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OBJECTIVE: Heart failure is a progressive and debilitating disease. Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy may improve the function of cardiac muscle cells. This study aimed to test the hypothesis that intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy can improve outcomes and reduce the number of recurrent and terminal events in advanced heart failure patients with reduced ejection fraction. METHODS: A total of 768 heart failure patients with reduced ejection fraction and New York Heart Association classification II to IV were included in this prospective cohort study. Patients either underwent intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy (CA group, n=384) or received oral placebo (PA group; n=384). Data regarding recurrent and terminal event(s), treatment-emergent adverse effects, and outcome measures were collected and analyzed. RESULTS: After a follow-up period of 18 months, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduced the number of hospital admissions (p=0.001), ambulatory treatments (p=0.0004), and deaths (p=0.024). Additionally, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy improved the left ventricular ejection fraction (p<0.0001) and Kansas City Cardiomyopathy Questionnaire score (p<0.0001). The number of recurrent and terminal events/patients were higher in the PA group than in the CA group after the follow-up period of 18 months (p=0.015). The effect of the intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy was independent of the confounding variables. No new arrhythmias were reported in the CA group. CONCLUSIONS: Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduces the number of recurrent and terminal events and improves the clinical course of advanced heart failure patients with reduced ejection fraction.
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Humans , Male , Female , Sarcoplasmic Reticulum , Heart Failure , Stroke Volume , Genetic Therapy , Calcium , Prospective Studies , Ventricular Function, Left , Sarcoplasmic Reticulum Calcium-Transporting ATPasesABSTRACT
Objective To observe the enzymatic changes of myocardial sarcoplasmic reticulum in rats after injecting endotoxin (LPS), and provide basic research results for the future study of myocardial sarcoplasmic reticulum dysfunction caused by LPS in rats. Methods Ten SD rats were randomly divided into blank control group and LPS injection group with 5 rats in each group. In the LPS injection group, endotoxin was injected into the tail vessels of the rats. Results The heart rate (HR) of the LPS injection group increased and was faster than that in the blank control group [(204±18) beat per min vs. (139±10) beat per min on the first day, and (199±22) beat per min vs. (143±17) beat per min on the next day, both P values were less than 0.05]. The mean arterial pressure (MAP) was lower than that of the blank control group on the first day [(87±12) mmHg vs. (102±7) mmHg, P<0.05]. Under light and electron microscope, the myocardial cells of rats with LPS injection were loosely arranged, with intercellular infiltration with inflammatory cells, muscle fibers broken, and difficult to identify the morphology of mitochondria and sarcoplasmic reticulum. Quantitative PCR results showed that after endotoxin injection, troponin (CASQ1), sodium-calcium exchanger (NCX), calmodulin phosphatase 1 (ppplCa), phospholipid protein (PLN), sarcoplasmic reticulum Ca2+-ATPase (SERCA2) increased significantly (P<0.05). Conclusion Endotoxin can inhibit cardiomyocyte function by affecting the activity of sarcoplasmic reticulum calcium regulatory protein-related enzymes through various mechanisms.
