BACKGROUND: Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is a key protein that maintains myocardial Ca 2+ homeostasis. The present study aimed to investigate the mechanism underlying the SERCA2a-SUMOylation (small ubiquitin-like modifier) process after ischemia/reperfusion injury (I/RI) in vitro and in vivo . METHODS: Calcium transient and systolic/diastolic function of cardiomyocytes isolated from Serca2a knockout (KO) and wild-type mice with I/RI were compared. SUMO-relevant protein expression and localization were detected by quantitative real-time PCR (RT-qPCR), Western blotting, and immunofluorescence in vitro and in vivo . Serca2a-SUMOylation, infarct size, and cardiac function of Senp1 or Senp2 overexpressed/suppressed adenovirus infected cardiomyocytes, were detected by immunoprecipitation, triphenyltetrazolium chloride (TTC)-Evans blue staining, and echocardiography respectively. RESULTS: The results showed that the changes of Fura-2 fluorescence intensity and contraction amplitude of cardiomyocytes decreased in the I/RI groups and were further reduced in the Serca2a KO + I/RI groups. Senp1 and Senp2 messenger ribose nucleic acid (mRNA) and protein expression levels in vivo and in cardiomyocytes were highest at 6 h and declined at 12 h after I/RI. However, the highest levels in HL-1 cells were recorded at 12 h. Senp2 expression increased in the cytoplasm, unlike that of Senp1. Inhibition of Senp2 protein reversed the I/RI-induced Serca2a-SUMOylation decline, reduced the infarction area, and improved cardiac function, while inhibition of Senp1 protein could not restore the above indicators. CONCLUSION I/RI activated Senp1 and Senp2 protein expression, which promoted Serca2a-deSUMOylation, while inhibition of Senp2 expression reversed Serca2a-SUMOylation and improved cardiac function.
Animals ; Mice ; Calcium/metabolism* ; Cysteine Endopeptidases/metabolism* ; Myocardial Reperfusion Injury/metabolism* ; Myocardium/metabolism* ; Myocytes, Cardiac/metabolism* ; Proteins/metabolism* ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics*

OBJECTIVE: To explore the molecular basis for a pedigree affected with Darier-White disease. METHODS: Genomic DNA was isolated from 3 patients and 1 unaffected member from the pedigree, as well as 80 healthy controls. Targeted sequence capture and next-generation sequencing were used to screen mutations of skin disease-related genes. Candidate mutations were verified by Sanger sequencing, and co-segregation analysis was carried out to confirm the pathogenicity of mutation. Conservation analysis and protein structure and function were also predicted with Bioinformatic tools. RESULTS: A heterozygous mutation c.2246G>T (p.G749V) was identified in exon 15 of ATP2A2 gene in all 3 patients from the pedigree, but not in the unaffected member or 80 healthy controls. The corresponding amino acid was highly conserved, and mutation of which can lead to structural and functional changes of the protein. CONCLUSION The c.2246G>T missense mutation of the ATP2A2 gene probably underlies the Darier-White disease in this pedigree by causing damages to the structure and function of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).
Darier Disease ; genetics ; Heterozygote ; Humans ; Mutation, Missense ; Pedigree ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics

OBJECTIVETo detect mutations of ATP2A2 gene in a pedigree and a sporadic case with Darier disease (DD) and explore the underlying molecular mechanism.

METHODSClinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from blood samples of four members from the pedigree (including three patients and one healthy member), the sporadic case and 100 healthy controls. PCR was performed to amplify all coding exons of the ATP2A2 gene. And the products were directly sequenced to detect mutations.

RESULTSA missense mutation c.1484C>T (p.S495L) in exon 12 was detected in all patients of the pedigree. For the sporadic case, a novel splicing mutation c.325-2A>G was detected at the junction between intron 4 and exon 5. The same mutations were not found in the 100 healthy controls.

CONCLUSIONMutations of the ATP2A2 gene may lead to the occurrence of DD in both familial and sporadic cases with DD.

Aged ; Alternative Splicing ; genetics ; Base Sequence ; Child ; DNA Mutational Analysis ; Darier Disease ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Male ; Mutation, Missense ; Pedigree ; Point Mutation ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics

OBJECTIVETo 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.

METHODSSix 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.

RESULTSThe 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.

CONCLUSIONrAAV-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.

Animals ; Calcium-Binding Proteins ; genetics ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; Male ; Phosphorylation ; RNA, Antisense ; administration & dosage ; Rats ; Rats, Wistar ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Ventricular Function, Left ; drug effects

OBJECTIVEChronic myocardial ischemia (CMI) has become the most important cause of heart failure (HF) all over the world. The aim of the current study was to investigate the effects of Sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) gene transfer on cardiac function and endoplasmic reticulum stress (ERS) associated myocardial apoptosis in a minipig HF animal model induced by CMI.

