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OBJECTIVES@#To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism.@*METHODS@#FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-InTM T-RExTM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation.@*RESULTS@#After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine.@*CONCLUSIONS@#This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.
Subject(s)
Humans , Calcium-Binding Proteins/metabolism , Cell Line , Myocardium/metabolism , PhosphorylationABSTRACT
Objective:To investigate the role of Na+/Ca2+ exchanger (NCX) in myocardial ischemiareperfusion injury and the underlying mechanisms.Methods:Forty Sprague-Dawley rats were divided into 4 groups randomly:a control group,a KBR7943 group,an ischemia-reperfusion group (IR group),and an IR plus KB-R7943 group (KB-R7943+IR group).Isolated Sprague Dawley male rat hearts underwent Langendorffperfusion.The ratio of left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP),the infarct size of myocardium,and the lactate dehydrogenase (LDH) activity in the coronary flow was determined.HE staining was used to assess the change of myocardial morphology.Western blot was used to determine the levels of cleaved caspase-3,cytochrome c and the phosphorylation of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and the Thr17 site ofphospholamban.Results:Compared with the control group,IR group significantly induced an enlarged infarct size,reduction of the ratio of LVDP,up-regulation of cytochrome c,cleaved caspase-3,p-CaMKⅡ and p-phospholamban,and increased in the activity of LDH,the level of LVEDP (P＜0.01) and the disordered myocardial morphology.These effects were significantly attenuated in the presence of KB-R7943 treatment (10 μmol/L).Conclusion:NCX mediates myocardial ischemia-reperfusion-induced cell apoptosis and necrosis through activation of CaMKⅡ.
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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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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 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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<p><b>OBJECTIVE</b>To study the effect of phospholamban antisense RNA (asPLB) on sarcoplasmic reticulum Ca2+-ATPase activity and cardiac function in rats with diabetes mellitus (DM) mediated by recombinant adeno-associated virus (rAAV) vector.</p><p><b>METHODS</b>Six weeks after the induction of DM by streptozotocin injected intraperitoneally, the rats were divided into three groups, namely: DM-rAAV-asPLB group, DM-saline group and DM group (control group). The rats in the DM-rAAV-asPLB group were intramyocardially injected with rAAV-asPLB, the rats in the DM-saline group were injected with saline, and those in the control group did not receive any treatment. Six weeks after gene transfer, the expressions of PLB protein and PLB phosphorylation were detected by Western-blot, while the activity of sarcoplasmic reticulum (SR) Ca2+-ATPase and left ventricular function were measured.</p><p><b>RESULTS</b>The PLB protein expression level was significantly higher whereas the PLB phosphorylation, SR Ca2+-ATPase activity and left ventricular function were significantly lower in the DM-saline group than in the control group. No significant difference was found in PLB protein expression level, PLB phosphorylation or SR Ca2+-ATPase activity between the DM-rAAV-asPLB group and the control group. The left ventricular function in the DM-rAAV-asPLB group was poorer than in the control group and was better than in the DM-saline group.</p><p><b>CONCLUSION</b>rAAV-asPLB can down-regulate PLB protein expression and up-regulate PLB phosphorylation and SR Ca2+-ATPase activity, thus contributing to the improvement of in vivo left ventricular function.</p>
Subject(s)
Animals , Male , Rats , Calcium-Binding Proteins , Genetics , Metabolism , Diabetes Mellitus, Experimental , Metabolism , Phosphorylation , RNA, Antisense , Rats, Wistar , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Metabolism , Ventricular Function, LeftABSTRACT
It is discovered that dilated cardiomyopathy (DCM) is not only affected by environmental factors, but also has a significant genetic basis. Phospholamban ( PLN ), one of these related genes, is a prominent regulator of myocardial contractility, It is an important regulatory factor for heart muscle contraction. It can regulate the activity of sarcoplasmic reticulum Ca2+ ATPase ( SERCA2a), and inhibit SERCA2a activity through restricting the Ca2+ flow in myocardial cells. The activity of SERCA2a can be improved by lowering the expression of PLNmRNA, and the affinity of SERCA2a is strengthened, so that the heart's activity is improved.
