Calcium Transport ATPase Of Sarcoplasmic Reticulum Research Articles

Simulation and FRET Analyses of SERCA, Phospohlamban, and Sarcolipin Complexes

We have used molecular modeling and experimental FRET constraints of fluorescent protein fusion constructs to study molecular interactions and small-molecule effects on SERCA and its transmembrane regulatory peptide phospholamban (PLB) and sarcolipin (SLN). The sarcoplasmic reticulum calcium transport ATPase (SERCA) is reversibly inhibited in heart and muscle by PLB and SLN, with phosphorylation-induced relief of inhibition. We previously utilized fluorescent fusion-protein biosensors and FRET assays to screen for small-molecule modulators of SERCA structural dynamics and activation (Schaaf et al., Biosensors 2018). In order to interpret FRET results (dynamic population distributions) in a three-dimensional, structural context, we performed molecular modeling and conformational sampling simulations. Starting points for molecular simulations were SERCA x-ray crystal structures in the following biochemical states: E1-2Ca (1SU4), E1P-2Ca-ADP (2ZBD), E2P-ADP (1WPG), E2 (1IWO), E1-2Ca+SLN-bound (3W5A), and E1-2Ca+PLB-bound (4KYT). Simulations generated an ensemble of conformations from which the inter-probe distance (R) and the orientation factor (κ2) were calculated. These two simulation-based parameters were then used to calculate donor lifetime change due to FRET from GFP to RFP tags. Simulation results were correlated to distances measured experimentally using subnanosecond-resolved fluorescence. Spectroscopy studies were performed in the Biophysical Technology Center, University of Minnesota Department of Biochemistry, Molecular Biology, and Biophysics. Simulations were run at the University of Minnesota Supercomputing Institute. This work is supported by NIH grants to DDT (GM27906, HL129814, AG026160).

Effect of tapering after a period of high-volume sprint interval training on running performance and muscular adaptations in moderately trained runners.

The effect of tapering following a period of high-volume sprint interval training (SIT) and a basic volume of aerobic training on performance and muscle adaptations in moderately trained runners was examined. Eleven (8 men, 3 women) runners [maximum oxygen uptake (V̇o2max): 56.8 ± 2.9 ml·min-1·kg-1; mean ± SD] conducted high-volume SIT (HV; 20 SIT sessions; 8-12 × 30 s all-out) for 40 days followed by 18 days of tapering (TAP; 4 SIT sessions; 4 × 30 s all-out). Before and after HV as well as midway through and at the end of TAP, the subjects completed a 10-km running test and a repeated running test at 90% of vV̇o2max to exhaustion (RRT). In addition, a biopsy from the vastus lateralis muscle was obtained at rest. Performance during RRT was better ( P < 0.01) at the end of TAP than before HV (6.8 ± 0.5 vs. 5.6 ± 0.5 min; means ± SE), and 10-km performance was 2.7% better ( P < 0.05) midway through (40.7 ± 0.7 min) and at the end of (40.7 ± 0.6 min) TAP than after HV (41.8 ± 0.9 min). The expression of muscle Na+-K+-ATPase (NKA)α1, NKAβ1, phospholemman (FXYD1), and sarcoplasmic reticulum calcium transport ATPase (SERCA1) increased ( P < 0.05) during HV and remained higher during TAP. In addition, oxygen uptake at 60% of vV̇o2max was lower ( P < 0.05) at the end of TAP than before and after HV. Thus short-duration exercise capacity and running economy were better than before the HV period together with higher expression of muscle proteins related to Na+/K+ transport and Ca2+ reuptake, while 10-km performance was not significantly improved by the combination of HV and tapering. NEW & NOTEWORTHY Short-duration performance became better after 18 days of tapering from ~6 wk of high-volume sprint interval training (SIT), whereas 10-km performance was not significantly affected by the combination of high-volume SIT and tapering. Higher expression of muscle NKAα1, NKAβ1, FXYD1, and SERCA1 may reflect faster Na+/K+ transport and Ca2+ reuptake that could explain the better short-duration performance. These results suggest that the type of competition should determine the duration of tapering to optimize performance.

Gastrodin Pretreatment Impact on Sarcoplasmic Reticulum Calcium Transport ATPase (SERCA) and Calcium Phosphate (PLB) Expression in Rats with Myocardial Ischemia Reperfusion.