METHODSHF was induced in minipigs by implantation of ameroid constrictor in the initial segment of left anterior descending (LAD) branch of coronary artery. After confirmation of myocardial perfusion defects and cardiac function impairment by myocardial perfusion imaging and echocardiography, animals were divided into 4 groups (n = 4 each): HF group, HF + enhanced green fluorescent protein (EGFP) group, HF + SERCA2a group, and shamed animals as control group. A total amount of 1 × 10(12) v.g. of rAAV1-EGFP or rAAV1-SERCA2a were injected intramyocardially to each animal of HF + EGFP and HF + SERCA2a groups. Sixty days after gene transfer, protein level and activity of SERCA2a were examined, cardiac functions and changes of serum inflammatory and neuro-hormonal factors were determined. Apoptotic index of the ischemic myocardium, protein levels of ER stress marker glucose regulated protein 78 (GRP 78) and ER stress specific apoptotic marker caspase-12 were also assayed.

RESULTSAt the study end, echocardiographic and hemo dynamic measurements indicated a significant improvement of both cardiac systolic and diastolic function in HF + SERCA2a group compared with HF/HF + EGFP groups [LVEF (60.2 ± 8.6)% vs (44.2 ± 7.1)% and (46.8 ± 6.7)%, Ev/Av 1.28 ± 0.24 vs 0.77 ± 0.17 and 0.80 ± 0.21, +dp/dt(max) (2713.9 ± 434.0) mm Hg/s (1 mm Hg = 0.133 kPa) vs (1892.3 ± 434.2) mm Hg/s and (1931.2 ± 397.4) mm Hg/s, -dp/dt(max) (1422.1 ± 334.4) mm Hg/s vs (848.3 ± 308.3) mm Hg/s and (849.5 ± 278.3) mm Hg/s, P < 0.05], along with increase in both SERCA2a protein level (1.13 ± 0.26 vs 0.73 ± 0.17 and 0.64 ± 0.18, P < 0.05) and activity [(16.2 ± 5.5) IU/ml vs (7.9 ± 3.1) IU/ml and (7.5 ± 2.8) IU/ml, P < 0.05] compared with HF/HF + EGFP groups. Serum concentrations of inflammatory factor tumor necrotic factor α [(382.3 ± 114.4) ng/L vs (732.3 ± 201.4) ng/L and (689.8 ± 192.5) ng/L, P < 0.05], neural-hormonal factors brain natriuretic peptide [(142.6 ± 45.3) ng/L vs (422.3 ± 113.6) ng/L and (393.7 ± 103.3) ng/L, P < 0.01], endothelin-1 [(111.4 ± 37.5) ng/L vs (193.5 ± 54.3) ng/L and (201.0 ± 72.1) ng/L, P < 0.05] and angiotensin II [(189.7 ± 65.2) µg/L vs (538.3 ± 135.2) µg/L and (525.5 ± 144.1) µg/L, P < 0.01] were also significantly decreased in HF + SERCA2a group compared with HF/HF + EGFP groups. The apoptotic index [(12.71 ± 4.11)% vs (23.22 ± 7.23)% and (24.31 ± 6.38)%, P < 0.05], protein levels of GRP 78 (1.27 ± 0.33 vs 3.23 ± 1.14 and 4.18 ± 1.13, P < 0.05) and protein level ratios of cleaved caspase-12 to total caspase-12 [(4.62 ± 1.93)% vs (9.71 ± 2.70)% and (10.14 ± 2.81)%, P < 0.05] were also significantly reduced in the ischemic myocardium of HF + SERCA2a group compared with the HF/HF + EGFP groups.

CONCLUSIONOverexpression of SERCA2a significantly improved cardiac systolic and diastolic function in this HF model partly through attenuation of ER stress related myocardial apoptosis, suggesting its therapeutic potential for CMI related heart failure.

