首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca2+ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca2+ handling, using a canine model of chronic HF. The cytosolic Ca2+ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca2+ content. Direct measurements of [Ca2+]SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca2+] is markedly lowered in HF, suggesting that alterations in Ca2+ transport rather than fractional SR volume reduction accounts for the diminished Ca2+ release capacity of SR in HF. SR Ca2+ ATPase (SERCA2)-mediated SR Ca2+ uptake rate was not significantly altered, and Na+/Ca2+ exchange activity was accelerated in HF myocytes. At the same time, SR Ca2+ leak, measured directly as a loss of [Ca2+]SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca2+]SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca2+ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca2+ leak is a major factor in the abnormal intracellular Ca2+ handling that critically contributes to the reduced SR Ca2+ content of failing cardiomyocytes.  相似文献   

2.
Sarcoplasmic reticulum contains the internal Ca2+ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca2+ level is the same all throughout the SR; however, whether the Ca2+ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca2+ buffer capacity of the SR by comparing the reduction in SR Ca2+ levels with the corresponding increase in [Ca2+]i during activation of either IP3Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca2+ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca2+ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca2+ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca2+ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca2+ influx, indicate the relevance of luminal SR Ca2+ buffer capacity in the [Ca2+]i response. To further study the importance of luminal SR Ca2+ buffer capacity in the release process we used low levels of heparin to partially inhibit IP3Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca2+ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca2+-binding proteins in the high-capacity, low-affinity conformation, particularly for IP3R-mediated Ca2+ release.  相似文献   

3.
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   

4.
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   

5.
Ca2+ transport by the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) is sensitive to monovalent cations. Possible K+ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca2+ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca2+ transport correlated in most cases with their direct effects on SERCA. Choline+, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca2+ transport. Of the monovalent cations tested, only Cs+ significantly affected the Hill coefficient of Ca2+ transport (nH). An increase in nH from ∼2 in K+ to ∼3 in Cs+ was seen in all of the forms of SERCA examined. The effects of Cs+ on the maximum velocity of Ca2+ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K+ binding sites of the different forms of the protein.  相似文献   

6.
This study investigates sarcoplasmic reticulum (SR) calcium-(Ca2+) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca2+ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca2+ uptake of +33%. Specific Ca2+ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca2+ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca2+ re-uptake function of the SR under defined culture conditions.  相似文献   

7.
The sarcoplasmic reticulum Ca2+ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca2+ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca2+ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔHcal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg2+ or ruthenium red, conditions that close the ryanodine Ca2+ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔHcal values of ATP hydrolysis increased significantly. Neither Mg2+ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔHcal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca2+ channel is opened or closed.  相似文献   

8.
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   

9.
NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) is widely used as a large-conductance Ca2+-activated K+ (BKCa) channel opener. It was previously reported that activation of BKCa channels by NS1619 could protect the cardiac muscle against ischaemia and reperfusion injury. This study reports the effects of NS1619 on intracellular Ca2+ homeostasis in H9C2 and C2C12 cells as well as its molecular mechanism of action. The effects of NS1619 on Ca2+ homeostasis in C2C12 and H9C2 cells were assessed using the Fura-2 fluorescence method. Ca2+ uptake by sarcoplasmic reticulum (SR) vesicles isolated from rat skeletal muscles and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity were measured. The effect of NS1619 on the isometric force of papillary muscle contraction in the guinea pig heart was also examined. H9C2 and C2C12 cells treated with NS1619 released Ca2+ from internal stores in a concentration-dependent manner. Ca2+ accumulation by the SR vesicles was inhibited by NS1619 treatment. NS1619 also decreased the activity of SERCA derived from rat skeletal muscle. The calcium release from cell internal stores and inhibition of SERCA by NS1619 are pH dependent. Finally, NS1619 had a profound effect on the isometric force of papillary muscle contraction in the guinea pig heart. These results indicate that NS1619 is a potent modulator of the intracellular Ca2+ concentration in H9C2 and C1C12 cells due to its interaction with SRs. The primary target of NS1619 is SERCA, which is located in SR vesicles. The effect of NS1619-mediated SERCA inhibition on cytoprotective processes should be considered.  相似文献   

10.
Cardiac contractility is regulated through the activity of various key Ca2+-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca2+ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca2+ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca2+ stores.  相似文献   

11.
The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca2+ release during systole. However, it remains obscure how the dyadic organization affects SR Ca2+ handling during diastole. By measuring intraluminal SR Ca2+ ([Ca2+]SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca2+ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca2+ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca2+-ATPase (SERCA). Detubulation decreased [Ca2+]SR decline during rest, thus making the leaked SR Ca2+ more accessible for SERCA. These results suggest that Ca2+ extrusion systems are localized in T-tubules. Inhibition of Na+-Ca2+ exchanger (NCX) slowed [Ca2+]SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca2+]SR decline. Despite a significant SR Ca2+ leak, Ca2+ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca2+ spark activity independent of SR Ca2+ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca2+ that leaks from the SR. This can be explained if the majority of SR Ca2+ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca2+] by NCX play a critical role in suppressing Ca2+ sparks during rest.  相似文献   