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Objective To evaluate the effect of sevoflurane preconditioning on the function of sar-coplasmic reticulum in cardiomyocytes during ischemia-reperfusion (I∕R) in isolated rat hearts. Methods Healthy adult male Sprague-Dawley rats, weighing 270-300 g, were anesthetized with intraperitoneal pen-tobarbital. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 37 ℃ . Twenty-four isolated rat hearts successfully perfused in the Langendorff ap-paratus were divided into 3 groups (n= 8 each) using a random number table: control group (group C), I∕R group and sevoflurane preconditioning group (group SP). Myocardial ischemia was induced by interrup-ting perfusion for 30 min followed by 120 min reperfusion. In group SP, the hearts were perfused with 2. 4％ sevoflurane for 10 min starting from 10 min before ischemia. Left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were recorded at 5, 10, 15, 30 and 60 min of reperfusion. Coronary effluent was collected at 10 min before reperfusion for measurement of the levels of lactate dehydrogenase (LDH) and cardiac troponin I (cTnI). Myocardial specimens were obtained at 120 min of reperfusion for examination of the pathological changes (using HE staining) and for determination of myocardial infarct size (by TTC staining), sarcoendoplasmic reticulum Ca2+-ATPase (SERCA2a) activity (with a spectrophotometer) and expression of Bcl-2, Bax and SERCA2a ( by Western blot). Results Compared with group C, LVDP was significantly decreased and LVEDP was increased at each time point, myocardial infarct size was increased, the levels of LDH and cTnI in coronary effluent were increased, the expression of Bax was up-regulated, the expression of Bcl-2 and SERCA2a was down-regulated, and the activity of SERCA2a was decreased in group I∕R (P<0. 01). Compared with group I∕R, LVDP was signifi-cantly increased and LVEDP was decreased at each time point, myocardial infarct size was decreased, the levels of LDH and cTnI in coronary effluent were decreased, the expression of Bax was down-regulated, the expression of Bcl-2 and SERCA2a was up-regulated, the activity of SERCA2a was increased (P<0. 01), and the pathological changes were significantly attenuated in group SP. Conclusion The mechanism by which sevoflurane preconditioning reduces I∕R injury in isolated rat hearts may be related to improving the function of sarcoplasmic reticulum in cardiomyocytes.
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Objective To investigate the effect of 5-AZA-2'-dC on Angiotensin Ⅱ (Ang Ⅱ)-induced cardiomyocyte hypertrophy.Methods Cultured cells derived from neonatal heart of rat were divided into 5 groups:normal control,hypertrophic group,5-AZA-2'-dC treatment group,and 5-AZA-2'-dC pretreatment group.Neonatal rat cardiomyocyte hypertrophic response was assayed by the size of cardiomyocytes and atrial natriuretic polypeptide (ANP) expressive level.The level of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a),total calmodulin kinase Ⅱ (CaMK Ⅱ) and phospho-CaMK Ⅱ (p-CaMK Ⅱ) detected by Western blot.The intracellular calcium changes of cardiomyocytes were imaged by confocal fluorescent microscopy.Results Cells treated with Ang Ⅱ at 10-6 mol/L for 48 h were chosen as hypertrophic cardiomyocyte model.The mRNA expression and protein level of ANP were significantly decreased in the treatment and pretreatment groups compared with hypertrophic group.The protein level of SERCA2a was significantly decreased in the hypertrophic group,and increased in the treatment and pretreatment group compared with hypertrophic group.The protein level of SERCA2a was significantly decreased in the hypertrophic group,and increased in the treatment and pretreatment group compared with hypertrophic group,whereas phospho-CaMK Ⅱ showed an opposite change tendency.The time required for increasing and declining to half of the intracellular calcium peak value were both delayed in hypertrophic group,as the treatment and pretreatment groups showed shorter time required compared with hypertrophic group.Conclusion 5-AZA-2'-dC could inhibit Ang Ⅱ-induced cardiomyocyte hypertrophy which might be related to regulate SERCA2a expression.Increased SERCA2a expression may maintain the calcium homeostasis through shortening the time of transfer Ca2+ from the cytosol of the cell to the lumen of the sarcoplasmic reticulum.
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Ryanodine receptors (RyRs) are tetrameric Ca2+ release channels of sarcoplasmic reticulum (SR). This review attempts to detail the key mechanism of RyR channel gating and to discuss the hypothesis that skeletal muscle fatigue, defined as reduced force production, would result from functional changes in both individual RyR channel opening and coupling among RyR channels. Previous studies have shown that RyR channels in skeletal muscle open simultaneously, called coupled gating, because of physical interaction among channels. In this review, mechanisms underlying muscle fatigue are discussed with consideration of the coupling effect. Fatigue mechanisms are thought to be different between acute exercise and long-term exercise training. The impairments in individual channel opening and coupling between RyR channels can occur after acute exercise, leading to decreased SR Ca2+ release and force depression. On the contrary, during long-term exercise training, individual channel opening would be enhanced but coupling between channels would be impaired. If this were to continue for long periods, SR Ca2+ content would reduce, leading to less Ca2+ release and lower force production.