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Objective To explore whether ultrasound-mediated microbubbles destruction could enhance anti-sense RNA transfection and expression. Methods Phospholamban antisense RNA eukaryon vector PcDNA 4. 1-asPLB was successfully constructed and it was transfected into cardiac myocytes by various methods including calcium phosphate precipitation, ultrasound exposure and ultrasound-mediated microbubbles destruction. The expression of PLB and sarcoplasmic retculum Ca2+ ATPase (SERCA2a) in cardiac myocytes was tested by RT-PCR and western blot. Results The transfection and expression of PcDNA 4. 1-asPLB increased significantly in cells treated with ultrasound-mediated microbubbles destruction compared to other transfer groups( P ＜0.05). The expression of PLB was inhibited specifically after cardiac myocytes were transfected with PcDNA 4. 1-asPLB. There was no change of PLB expression after cardiac myocytes transfected with PcDNA 4. 1 ( P ＜0.05). Though the expression of SERCA2a never exhibited any changes after PcDNA 4. 1-asPLB transfection, the PLB/SERCA2a ratio decreased markedly. Conclusions As a highly effective antisense RNA transfer method, ultrasound-mediated microbubbles destruction can enhance the transfection and expression of the PcDNA 4. 1-asPLB significantly. The PcDNA4. 1-asPLB transfection inhibits the expression of PLB and result in decrease of PLB/SERCA2a ratio in cardiac myocytes.
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We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50 percent diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50 percent food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.
Subject(s)
Animals , Male , Rats , Calcium-Binding Proteins/metabolism , Down-Regulation/physiology , Food Deprivation/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Calcium-Binding Proteins/genetics , Down-Regulation/genetics , Rats, Inbred WKY , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/geneticsABSTRACT
A growing body of evidence, including studies using genetically engineered mouse models, has shown that Ca2+ cycling and Ca2+ -dependent signaling pathways play a pivotal role in cardiac hypertrophy and heart failure. In addition, recent studies identified that mutations of the genes encoding sarcoplasmic reticulum (SR) proteins cause human cardiomyopathies and lethal ventricular arrhythmias. The regulation of Ca2+ homeostasis via the SR proteins may have potential therapeutic value for heart diseases such as cardiomyopathy, heart failure and arrhythmias.
Subject(s)
Animals , Humans , Animals, Genetically Modified , Arrhythmias, Cardiac/genetics , Calcium/metabolism , Calcium Channels/genetics , Calcium-Binding Proteins/genetics , Cardiac Output, Low/genetics , Cardiomyopathies/genetics , Heart Diseases/etiology , Mutation/genetics , Sarcoplasmic Reticulum/metabolismABSTRACT
In the present study, the postnatal developmental changes in the expressional levels of cardiac sarcoplasmic reticulum (SR) Ca2+ regulatory proteins, i.e. Ca2+-ATPase, phospholamban, and Ca2+ release channel, were investigated. Both SR Ca2+-ATPase and phospholamban mRNA levels were about 35% of adult levels at birth and gradually increased to adult levels. Protein levels of both SR Ca2+-ATPase and phospholamban, which were measured by quantitative immunoblotting, were closely correlated with the mRNA levels. The initial rates of Ca2+ uptake at birth were about 40% of adult rates and also increased gradually during the myocardial development. Consequently, the relative phospholamban/Ca2+-ATPase ratio was 1 in developmental hearts. Ca2+ release channel (ryanodine receptor) mRNA was about 50-60% at birth and increased gradually to adult level throughout the postnatal rat heart development. 3[H]ryanodine binding increased gradually during postnatal myocardial development, which was closely correlated with ryanodine mRNA expression levels during the development except the ryanodine mRNA level at birth. These findings indicate that cardiac SR Ca2+- ATPase, phospholamban, and Ca2+ release channel are expressed coordinately, which may be necessary for intracellular Ca2+ regulation during the rat heart development.
