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1.
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.  相似文献   

2.
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)  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
The Ca2+ content in the sarcoplasmic reticulum (SR) determines the amount of Ca2+ released, thereby regulating the magnitude of Ca2+ transient and contraction in cardiac muscle. The Ca2+ content in the SR is known to be regulated by two factors: the activity of the Ca2+ pump (SERCA) and Ca2+ leak through the ryanodine receptor (RyR). However, the direct relationship between the SERCA activity and Ca2+ leak has not been fully investigated in the heart. In the present study, we evaluated the role of the SERCA activity in Ca2+ leak from the SR using a novel saponin-skinned method combined with transgenic mouse models in which the SERCA activity was genetically modulated. In the SERCA overexpression mice, the Ca2+ uptake in the SR was significantly increased and the Ca2+ transient was markedly increased. However, Ca2+ leak from the SR did not change significantly. In mice with overexpression of a negative regulator of SERCA, sarcolipin, the Ca2+ uptake by the SR was significantly decreased and the Ca2+ transient was markedly decreased. Again, Ca2+ leak from the SR did not change significantly. In conclusion, the selective modulation of the SERCA activity modulates Ca2+ uptake, although it does not change Ca2+ leak from the SR.  相似文献   

8.
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.  相似文献   

9.
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 Vmax 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.  相似文献   

10.
Calumenin is a multiple EF-hand Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265–269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca2+-cycling proteins. But it enhanced Ca2+ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca2+ uptake showed increased Ca2+ sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 μm Ca2+, suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132–222 amino acids of calumenin and 853–892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca2+-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca2+-cycling of cardiomyocytes.  相似文献   

11.
Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (Dmit) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca2+ signaling, T-T remodeling, Dmit, citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine).We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca2+ (19%) and blunted Ca2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca2+ transients. However, dRel/dt persisted during Ca2+ transient relaxation due to abundant late Ca2+ sparks. Isoproterenol significantly up-regulated systolic Ca2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca2+-dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca2+ leak. Furthermore, Dmit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca2+ and contractility dysregulation. We conclude that decreased SR Ca2+, due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD.  相似文献   

12.
We review the recent development of novel biochemical and spectroscopic methods to determine the site-specific phosphorylation, expression, mutation, and structural dynamics of phospholamban (PLB), in relation to its function (inhibition of the cardiac calcium pump, SERCA2a), with specific focus on cardiac physiology, pathology, and therapy. In the cardiomyocyte, SERCA2a actively transports Ca2+ into the sarcoplasmic reticulum (SR) during relaxation (diastole) to create the concentration gradient that drives the passive efflux of Ca2+ required for cardiac contraction (systole). Unphosphorylated PLB (U-PLB) inhibits SERCA2a, but phosphorylation at S16 and/or T17 (producing P-PLB) changes the structure of PLB to relieve SERCA2a inhibition. Because insufficient SERCA2a activity is a hallmark of heart failure, SERCA2a activation, by gene therapy (Andino et al. 2008; Fish et al. 2013; Hoshijima et al. 2002; Jessup et al. 2011) or drug therapy (Ferrandi et al. 2013; Huang 2013; Khan et al. 2009; Rocchetti et al. 2008; Zhang et al. 2012), is a widely sought goal for treatment of heart failure. This review describes rational approaches to this goal. Novel biophysical assays, using site-directed labeling and high-resolution spectroscopy, have been developed to resolve the structural states of SERCA2a-PLB complexes in vitro and in living cells. Novel biochemical assays, using synthetic standards and multidimensional immunofluorescence, have been developed to quantitate PLB expression and phosphorylation states in cells and human tissues. The biochemical and biophysical properties of U-PLB, P-PLB, and mutant PLB will ultimately resolve the mechanisms of loss of inhibition and gain of inhibition to guide therapeutic development. These assays will be powerful tools for investigating human tissue samples from the Sydney Heart Bank, for the purpose of analyzing and diagnosing specific disorders.  相似文献   

13.
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 Pi 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.  相似文献   