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Objective: To explore sarcoplasmic reticulum ryanodine receptor2 (RyR 2) expression and calcium releasing function in chronic heart failure (CHF) rabbits and to study the impact of long term valsartan treatment in relevant animals. Methods: HF model was established by volume overloading with pressure overloading in experimental rabbits. 27 rabbits were divided into 3 groups: Sham group, HF group and HF+valsartan group. n=9 in each group and the animals were treated for 7 weeks. Left ventricular structure, hemodynamic parameters, expression and functional changes of myocardiocyte sarcoplasmic reticulum RyR 2 were observed and compared among different groups. Results: Compared with Sham group, HF group had increased left ventricular mess index (LVMI), left ventricular end diastolic pressure (LVEDP) and decreased left ventricular shortening fraction, LVEF, all P<0.05. Compared with HF group, HF+valsartan group showed decreased LVMI, LVEDP and increased left ventricular shortening fraction, LVEF, all P<0.05. Sarcoplasmic reticulum RyR 2 expression and calcium releasing function were lower in HF group than Sham group, P<0.05; while they were both higher in HF+valsartan group than HF group, P<0.05. Conclusion: Long term application of valsartan could improve the cardiac function which might be related to increased myocardial sarcoplasmic reticulum RyR 2 expression and calcium releasing function in experimental CHF rabbits.
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Objective To investigate the effects of bisoprolol on myocardial SERCA2a activity in rats with heart fail-ure.Methods Male SD rats were randomly divided into normal control group (control group), sham operation group ( sham group ) , model group , bisoprolol group ( Bis group ) , captopril group ( Cap group ) and bisoprolol plus captopril group[(Bis+Cap)group], heart failure rat model was induced by intraperitoneal injections of doxorubicin .Distilled water, bisoprolol, captopril or bisoprolol plus captopril were administrated by gastrogavage for 35 days, respectively. Indices of cardiac function and plasma levels of B-type natriuretic peptide ( BNP) were measured , myocardial expres-sion of miR-25-3p was detected by Stem-loop RT-qPCR, myocardial levels of SERCA2a and phospholamban (PLB) were detected by Western blot , myocardial SERCA2a activity was determined by the inorganic phosphorus method . Results Cardiac function in model group decreased significantly while plasma levels of BNP were significantly higher than those of control group ( P<0.01 ) .Myocardial expression of miR-25-3p in model group was significantly higher while myocardial levels of SERCA 2a and PLB,SERCA2a activity were significantly lower than those of con-trol group(P<0.01).Cardiac function in Bis group , Cap group and Bis +Cap group improved significantly while plasma levels of BNP were significantly lower than those of model group ( P<0.01 ) .Myocardial expression of miR-25-3p in Bis group, Cap group and Bis +Cap group were significantly lower while myocardial levels of SERCA2a and PLB were significantly higher than those in model group (P<0.01).The SERCA2a/PLB ratio and SERCA2a activity in Bis group and Bis +Cap group were significantly higher than those of model group ( P<0.05 ) .Conclu-sions Bisoprolol therapy improves cardiac function in rats with heart failure , which may be related to inhibition of myocardial miR-25-3p, increasing myocardial SERCA2a and PLB levels, enhancing SERCA2a activity.
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Intracellular calcium (Ca²⁺) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H₂O₂) on intracellular Ca²⁺ accumulation in mouse pancreatic acinar cells. Perfusion of H₂O₂ at 300 µM resulted in additional elevation of intracellular Ca²⁺ levels and termination of oscillatory Ca²⁺ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca²⁺. Antioxidants, catalase or DTT, completely prevented H₂O₂-induced additional Ca²⁺ increase and termination of Ca²⁺ oscillation. In Ca²⁺-free medium, H₂O₂ still enhanced CCh-induced intracellular Ca²⁺ levels and thapsigargin (TG) mimicked H₂O₂-induced cytosolic Ca²⁺ increase. Furthermore, H₂O₂-induced elevation of intracellular Ca²⁺ levels was abolished under sarco/endoplasmic reticulum Ca²⁺ ATPase-inactivated condition by TG pretreatment with CCh. H₂O₂ at 300 µM failed to affect store-operated Ca²⁺ entry or Ca²⁺ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca²⁺ uniporter blocker, failed to attenuate H₂O₂-induced intracellular Ca²⁺ elevation. These results provide evidence that excessive generation of H₂O₂ in pathological conditions could accumulate intracellular Ca²⁺ by attenuating refilling of internal Ca²⁺ stores rather than by inhibiting Ca²⁺ extrusion to extracellular fluid or enhancing Ca²⁺ mobilization from extracellular medium in mouse pancreatic acinar cells.
