left ventricles. However, mRNA and protein levels of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) regulatory protein sarcolipin, which is predominantly expressed in normal atria, were increased 12- and 6-fold, respectively. Immunofluorescence analysis confirmed the absence of sarcolipin in normal left ventricles and its marked upregulation in MR left ventricles. These results demonstrate alterations in multiple pathways associated with β-adrenergic signaling and sarcolipin in the left ventricles of patients with isolated MR and LV ejection fraction>50%, suggesting a beneficial role for β-adrenergic blockade in isolated MR.

Abstract 354: Nitrate Improves Postresuscitation Myocardial Dysfunction by Preserving Myocardial Nitric Oxide and Phospholamban Protein

Objectives: Sarcoplasmic reticulum (SR) Ca 2+ handling proteins have been shown to play an important role in myocardial dysfunction after acute ischemic/reperfusion injury. We assessed the hypothesis that nitrite would improve postresuscitation myocardial dysfunction by increasing NO generation, and the mechanism is related to the modulation of SR Ca 2+ handling proteins. Methods: We performed a randomized prospective animal study using male Sprague-Dawley rats. Cardiac arrest was induced by intravenous bolus of potassium chloride (40 μg/g). Nitrite (1.2nmol/g) or placebo was administered when chest compression was started. No cardiac arrest was induced in sham group. Hemodynamic parameters were monitored invasively for one hour after ROSC. Echocardiogram was performed to evaluate cardiac function. Myocardial sample was harvested 5 minutes and 1 hour after ROSC respectively. The expression of sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a), phospholamban (PLB) and rynodine receptors (RyRs) were analyzed by Western blot analysis. Myocardial NO five minutes after ROSC were measured using Nitric Oxide Assay Kit. Results: Myocardial function measured as the maximal rate of LV pressure increase (dP/dtmax), the maximal rate of LV pressure decline (-dP/dtmax), ejection fraction, fractional shortening was significantly impaired in nitrite and placebo groups after resuscitation, and cardiac function in nitrite group was significantly greater than placebo group. Nitrite administration increased the level of nitric oxide in myocardium 5 min after resuscitation when compared to the other two groups. The levels of phosphorylated phospholamban were decreased after resuscitation, and nitrite preserved the phosphorylation of phospholamban in myocardium after resuscitation when compared to placebo group. No significant differences were found in the expressions of myocardial sarcoplasmic reticulum Ca 2+ ATPase and rynodine receptors after resuscitation. Conclusions: Nitrite administered during resuscitation improves postresusctation myocardial dysfunction as an exogenous donor of nitric oxide, and the modulation of phosphorylated phospholamban, an important SR Ca 2+ uptake protein, is involved in the mechanism.

Cellular and Molecular Mechanisms of Atrial Arrhythmogenesis in Patients With Paroxysmal Atrial Fibrillation

Electrical, structural, and Ca2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+ -release events in paroxysmal AF (pAF) are unknown. Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+ /Ca2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+ -transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+ -transient amplitude, integrated Na+/Ca2+ -exchange current) were larger in pAF. Ca2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+ -release events, causing delayed after-depolarizations/triggered activity in pAF. Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Preserved Cardiac Function despite Marked Impairment of cAMP Generation

ObjectivesSo many clinical trials of positive inotropes have failed, that it is now axiomatic that agents that increase cAMP are deleterious to the failing heart. An alternative strategy is to alter myocardial Ca2+ handling or myofilament response to Ca2+ using agents that do not affect cAMP. Although left ventricular (LV) function is tightly linked to adenylyl cyclase (AC) activity, the beneficial effects of AC may be independent of cAMP and instead stem from effects on Ca2+ handling. Here we ask whether an AC mutant molecule that reduces LV cAMP production would have favorable effects on LV function through its effects on Ca2+ handling alone.Methods and ResultsWe generated transgenic mice with cardiac-directed expression of an AC6 mutant (AC6mut). Cardiac myocytes showed impaired cAMP production in response to isoproterenol (74% reduction; p<0.001), but LV size and function were normal. Isolated hearts showed preserved LV function in response to isoproterenol stimulation. AC6mut expression was associated with increased sarcoplasmic reticulum Ca2+ uptake and the EC50 for SERCA2a activation was reduced. Cardiac myocytes isolated from AC6mut mice showed increased amplitude of Ca2+ transients in response to isoproterenol (p = 0.0001). AC6mut expression also was associated with increased expression of LV S100A1 (p = 0.03) and reduced expression of phospholamban protein (p = 0.01).ConclusionLV AC mutant expression is associated with normal cardiac function despite impaired cAMP generation. The mechanism appears to be through effects on Ca2+ handling — effects that occur despite diminished cAMP.