14.
This comparative study investigates the relationship between sarcoplasmic reticulum (SR) calcium(Ca2+)-ATPase transport activity and phospholamban (PLB) phosphorylation in whole cardiac homogenates of spo`ntaneously hypertensive rats (SHR) and their parent, normotensive Wistar Kyoto (WKY) strain during early postnatal development at days 1, 3, 6, 12 and at day 40 to ascertain any difference in SR Ca2+ handling before the onset of hypertension. At day 1, the rate of homogenate oxalate-supported Ca2+ uptake was significantly higher in SHR than in WKY (0.25 ± 0.02 vs 0.12 ± 0.01 nmoles Ca2+/mg wet ventricular weight/min, respectively; p < 0.001). This interstrain difference disappeared with further developmental increase in SR Ca2+ transport. Western Blot analysis and a semiquantitative ELISA did not reveal any difference in the amount of immunoreactive PLB (per mg of total tissue protein) between strains at any of the ages studied. In addition, levels of phosphorylated PLB formed in vitro in the presence of radiolabelled ATP and catalytic (C) subunit of protein kinase A did not differ between SHR and WKY at days 1, 3, 6 and 12. At day 40, C subunit-catalyzed formation of 32P-PLB was reduced by 66% (p < 0.001) in SHR when compared to age-matched WKY In the early postnatal period between day 1 and 12 SR Ca2+-transport values were linearly related to the respective 32P-PLB levels of both SHR and WKY rats. The results indicate that cardiac SR of SHR can sequester Ca2+ at a much higher rate immediately after birth compared to WKY rats. The disappearance of this interstrain difference with further development suggests that some endogenous neuroendocrine or nutritional factor(s) from the hypertensive mother may exert an influence upon the developing heart in utero resulting in a transiently advanced maturation of the SR Ca2+ transport function in SHR pups at the time of birth.  相似文献   

15.
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.  相似文献   

16.
Junctate is a newly identified sarcoplasmic reticulum (SR) Ca2+ binding protein, but its function in cardiac muscle has remained unclear. Our previous study showed that chronic over-expression of junctate in transgenic mice led to altered SR functions and development of severe hypertrophy. In this study, we identified the interaction of junctate with SERCA2a by co-immunoprecipitation and GST-pull-down assay. This interaction was inhibited by higher Ca2+ concentration. Immunolocalization assays also showed that junctate and SERCA2a were co-localized in the SR of cardiomyocytes. Direct binding of the C-terminal region of junctate (amino acids 79-270) and luminal domain of SERCA2a (amino acids 70-89) was observed by deletion mutation experiments. Adenovirus-mediated transient over-expression of junctate in cardiomyocytes showed a reduced decay time of Ca2+ transients and increased oxalate-supported SERCA2 Ca2+ uptake, suggesting an increased activity of SERCA2a. Taken together, according to our data, junctate may play an important role in the regulation of SR Ca2+ cycling through the interaction with SERCA2a in the murine heart.  相似文献   

17.
Summary Relatively pure mitochondrial-free Sarcoplasmic reticulum (SR) was isolated from crab and cockroach skeletal muscle, and the SR calcium uptake was studied in control and insecticide-treated conditions by incubation in Ca45 media followed by millipore filtration and counting by liquid scintillation methods. All insecticides used (parathion, DDT, BHC and tri-cresyl phosphate) at 1 mM concentrations caused a slight fall in the intrinsic Ca2+ content of the SR, but this Ca2+ loss was insufficient to explain the contracture induction effect of these agents.All insecticides used (at 1 mM) caused a massive inhibition of SR Ca2+ uptake, and all were able to release Ca2+ previously bound to the SR during incubation in the presence of ATP. It is concluded that the contracture-induction effect of these insecticides is due to their inhibition of SR Ca2+ uptake, resulting in a massive rise in myoplasmic free Ca2+.  相似文献   

18.
The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca2+ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca2+ uptake and E–C coupling. Ca2+ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca2+ uptake as low as 5 °C. As a consequence, the Q10 values in coho salmon were low over a range of different temperature intervals. Maximal Ca2+ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca2+ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca2+ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.  相似文献   

19.
Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca2+) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca2+ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo-3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca2+ decay time, Ca2+ sparks, and Ca2+ leakage but lower [Ca2+]i transients and sarcoplasmic reticulum Ca2+ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca2+ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB-Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na+ current in CKD significantly altered cardiac Ca2+ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.  相似文献   

20.

Background

Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias.

Methods

Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca2+ was measured using Indo-1AM and Ca2+ alternans (Ca-ALT) was induced with a standard ramp pacing protocol.

Results

As predicted, SR Ca2+ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca2+ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20).

Conclusions

Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic.  相似文献   

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