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Animals , Mice , Acinar Cells , Antioxidants , Calcium , Carbachol , Catalase , Cell Membrane , Cytosol , Extracellular Fluid , Hydrogen Peroxide , Hydrogen , Ion Transport , Pancreatitis , Perfusion , Reactive Oxygen Species , Reticulum , Ruthenium Red , ThapsigarginABSTRACT
Obesity and diabetes has become a major epidemic across the globe. Controlling obesity has been a challenge since this would require either increased physical activity or reduced caloric intake; both are difficult to enforce. There has been renewed interest in exploiting pathways such as uncoupling protein 1 (UCP1)-mediated uncoupling in brown adipose tissue (BAT) and white adipose tissue to increase energy expenditure to control weight gain. However, relying on UCP1-based thermogenesis alone may not be sufficient to control obesity in humans. On the other hand, skeletal muscle is the largest organ and a major contributor to basal metabolic rate and increasing energy expenditure in muscle through nonshivering thermogenic mechanisms, which can substantially affect whole body metabolism and weight gain. In this review we will describe the role of Sarcolipin-mediated uncoupling of Sarcoplasmic Reticulum Calcium ATPase (SERCA) as a potential mechanism for increased energy expenditure both during cold and diet-induced thermogenesis.
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Humans , Adipose Tissue, Brown , Adipose Tissue, White , Basal Metabolism , Diabetes Mellitus , Energy Intake , Energy Metabolism , Hand , Metabolism , Motor Activity , Muscle, Skeletal , Obesity , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Thermogenesis , Weight GainABSTRACT
Objective To investigate of the effect and mechanisman of SERCA2 on the phenotype modulation of HASMCs. Methods HASMCs were starved for 5 days and divided into different groups ,then we observed morphology change of the cells from the microscope and detected a-actin、SERCA2 and p-ERK by Western Blot,cells proliferation was observed by CCK-8 method. Results Compared with the control group,PDGF could reduce a-actin of HASMCs and increased the cells proliferation ability ,TSG could significantly inhibit the effect (P<0.01), PDGF could also significantly inhibit SERCA2 protein and increased the expression p-ERK (P<0.01), while U0126 significantly inhibited the effect (P < 0.01). Conclusion PDGF may induce HASMCs phenotype modulation through the regulation of SERCA2 and p-ERK.
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Objective To evaluate the effects of acute peritonitis on rocuronium?induced neuromus?cular blockade in abdominal muscles and function of the sarcoplasmic reticulum of rats. Methods Thirty?six pathogen?free male Sprague?Dawley rats, weighing 220-250 g, were divided into 2 groups using a ran?dom number table: control group (group C, n=12) and acute peritonitis group (group P, n=24). After the rats were anesthetized with pentobarbital sodium, acute peritonitis was induced by artificial gastric per?foration in group P. At 1 and 2 h after operation, the changes in the intra?abdominal pressure (IAP) with different volumes were detected, and blood samples were collected from the orbital veins for determination of serum levels of interleukin?6, tumor necrosis factor?alpha and interleukin?13. Rocuronium 3. 5 mg∕kg was then injected via the caudal vein. The IAP was recorded at 1, 5 and 10 min after administration. The intra?cellular free Ca2+ concentration was assessed using fura?2, and the maximal Ca2+ uptake and release rate in the sarcoplasmic reticulum were calculated. Results Compared with group C, the serum levels of interleu?kin?6 and tumor necrosis factor?alpha at 2 h after operation and IAP at 1 and 2 h after operation were signifi?cantly increased, the IAP was increased at 1, 5 and 10 min after administration of rocuronium, and the maximal Ca2+ uptake rate and amount of calcium uptake in the sarcoplasmic reticulum were decreased in group P ( P<0.01) . Conclusion Acute peritonitis decreases rocuronium?induced neuromuscular blockade in abdominal muscles, which may be related to the impaired Ca2+uptake function of the sarcoplasmic reticu?lum of rats.