Subject(s)
Adult , Animals , Humans , Rats , Adenosine Triphosphatases , Heart , Immunoblotting , Parturition , RNA, Messenger , Ryanodine , Sarcoplasmic ReticulumABSTRACT
AIM:To investigate the alterations of phospholamban(PLB)expression and cardiac sarcoplasmic reticulum(SR)Ca 2+-ATPase activity,and the change of cardiac function in rats with diabetes mellitus(DM).METHODS:The diabetes mellitus in male Wistar rats was induced by intraperitoneal injection of streptozotocin.The levels of PLB mRNA and PLB protein,the activity of SR Ca 2+-ATPase and the left ventricular hemodynamics parameters were measured 4 weeks,6 weeks and 8 weeks after DM was induced in rats,while the normal rats served as control group.RESULTS:There was no significant difference in PLB mRNA level and protein level between 4-week-DM rats and normal control rats.6-week-DM rats and 8-week-DM rats had markedly increased PLB mRNA and protein level compared with normal control rats.SR Ca 2+-ATPase activity was not significantly changed in 4-week-DM rats compared with normal control rats,and was markedly depressed in 6-week-DM rats and 8-week-DM rats.LVSP,LVEDP and ?dp/dt max were not significantly changed in 4-week-DM rats compared with normal control rats.In 6-week-DM rats and 8-week-DM rats,LVSP and ?dp/dt max were decreased,LVEDP was increased compared with normal control rats.CONCLUSION:The elevated levels of PLB mRNA and PLB protein contribute to SR Ca 2+-ATPase activity reduction,which leads to cardiac dysfunction in DM rats.
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In the present study, the underlying mechanisms for diabetic functional derangement and insulin effect on diabetic cardiomyopathy were investigated with respect to sarcoplasmic reticulum (SR) Ca2+-ATPase and phospholamban at the transcriptional and translational levels. The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased in streptozotocin-induced diabetic rat cardiac SR, however, even minimal amount of insulin could reverse both parameters. Levels of both mRNA and protein of phospholamban were significantly increased in diabetic rat hearts, whereas the mRNA and protein levels of SR Ca2+-ATPase were significantly decreased. In case of phospholamban, insulin treatment reverses these parameters to normal levels. Minimal amount of insulin could reverse the protein levels; however, it could not reverse the mRNA level of SR Ca2+-ATPase at all. Thus, the decreased SR Ca2+ uptake appear to be largely attributed to the decreased SR Ca2+-ATPase level, which is further impaired due to the inhibition by the increased level of phospholamban. These results indicate that insulin is involved in the control of intracellular Ca2+ in the cardiomyocyte through multiple target proteins via multiple mechanisms for the decrease in the mRNA for both SR Ca2+-ATPase and phospholamban which are unknown and needs further study.
Subject(s)
Animals , Rats , Diabetic Cardiomyopathies , Heart , Insulin , Myocytes, Cardiac , RNA, Messenger , Sarcoplasmic ReticulumABSTRACT
AIM: To observe the changes of sarcoplasmic reticulum Ca~(2+)-ATPase (SERCA), phospholamban (PLB) during heart failure after acute myocardial infarction (AMI) in rats and the effect of carvedilol. METHODS: Rats were randomly assigned to normal control group, sham-operation group, AMI group and carvedilol (CAR) group. 6 weeks later, in vivo hemodynamic, morphometry and SERCA, PLB mRNA and protein expression of myocytes were measured in all animals. RESULTS: In comparison with sham-operation group, LV end diastolic pressure (LVEDP) and weight of ventricles were increased, while maximal rate of rise and fall (?dp/dt) of LV pressure were decreased in AMI group. After treatment with carvedilol, these parameters were all improved. The mRNA and protein expression of SERCA were downregulated (P0.05). CONCLUSIONS: The changes of SERCA and PLB may be the important mechanism of contractile dysfunction in heart failure after AMI. Carvedilol is effective in preventing LV dysfunction after AMI. The molecular mechanism may be related with normalization of SERCA expression.
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AIM: To investigate the effect of phospholamban antisense RNA (asPLB) on the activity of sarco-endoplasmic reticulum (SR) Ca 2+-ATPase, and the change of intracellular free Ca 2+ concentration ([Ca 2+]i) in rat cardiomyocytes by adeno-associated virus(AAV) vector. METHODS: rAAV-asPLB and rAAV-LacZ were constructed by AAV Helper-Free System. RT-PCR and Western blotting were used to determine the mRNA and protein expression of PLB. The activity of SR Ca 2+-ATPase and the [Ca 2+]i were measured. RESULTS: Compared to controls, the PLB mRNA and protein expression reduced in rat cardiomyocytes transfected with rAAV-asPLB. The activity of Ca 2+-ATPase was increased. In rest state, the level of [Ca 2+]i in rAAV-asPLB transfected group was decreased. The level of [Ca 2+]i was increased when induced by isoproterenol. CONCLUSION: rAAV-asPLB vector disrupts the expression of PLB, enhances the activity of Ca 2+-ATPase, reduces the resting [Ca 2+]i and enhances the isoproterenol-induced [Ca 2+]i.