BackgroundCalcium overload, inflammation, and apoptosis play important roles in myocardial ischemia-reperfusion injury (MIRI). Gastrodin pretreatment can alleviate MIRI. This study observed sarcoplasmic reticulum calcium transport ATPase (Ca2+-ATPase, SERCA) and calcium phosphate (PLB) protein expression in the ventricular remodeling process after myocardial infarction to explore the effect of gastrodin pretreatment on MIRI.Material/MethodsHealthy 7-week-old male SD rats were randomly divided into a sham group (A), a model group (B), and gastrodin pretreatment groups C, D, and E (100, 200, and 400 mg/kg, respectively) with 20 in each group. Anterior descending coronary artery ligation method was used to establish a rat MIRI model with 30-min ischemia and 120-min reperfusion. Cardiac electrophysiological activity was recorded. Serum IL-6 and IL10 levels were determined by ELISA. SERCA activity was tested by colorimetric phosphorus method. SERCA, PLB, and pSer-PLB protein expression were detected by Western blot.ResultsCompared with the sham group, IL-6 and IL-10 levels were elevated, SERCA2a expression was downregulated, and PLB protein was elevated in the model group (P<0.05). pSer16-PLB showed no significant difference among groups, and the ratio of pSer16-PLB/PLB obviously decreased (P<0.05). IL-6 level gradually declined and IL-10 increased in the gastrodin group following concentration elevation. SERCA 2a expression rose in the gastrodin group in a dose-dependent manner (P<0.05). Elevated PLB protein expression showed no significant difference, while pSer16-PLB protein increased (P<0.05), leading to elevated pSer16 PLB/PLB ratio (P<0.05).ConclusionsGastrodin pretreatment alleviates MIRI and inflammation injury by regulating SERCA and PLB expression to decrease calcium overload.

The arrhythmogenic effect of self-assembling nanopeptide hydrogel scaffolds on neonatal mouse cardiomyocytes

The chaotic spatial disarray due to extracellular matrix expansion disrupts cardiomyocytes interaction and causes arrhythmia. We hypothesized that disordered nanopeptide scaffolds can mimic the chaotic spatial disarray related to cardiac fibrosis and have arrhythmogenic effects on cardiomyocytes. Primary mouse cardiomyocytes were cultured in 2D traditional and 3D nanopeptide hydrogel scaffold systems. Cardiomyocytes in 3D scaffolds showed irregular spontaneous contractile activity as compared with 2D culture controls. Calcium fluorimetric imaging revealed that basal intracellular calcium level increased 1.42-fold in cardiomyocytes cultured in the 3D scaffold, in vitro. The mRNA levels of sarcoplasmic reticulum calcium transport ATPase, ryanodine 2 receptor and connexin 43 elevated 2.14-fold, 2.33-fold and 2.62-fold in 3D compared with 2D. Immunofluorescence imaging revealed lateralization of the distribution of connexin 43 in 3D group. These findings suggest that 3D hydrogel culture system provides a model for the development of cardiac dysrhythmia. These limitations should be considered during cardiac tissue engineering. From the Clinical EditorThis team of scientists has established a unique 3D hydrogel culture system as a model for the development of cardiac dysrhythmia.

Abstract 18881: Improper Elastic Fiber Assembly Drives Cardiac Pathology

Elastin is ubiquitously expressed in the cardiovascular system and essential for maintaining aortic wall integrity and function. Little is known however, about the role of elastin in the heart. To explore the influence of elastin on cardiac pathology we knocked down an essential protein for elastogenesis, fibulin-4, in human induced pluripotent stemcell (iPS)-derived cardiomyocytes. Strikingly, 90% reduction of fibulin-4 expression by shRNA, increased cardiomyocyte size by 35% and enhanced expression of atrial natriuretic peptide (ANP) and of TGFβ-regulated genes such as connective tissue growth factor and plasminogen activator inhibitor-1 in these cultured cardiomyocytes (all p&lt;0.05). Accordingly, mice with a 75% reduction in fibulin-4 expression (Fibulin-4R/R), developed cardiac hypertrophy, dilation and dysfunction (ejection fraction [76±2 to 21±3 %]) as well as aortic aneurysms. This was accompanied by elevated myocardial TGFβ signalling and ANP, while expression of sarcoplasmic reticulum calcium transport ATPase and maximal force generating capacity of single membrane-permeabilized cardiomyocytes was reduced by 60% and 25%, respectively (both p&lt;0.05). Subsequently, we explored the influence of fibulin-4 and elastogenesis on the development of cardiac pathology by subjecting mice with a 50% reduction in fibulin-4 expression (Fibulin-4+/R), without apparent cardiovascular abnormalities, to 4 weeks of transverse aortic constriction (TAC). Following TAC, mortality was markedly increased in Fibulin-4+/R animals compared to wildtype mice [20% to 84%; p&lt;0.05]. Moreover, in surviving mice, reduced fibulin-4 expression aggravated TAC-induced LV systolic and diastolic dysfunction and pulmonary congestion without affecting valvular function. In conclusion, using gradually reductions in fibulin-4 expression in mice and iPS derived cardiomyocytes we show that improper elastic fiber assembly drives cardiac pathology.