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AIM:To investigate the effects of Xinkang recipe on myocardial miR-25-3p expression and sarco-plasmic reticulum calcium ATPase 2a ( SERCA2a) activity in heart failure rats .METHODS:Male SD rats were randomly divided into normal group , sham group , model group , Xinkang recipe group ( Xinkang group ) , and captopril group .The heart failure rat model was induced by intraperitoneal injection of doxorubicin .Distilled water , Xinkang recipe and capto-pril were administrated by gastric gavage for 35 d, respectively .The indexes of cardiac function and plasma level of brain natriuretic peptide (BNP) were measured.The SERCA2a activity was determined by the inorganic phosphorus method . The myocardial protein expression of SERCA 2a and phospholamban ( PLB) was detected by Western blot .The myocardial expression of miR-25-3p was detected by stem-loop RT-qPCR.RESULTS:Cardiac output (CO), left ventricular fraction-al shortening ( LVFS) and left ventricular ejection fraction ( LVEF) in Xinkang group and captopril group were significantly higher while the plasma levels of BNP were significantly lower than those in model group (P<0.01).The myocardial ex-pression levels of miR-25-3p in Xinkang group and captopril group were significantly lower while the myocardial protein le -vels of SERCA2a and PLB were significantly higher than those in model group (P<0.01).The SERCA2a/PLB ratio and SERCA2a activity in Xinkang group were significantly higher than those in model group (P<0.05), and no significant change was observed between captopril group and model group .CONCLUSION:Xinkang recipe therapy may improve car-diac function in heart failure rats , which may be related to inhibiting the expression of miR-25-3p, increasing the SER-CA2a/PLB ratio and enhancing SERCA 2a activity in the myocardium .
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OBJECTIVE To explore kinetic features and its underlying mechanism of the positive inotropic effect of liguzinediol(LZDO)in rats. METHODS ①An In vivo study was made to record the effect of LZDO 20 mg · kg-1 injected for 30 consecutive min from the left external jugular vein on pressure-volume relationships. ②Ex vivo study was used to record the antagonistic effect of LZDO on reduced contractility induced by caffeine. Caffeine and LZDO were perfused as follows:normal perfusion solution, caffeine 0.5 mmol · L-1,and then caffeine 0.5 mmol · L-1+LZDO 100 μmol · L-1. ③ Ca2+ transient from cardiomyocyte sarcoplasmic reticulum (SR) was measured to analyze the effect of LZDO on Ca2 +release blocked by thapsigargin. Thapsigargin and LZDO were perfused as follows:normal perfusion solution,thapsigargin 2 μmol · L-1,and then thapsigargin 2 μmol · L-1+LZDO 100 μmol · L-1.④The SR vesicles were prepared and the effect of LZDO(1,10 and 100μmol·L-1)on sarcoplasmic reticulum Ca2+ATPase(SERCA2a)activity was determined according to the ultramicro-Ca2+-ATP enzyme kit. RESULTS ① LZDO 20 mg · kg- 1 significantly reduced the end-systolic volume (Ves) and enhanced the end-systolic pressure (Pes),stroke volume (SV),ejection fraction (EF),cardiac output(CO),peak rate of rise of left ventricular pressure(+dp/dtmax)and stroke work(SW)(P<0.05). However,LZDO 20 mg · kg-1 did not significantly change the heart rate(HR )or the end-diastolic volume (Ved). ② Caffeine 0.5 mmol · L- 1 significantly enhanced HR,left ventricular developed pressure (LVDP ),and+dp∶dtmax at 5 min after caffeine and decreased at 30 min. However,LZDO 100μmol·L-1 restored the reduced HR,LVDP,and+dp/dtmax induced by caffeine at 30 min(P<0.05).③Thapsigargin 2μmol·L-1 significantly reduced the SR Ca2+transient from perfusion solution group(100±5)%to(51± 5)%(P<0.05) and LZDO 100 μmol · L-1 failed to restore the decreased Ca2+ transient〔(49 ± 4)%〕. Normalized Ca2+transients were reduced by thapsigargin 2μmol·L-1 and thapsigargin 2μmol·L-1+LZDO 100 μmol · L-1. ④ LZDO(10 and 100 μmol · L-1)significantly increased the activities of SERCA2a in perfusion solution group 0.98±0.10 to 1.17±0.20 and (1.43±0.09)μmol Pi·g-1·h-1,respectively(P<0.05). CONCLUSION LZDO can enhance SR Ca2+ gradient by activating the SERCA2a and might be developed to serve as a potential positive inotropic agent in clinical settings.