12.
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.  相似文献   

13.
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   

14.
The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca2+ release during systole. However, it remains obscure how the dyadic organization affects SR Ca2+ handling during diastole. By measuring intraluminal SR Ca2+ ([Ca2+]SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca2+ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca2+ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca2+-ATPase (SERCA). Detubulation decreased [Ca2+]SR decline during rest, thus making the leaked SR Ca2+ more accessible for SERCA. These results suggest that Ca2+ extrusion systems are localized in T-tubules. Inhibition of Na+-Ca2+ exchanger (NCX) slowed [Ca2+]SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca2+]SR decline. Despite a significant SR Ca2+ leak, Ca2+ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca2+ spark activity independent of SR Ca2+ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca2+ that leaks from the SR. This can be explained if the majority of SR Ca2+ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca2+] by NCX play a critical role in suppressing Ca2+ sparks during rest.  相似文献   

15.
Heart failure is one of the leading causes of sudden death in developed countries. While current therapies are mostly aimed at mitigating associated symptoms, novel therapies targeting the subcellular mechanisms underlying heart failure are emerging. Failing hearts are characterized by reduced contractile properties caused by impaired Ca2+ cycling between the sarcoplasm and sarcoplasmic reticulum (SR). Sarcoplasmic/endoplasmic reticulum Ca2+ATPase 2a (SERCA2a) mediates Ca2+ reuptake into the SR in cardiomyocytes. Of note, the expression level and/or activity of SERCA2a, translating to the quantity of SR Ca2+ uptake, are significantly reduced in failing hearts. Normalization of the SERCA2a expression level by gene delivery has been shown to restore hampered cardiac functions and ameliorate associated symptoms in pre-clinical as well as clinical studies. SERCA2a activity can be regulated at multiple levels of a signaling cascade comprised of phospholamban, protein phosphatase 1, inhibitor-1, and PKCα. SERCA2 activity is also regulated by post-translational modifications including SUMOylation and acetylation. In this review, we will highlight the molecular mechanisms underlying the regulation of SERCA2a activity and the potential therapeutic modalities for the treatment of heart failure. [BMB Reports 2013; 46(5): 237-243]  相似文献   

16.
Cytosolic calcium concentration ([Ca2+]c) is fundamental for regulation of many cellular processes such metabolism, proliferation, muscle contraction, cell signaling and insulin secretion. In resting conditions, the sarco/endoplasmic reticulum (ER/SR) Ca2+ ATPase's (SERCA) transport Ca2+ from the cytosol to the ER or SR lumen, maintaining the resting [Ca2+]c about 25–100 nM. A reduced activity and expression of SERCA2 protein have been described in heart failure and diabetic cardiomyopathy, resulting in an altered Ca2+ handling and cardiac contractility. In the diabetic pancreas, there has been reported reduction in SERCA2b and SERCA3 expression in β-cells, resulting in diminished insulin secretion. Evidence obtained from different diabetes models has suggested a role for advanced glycation end products formation, oxidative stress and increased O-GlcNAcylation in the lowered SERCA2 expression observed in diabetic cardiomyopathy. However, the role of SERCA2 down-regulation in the pathophysiology of diabetes mellitus and diabetic cardiomyopathy is not yet well described. In this review, we make a comprehensive analysis of the current knowledge of the role of the SERCA pumps in the pathophysiology of insulin-dependent diabetes mellitus type 1 (TIDM) and type 2 (T2DM) in the heart and β-cells in the pancreas.  相似文献   

17.
Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca2+-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca2+-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca2+-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.  相似文献   

18.
The Ca2+ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca2+ in intracellular membrane bound compartments, thereby lowering cytosolic Ca2+ to induce relaxation. The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.  相似文献   

19.
Beat-to-beat alternation in the cardiac intracellular Ca (Cai) transient can drive action potential (AP) duration alternans, creating a highly arrhythmogenic substrate. Although a steep dependence of fractional sarcoplasmic reticulum (SR) Ca release on SR Ca load has been shown experimentally to promote Cai alternans, theoretical studies predict that other factors are also important. Here we present an iterated map analysis of the coordinated effects of SR Ca release, uptake, and leak on the onset of Cai alternans. Predictions were compared to numerical simulations using a physiologically realistic AP model as well as to AP clamp experiments in isolated patch-clamped rabbit ventricular myocytes exposed to 1), the Ca channel agonist BayK8644 (100 nM) to increase SR Ca load and release fraction, 2), overexpression of an adenoviral SERCA2a construct to increase SR Ca uptake, and 3), low-dose FK506 (20 μM) or ryanodine (1 μM) to increase SR Ca leak. Our findings show that SR Ca release, uptake, and leak all have independent direct effects that promote (release and leak) or suppress (uptake) Cai alternans. However, since each factor affects the other by altering SR Ca load, the net balance of their direct and indirect effects determines whether they promote or suppress alternans. Thus, BayK8644 promotes, whereas Ad-SERCA2a overexpression, ryanodine, and FK506 suppress, Cai alternans under AP clamp conditions.  相似文献   

20.

Background

Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca2+ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca2+ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca2+ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively.

Methodology/Principal Findings

AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca2+ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca2+ load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.

Conclusions/Significance

Increased Ca2+ leak and cytosolic Ca2+ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca2+ cycling.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号