GW24-e1015 Nitrate improves postresuscitation myocardial dysfunction and the involvement of nitric oxide and phospholamban protein

ObjectivesThe abnormality of Sarcoplasmic reticulum (SR) Ca2+ handling proteins was shown to be essential in myocardial dysfunction after acute reperfusion injury, which might be attenuated by NO therapy. Cardiac arrest...

Solution NMR Studies on the Orientation of Membrane-Bound Peptides and Proteins by Paramagnetic Probes

Many peptides and proteins are attached to or immersed in a biological membrane. In order to understand their function not only the structure but also their topology in the membrane is important. Solution NMR spectroscopy is one of the most often used approaches to determine the orientation and localization of membrane-bound peptides and proteins. Here we give an application-oriented overview on the use of paramagnetic probes for the investigation of membrane-bound peptides and proteins. The examples discussed range from the large pool of antimicrobial peptides, bacterial toxins, cell penetrating peptides to domains of larger proteins or the calcium regulating protein phospholamban. Topological information is obtained in all these examples by the use of either attached or freely mobile paramagnetic tags. For some examples information obtained from the paramagnetic probes was included in the structure determination.

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 ((27)RSYQY), 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 Arg(27) and Tyr(31) are essential for SLN function. We also tested the effect of a truncated variant of SLN (Arg(27)stop) and extended chimeras of PLN with the five luminal residues of SLN added to its C terminus. The Arg(27)stop 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.

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 Ca(2+)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 Vmax of SERCA-mediated Ca(2+) uptake but not the pump affinity for Ca(2+); 2) SLN can bind to SERCA in the presence of high Ca(2+), but PLB can only interact to the ATP-bound Ca(2+)-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 Ca(2+) causes uncoupling of the SERCA pump and increased heat production.

Abnormal amounts of intracellular calcium regulatory proteins in SHRSP.Z-Leprfa/IzmDmcr rats with metabolic syndrome and cardiac dysfunction

Metabolic syndrome is known to increase the risk of abnormal cardiac structure and function, which are considered to contribute to increased incidence of cardiovascular disease and mortality. We previously demonstrated that ventricular hypertrophy and diastolic dysfunction occur in SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP fatty) rats with metabolic syndrome. The aim of this study was to investigate the possible mechanisms underlying abnormal heart function in SHRSP fatty rats. The amount of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a, phospholamban (PLB) protein, and Ser(16)-phosphorylated PLB was decreased in cardiomyocytes from SHRSP fatty rats compared with those from control Wistar-Kyoto rats at 18 weeks of age, and the PLB-to-SERCA2a ratio was increased. Left ventricular developed pressure was unchanged, and coronary flow rate and maximum rate of left ventricular pressure decline (-dP/dt) was decreased in SHRSP fatty rats. Treatment with telmisartan reversed the abnormalities of PLB amount, coronary flow rate, and -dP/dt in SHRSP fatty rats. These results indicate that abnormal amounts of intracellular Ca(2+) regulatory proteins in cardiomyocytes, leading to reduced intracellular Ca(2+) reuptake into the sarcoplasmic reticulum, may play a role in the diastolic dysfunction in SHRSP fatty rats and that these effects are partially related to decreased coronary circulation. Telmisartan may be beneficial in protecting against disturbances in cardiac function associated with metabolic syndrome.