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<p><b>OBJECTIVE</b>To compare the effect of Shen-Fu Injection (SFI) and epinephrine on the expression of sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) in a pig model with post-resuscitation myocardial dysfunction.</p><p><b>METHODS</b>Ventricular fibrillation (VF) was electrically induced in Wu-zhi-shan miniature pigs. After 8 min of untreated VF and 2 min of cardiopulmonary resuscitation (CPR), all animals were randomly administered a bolus injection of saline placebo (SA group, n=10), SFI (0.8 mg/kg, SFI group, n=10) or epinephrine (20 μg/kg, EPI group, n=10). After 4 min of CPR, a 100-J shock was delivered. If the defibrillation attempt failed to attain restoration of spontaneous circulation (ROSC), manual chest compressions were rapidly resumed for a further 2 min followed by a second defibrillation attempt. Hemodynamic variables were recorded, and plasma concentrations of catecholamines were measured. Adenylate cyclase (AC), cyclic adenosine monophosphate (cAMP) and the expressions of β1-adrenoceptor (AR) and SERCA 2a were determined.</p><p><b>RESULTS</b>Cardiac output, left ventricular dp/dtmax and negative dp/dtmax were significantly higher in the SFI group than in the SA and EPI groups at 4 and 6 h after ROSC. The expression of β1-AR and SERCA2a at 24 h after ROSC were significantly higher in the SFI group than in the SA and EPI groups (P<0.05 or P<0.01).</p><p><b>CONCLUSIONS</b>The administration of epinephrine during CPR decreased the expression of SERCA2a and aggravated postresuscitation myocardial function (P<0.01). SFI attenuated post-resuscitation myocardial dysfunction, and the mechanism might be related to the up-regulation of SERCA2a expression.</p>
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Animals , Male , Adenylyl Cyclases , Metabolism , Blotting, Western , Cardiac Output , Cardiopulmonary Resuscitation , Cyclic AMP , Metabolism , Dopamine , Metabolism , Drugs, Chinese Herbal , Pharmacology , Enzyme-Linked Immunosorbent Assay , Epinephrine , Blood , Heart Ventricles , Metabolism , Hemodynamics , Injections , Myocardium , Pathology , Norepinephrine , Blood , Receptors, Adrenergic, beta-1 , Metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Metabolism , Swine , Swine, Miniature , Up-RegulationABSTRACT
The objective of present work is to investigate metabolic alterations associated with heart failure, particularly one of its manifestations, a sustained hypocalcemia that causes hemodynamic changes contributed to subsequent myocardial injury. Comparative study was carried out using experimental models of pancreatic necrosis (PN) and crush syndrome (CS) accompanied by cardiac damage down to myocardial infarction. Study design: Wistar adult male rats randomly divided into groups (n=12/group). The controls are healthy intact animals. The pancreatic necrosis (PN) and crush syndrome (CS) groups were then randomly subdivided: PN group- into 3, 24 and 72 h groups concerning hemorrhage, early and late pancreatic necrosis respectively; CS group – into 2, 4, 24, and 48 h decompression stages. The rats were sacrificed to analyze spectra and calcium-binding properties of the membrane proteins isolated from the cardiomyocyte sarcoplasmic reticulum (SR). Development of pathological changes in the heart and pancreas were also monitored. Place and Duration of Study: Department of Pathological Biochemistry and Radioisotope Methods, H. Buniatyan Institute of Biochemistry of Natl. Acad. Sci (NAS), Republic of Armenia (RA). Experiments conducted between May 2011 and October 2013. Methodology: To study pathogenesis of hypocalcemia underlying myocardial damage a translocation of radioactive 45CaCI2 into cardiomyocytes and its intracellular distribution was examined. Binding of 45Ca2+ to the SR membrane proteins was measured after proteins separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and radioactivity from the gel plates was counted by a gas-flow meter Berthold–II. Isoelectric focusing of the protein isolated from the SR of cardiomyocyte was performed. Results: Statistically significant changes in mean radio labeled calcium incorporation into a total protein fraction of the cardiomyocyte SR from control (13682±271) were determined