Formation and Decay of the Vanadate Complex of the Sarcoplasmic Reticulum Calcium Transport Protein

The calcium free sarcoplasmic reticulum calcium transport ATPase incorporates in the presence of magnesium ions approx. 8 nmol monovanadate per mg protein, indicating the formation of a complex containing one vanadate residue per enzyme molecule. On ligand-removal or dilution, the saturated enzyme complex displays biphasic decay kinetics, while the unsaturated complex slowly dissociates monophasically. -Ligand competition by raising the concentrations of unlabeled vanadate results in a progressive decrease of the dissociation rate of the unsaturated enzyme. The complicated dissociation kinetics indicate a sequential mode of interaction between two ligand binding sites. The one to one stoichiometry of the complex suggests that the two sites are located at adjacent ATPase molecules. -It appears unlikely that the decay of the enzyme, vanadate complex is retarded by the formation of a stable quaternary complex between the enzyme, magnesium, mono- and polyvanadate.

Lipid requirement of the vanadate effect on the binding of calcium and ATP to the calcium transport ATPase of the sarcoplasmic reticulum.

Lipid deprivation of the sarcoplasmic reticulum calcium-transport ATPase neither affects the enzyme's affinity for ATP nor that of calcium. In contrast, vanadate binding is almost completely abolished. Lipid substitution by oleic acid which at a ratio of 0.3 mg/mg protein completely reactivates the calcium-dependent ATP hydrolysis restores vanadate binding. Concomitantly the mutual interactions between vanadate and calcium or ATP and ADP, respectively are restored. The vanadate-induced disappearance of the enzyme's ATP binding sites as well as its high-affinity binding sites for calcium follow the same time course. Conversely, the displacement of vanadate by calcium proceeds in parallel with the recovery of ADP binding. In lipid-restituted preparations as well as in native membranes vanadate induces the disappearance of external high-affinity and simultaneously the appearance of internal low-affinity calcium binding sites.

Reversal of decreased phosphorylation of sarcoplasmic reticulum calcium transport ATPase by 1,25-dihydroxycholecalciferol in experimental uremia.

When compared to that from sham-operated controls, sarcoplasmic reticulum isolated from skeletal muscle of uremic rabbits had a lower rate of calcium uptake and storing capacity. In vivo administration of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] restored the values in uremic animals toward normal. To obtain information about the mechanisms responsible for these differences, phosphorylation of the calcium transport ATPase was studied. The steady-state levels of phosphoprotein in uremic membranes were lower and returned to normal when the secosteroid was administered. Electrophoresis of the membranes phosphorylated with 32P-inosine triphosphate (32P-ITP) showed that the differences were related to a 100,000 dalton protein. The rate of phosphoprotein formation, determined with 32P-ITP and at 0 degrees C, was considerably lower in uremic than in control animals. Pretreatment with 1,25(OH)2D3 prevented this change. The hypothesis is advanced that the vitamin D metabolite affects the steady-state concentration and rate constant of formation of active sites in the Ca-ATPase. These results may partly explain the altered Ca transport function of the sarcoplasmic reticulum in experimental uremia.

A conformational transition of the sarcoplasmic reticulum calcium transport ATPase induced by vanadate.

Vanadate binding to sarcoplasmic reticulum vesicles results in the loss of the externally located high affinity calcium binding sites of the calcium transport ATPase. Conversely the occupation by calcium of the internally located low affinity sites in the vanadate enzyme complex leads to the release of vanadate. Since the total number of calcium binding sites is not diminished by vanadate binding but slightly increases we conclude that vanadate binding induces a transition of the enzymes external high to internal low affinity calcium binding sites. The transposition of external to internal calcium binding sites is accompanied by a definite change in the structure of the sarcoplasmic reticulum membranes. On vanadate binding the asymmetrically arranged electron dense protein particles become symmetrically distributed.

The influence of sodium trichloroacetate on the tryptophan fluorescence of sarcoplasmic reticulum ATPase

The chaotropic anion trichloroacetate quenches the tryptophan fluorescence of the sarcoplasmic reticulum calcium transport ATPase. Half-maximum quenching was observed at 50 mM trichloroacetate. In contrast to native preparations, in trichloroacetate-treated sarcoplasmic reticulum vesicles a decrease of the tryptophan fluorescence is observed on addition of millimolar concentrations of calcium. It is concluded that trichloroacetate renders the tryptophan fluorescence of the ATPase sensitive to the occupancy of its low-affinity sites.

Ionized and Bound Calcium Inside Isolated Sarcoplasmic Reticulum of Skeletal Muscle and Its Significance in Phosphorylation of Adenosine Triphosphatase by Orthophosphate

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5--15 mM) on ice gives calcium loads of 50--60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid], but almost completely released by ionophore X-537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non-saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half-saturation constant of 5,8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 microliter/mg protein, respectively. Calcium-dependent phosphorylation of sarcoplasmic reticulum calcium-transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium-dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively. It is further suggested that the saturation of the low-affinity calcium-binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium-dependent phosphorylation than in calcium-independent phosphorylation from orthophosphate. Maximum calcium-dependent phosphoprotein formation at 20 degrees C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.