Phosphorylated Phospholamban Stabilizes a Distinct Compact Conformation of Sarco(endo)Plasmic Reticulum Ca-ATPase

Sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is inhibited by the 52 amino acid protein phospholamban (PLB). Inhibition of SERCA is relieved when PLB is phosphorylated by protein kinase A (PKA). To better understand the mechanism of this regulatory mechanism on SERCA structure, we designed a “2-color” SERCA labeled with donor and acceptor fluorescent proteins in the A- and N-domain. Intramolecular fluorescence resonance energy transfer (FRET) between these two domains was quantified as an index of SERCA conformation. Fluorescence lifetime distribution analysis (FLDA) showed that SERCA sampled three discrete conformations in the absence of PLB or in the presence of non-phosphorylatable PLB (S16A). Co-transfection of SERCA with phosphomimetic PLB (S16E) revealed an additional state characterized by a highly compact conformation. This unique state was only observed under conditions of high intracellular calcium. The data support the hypothesis that PLB remains bound to SERCA after phosphorylation by PKA, inducing the pump to assume a very compact E1 conformation.

Orientation of Phospholamban in Lipid Bicelles Detected by Electron Paramagnetic Resonance

We have used electron paramagnetic resonance (EPR) to probe the structural dynamics of the integral membrane protein phospholamban (PLB), as a function of phosphorylation and addition of its regulatory target, the sarcoplasmic reticulum calcium ATPase (SERCA). We found previously that PLB remains bound to SERCA after phosphorylation, suggesting that a structural transition within the SERCA-PLB complex is responsible for relief of inhibition. Our current goal is to elucidate this mechanism through orientation and accessibility EPR, in order to support rational design of therapies to improve calcium transport in muscle cells. We used the monomeric mutant AFA-PLB, with the rigid electron spin label TOAC incorporated within the transmembrane domain, and reconstituted the protein in lipid bicelles and vesicles. EPR showed that the accessibilities of PLB spin labels to paramagnetic relaxation agents changed upon phosphorylation, indicating vertical (parallel to the membrane normal) movement of PLB. EPR on aligned bicelles showed that PLB changes its tilt relative to the membrane upon phosphorylation. Results will also be reported on structural changes in the presence of SERCA. This work was funded by grants from NIH (R01 GM27906 and T32 AR007612).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Characterization of Phospholamban Mutants Implicated in Dilated Cardiomyopathy

Contractility of heart muscle relies heavily on calcium ion messaging. Heart muscle relaxation takes place when this secondary messenger is transported against the concentration gradient from sarcoplasmic reticulum to myoplasm, performed by Ca2+-ATPase (SERCA). The function of this calcium pump is inhibited by membrane protein phospholamban (PLN), a process that is reversed upon phosphorylation of PLN's cytoplasmic domain at S16 or T17. In the recent years several dysfunctional PLN analogs have been identified in patients with impaired heart contractility. Interestingly, the mutations were particularly prominent at R9 hotspot, where substitutions of an arginine residue to Cys, Leu or His have been identified.We seek to deduce the molecular mechanism behind the interaction of these proteins, to gain the atomic insights into the regulation of contractility. For investigating the protein-protein and protein-membrane interactions, we employ an array of biophysical techniques, with an emphasis on NMR data. Conventional solution NMR techniques are used to probe the structure as well as fast/slow dynamics. Magic angle spinning solid-state NMR is applied for proteins with long correlation times to obtain the insights into the structure in the native lipid bilayer environment and the residues involved in protein-protein interaction. Oriented solid-state NMR gives information on the topology of the proteins with respect to the frame of the biomembrane.The ultimate goal is the development of ways to manipulate calcium signaling in vivo. By investigating the interactions between the mutant proteins involved in pathologies, such as dilated cardiomyopathy, we intend to build a picture of the pathways at the molecular level. This will permit developing methods for regulating the proteins with abnormal functioning, allowing to control their function under pathological conditions.