by3h of PN (23055±168, P<.001), 24 h of PN (22876±240, P<.01), and by 72 h (3851±271,P<.01), P vs. control. Similarly, these parameters were detected following CS by 2h decompression (24179±225, P<.01), 4-24 hours decompression (21666±124, P<.001) and 48 h decompression (2941±189, P<.001), P vs. control. We demonstrate that drop in the binding calcium level observed was partially due to impaired affinity to calcium of the cardiomyocyte SR calcium-binding proteins during development of both PN and CS despite a simultaneous manifestation of affinity to calcium of the SR 32-kDa protein. Conclusion: In the present study we have clearly shown that both experimental acute pancreatitis and long-term compression injury may cause similar changes,а loss the calcium-binding properties of the cardiomyocyte proteins, particularly those of SR serving as a main calcium depot under physiological circumstances and appear to be involved in common cellular and molecular mechanisms of myocardial injury contributing to hypocalcemia. Simultaneously, both PN and/or CS cause similar manifestations of the new calcium-binding properties of the cardiomyocyte SR 32-kDa membrane protein, and mirrored dynamic changes in its calcium affinity suggested by Scatchard plot analysis indicating a common mechanism that would be a transient attempt of certain heart cells to compensate hypocalcemia, and thus emerge from an otherwise pathological outcome. Thus, the above mentioned changes could be used to identify patients at high risk of cardiovascular disease in different pathologies.
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In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.61±0.26 vs NC=4.79±0.19 per 100 µm/s) and decreased its amplitude (HC=0.260±0.08 vs NC=0.324±0.10 ΔF/F0), full width at half-maximum amplitude (HC=1.05±0.08 vs NC=1.26±0.01 µm), total duration (HC=11.51±0.12 vs NC=14.97±0.24 ms), time to peak (HC=4.84±0.06 vs NC=6.31±0.14 ms), and time constant of decay (HC=8.68±0.12 vs NC=10.21±0.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.26±0.19 µm/s, amplitude=0.282±0.10 ΔF/F0, full width at half-maximum amplitude=1.14±0.01 µm, total duration=13.34±0.17 ms, time to peak=5.43±0.08 ms, and time constant of decay=9.43±0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.
Subject(s)
Animals , Male , Calcium/physiology , Heart Ventricles/metabolism , Hypertension/therapy , Motor Activity/physiology , Myocytes, Cardiac/metabolism , Physical Conditioning, Animal/methods , Calcium Signaling/physiology , Exercise Test/methods , Heart Ventricles/cytology , Hypertension/metabolism , Rats, Inbred SHR , Rats, Wistar , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolism , Tacrolimus Binding Proteins/genetics , Tacrolimus Binding Proteins/metabolismABSTRACT
Objective To investigate the relationship between sarcoplasmic reticulum Ca2+modulation proteins and postresuscitation myocardial dysfunction. Methods Thirty-eight SPF male Sprague-Dawley (SD) rats were randomly divided into control group(n=12)and cardiac arrest(CA)group(n=26). CA was induced by intravenous bolus of potassium chloride(40μg/g),and cardiopulmonary resuscitation(CPR)was conducted 8 minutes later. No CA was induced in control group except catheter placement for monitoring cardiopulmonary parameters after anesthesia. Invasive hemodynamic parameters were monitored for 1 hour after CPR. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after restoration of spontaneous circulation (ROSC),and sarcoplasmic reticulum Ca2+ ATPase (SERCA2a),phosphorylated phospholamban (p-PLB) and rynodine receptor(RyR)were determined by Western Blot. Results ROSC rate of CA group was 92.3%(24/26),and mean recovery time was (68 ±39)seconds. Cardiac function was significantly impaired in CA group at 1 hour after resuscitation, and ejection fraction, fraction shortening (FS), the maximal rate of left ventricular pressure increase/decline (±dp/dt max)were significantly decreased compared with those in control group 〔ejection fraction:0.548±0.060 vs. 0.809±0.043,F=71.692,P=0.000;FS:(34.4±4.4)%vs. (46.0±3.5)%,F=55.443,P=0.000;+dp/dt max(mmHg/s):4 718±743 vs. 7 098±394,P0.05). Conclusions The impairment of the p-PLB is closely related to postresuscitation myocardial dysfunction.
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Objective: To investigate the effect of diabetes on the intensity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a)-SUMOylation and SERCA2a activity of myocardium in experimental rats. Methods: The 8 weeks old SD rats were divided into 2 groups, Diabetic group, with diet-induced type 2 diabetic rats and Control group, with normal rats. The systolic and diastolic cardiac functions were evaluated by echocardiography and left ventricular pressure measurement. The intensity of SERCA2a-SUMOylation was examined by co-immunoprecipitation and SUMOylation kit. Results: Compared with Control group, Diabetic group had decreased systolic and diastolic cardiac functions, especially for diastolic function;decreased SERCA2a protein expression and intensity of SUMOylation;decreased SUMOylation E2 (Ubc9 ) protein expression. The protein levels of SUMO1, SAE1 and SAE2 were similar between 2 groups. Conclusion: The intensity of SERCA2a-SUMOylation and Ubc9 decreased in diabetic myocardium which implies that SERCA2a-SUMOylation and Ubc9 were closely related to the damage of diabetic myocardium in experimental rats.
ABSTRACT
Ca2+ sparks represent synchronous opening of the ryanodine receptor (RyR) Ca2+ release channels located at the sarcoplasmic reticulum (SR) membrane. Whereas a quantal nature of Ca2+ sparks has been defined in cardiac muscle, the regulation of Ca2+ sparks in skeletal muscle has not been well-studied. Osmotic-stress applied to an intact skeletal muscle fiber can produce brief Ca2+ sparks and prolonged Ca2+ burst events. Here, we show that termination of Ca2+ bursts occurs in a step wise and quantal manner. Ca2+ burst events display kinetic features that are consistent with the involvement of both stochastic attrition and coordinated closure of RyR channels in the termination of SR Ca2+ release. Elemental unitary transition steps could be defined with a mean DF/F0 of ~0.28, corresponding to the gating of 1-2 RyR channels. Moreover, the amplitude of the elemental transition steps declines at the later stage of the burst event. In tandem Ca2+ burst events where two Ca2+ bursts occur at the same position within a fiber in rapid succession, the trailing event is consistently of lower amplitude than the initial event. These two complementary results suggest that SR Ca2+ release may be associated with local depletion of SR Ca2+ stores in mammalian skeletal muscle.
Subject(s)
Animals , Calcium/metabolism , Calcium Signaling , Male , Mammals , Mice , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscle, Skeletal/metabolism , Osmotic Pressure , Time FactorsABSTRACT
ATP2A2 is a member of ATP2As family, it encodes SERCA2b, a sarco (endo) plasmic reticulum calcium transport ATPases (SERCAs). As the main function of SERCA2b is to transport calcium from the cytosol to the sarco(endo) plasmic reticulum, it plays a vital role in numerous calcium-related signaling pathways involving control of tumor growth, differentiation, angiogenesis, metastasis and apoptosis. Recent studies have identified the accurate change of ATP2A2 expression in some tumors, which makes the first step in investigating how ATP2A2 participates in tumorigenesis and whether it can be taken as a new tumor marker and target for treatment. Here we made a comprehensive review on the role of ATP2A2 in tumorigenesis, and it is believed that the abnormal expression of ATP2A2 can damage the calcium homeostasis between cytosol and sarco (endo) plasmic reticulum, accelerating malignant proliferation, migration and angiogenesis of the tumor. Moreover, we also discussed the prospect of research and application of ATP2A2.