Animals ; Chronic Disease ; Disease Models, Animal ; Genetic Therapy ; Heart Failure ; therapy ; Myocardial Ischemia ; therapy ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; Swine ; Swine, Miniature

The present study is aimed to study the effect of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) gene transfer on the contractile function of isolated cardiomyocytes of canines. The cardiomyocytes were isolated with collagenases. The isolated cardiac cells were divided into untransfected group, empty vector group and SERCA2a-transfected group. Recombinant adenovirus vector carrying enhanced green fluorescent protein gene was used for SERCA2a gene delivery. The expression of SERCA2a protein in cardiomyocytes was determined by Western blot. Contractile function of cardiomyocytes was measured with motion edge-detection system of single cell at 48 h after transfection. The results showed, compared with untransfected group, SERCA2a protein level, percentage of peak contraction amplitude under normal condition, percentages of peak contraction amplitude under Ca(2+) or isoproterenol stimulation, time-to-peak contraction (TTP) and time-to-50% relaxation (R50) in SERCA2a-transfected group all increased significantly. While all the above indices in empty vector group did not show any differences with those in untransfected group. These results suggest that the overexpression of SERCA2a by gene transfer may enhance the contraction function of canine myocardial cells.
Adenoviridae ; genetics ; metabolism ; Animals ; Dogs ; Male ; Myocardial Contraction ; drug effects ; physiology ; Myocytes, Cardiac ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism ; Transfection

BACKGROUNDHeart failure (HF) is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral (rAAV) gene transfer of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) can be effective in treating rats with chronic heart failure (CHF). The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model.

METHODSHF was induced in beagles by rapid right ventricular pacing (230 beats/min) for 30 days. A reduced rate ventricular pacing (180 beats/min) was continued for another 30 days. The beagles were assigned to four groups: (a) control group (n = 4); (b) HF group (n = 4); (c) enhanced green fluorescent protein group (n = 4); and (d) SERCA2a group (n = 4). rAAV1-EGFP (1 x 10(12) microg) and rAAV1-SERCA2a (1 x 10(12) microg) were delivered intramyocardially. SERCA2a expression was assessed by Western blotting and immunohistochemistry.

RESULTSFollowing 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group (P < 0.05). Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure (+dp/dt(max)), and the maximal rate of decline of left ventricular pressure (-dp/dt(max)) (P < 0.05). Left ventricular end-diastole pressure significantly decreased (P < 0.05). The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group (P < 0.05).

CONCLUSIONSIntramyocardial injection of rAAV1-SERCA2a can improve the cardiac function in beagles induced with HF. We expect further studies on SERCA2a's long-term safety, efficacy, dosage and the optimization before using it in humans with HF.

Animals ; Blotting, Western ; Disease Models, Animal ; Dogs ; Echocardiography ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; metabolism ; Heart ; physiology ; Heart Failure ; therapy ; Hemodynamics ; Immunohistochemistry ; Myocardium ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; physiology

OBJECTIVETo investigate the effect of astragalus (As) on calcium accumulation and SERCA2a gene expression in left ventricular tissues in rats with pressure overload-induced cardiac hypertrophy.

METHODcardiac hypertrophy was induced by clipping the abdominal aorta in rats. Male SD rats were allocated to six groups: sham-operrated (Sham), aortic stenosis (Model), model +As-L (5 g x kg(-1) x d(-1)), model+As-M (10 g x kg(-1) x d(-1)), model+As-H (20 g x kg(-1) x d(-1)) and model + captopril (0.05 mg x kg(-1) x d(-1), a positive control). The drugs were administered orally from the 13 th week after surgery. Rats were examined after 12 week treatment with drugs. The cardiac hypertrophy was evaluated by left ventricular mass index (LVMI, left ventricular weight/ body weight). The calcium content in left ventricular tissue was measured by atomic absorption spectrometry. SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and protein, respectively.

RESULTThe increase of LVMI was dose-dependently lessened by As (P < 0.01, P < 0.001). The effect of As-H was similar to that of Captopril. As markedly attenuated calcium accumulation in myocardial tissure (P < 0.01). RT-PCR and Western blot results demonstrated that SERCA2a gene expressions were downregulated (P < 0.05) significantly in model group compared with sham group. As-H upregulated SERCA2a gene expressions (P < 0.05), whereas Captopril had no effect on that.

CONCLUSIONThe inhibition of As on left ventricular hypertrophy induced by pressure overload in rats may partly contribute to its attenuation of calcium accumulation and up-regulation of SERCA2a gene expressions in left ventricular tissues.

Animals ; Astragalus Plant ; chemistry ; Blotting, Western ; Calcium ; metabolism ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Gene Expression Regulation ; drug effects ; Heart ; drug effects ; Hypertrophy, Left Ventricular ; metabolism ; physiopathology ; Male ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism

PURPOSE: The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca(2+) signaling. However, whether the changes in Ca(2+) signaling and Ca(2+) signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2(+/-) mouse parotid gland acinar cells, Ca(2+) signaling, expression levels of Ca(2+) signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2(+/-) mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca(2+) ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP(3)Rs), but the localization and activities of IP3Rs were not altered. In SERCA2(+/-) mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca(2+) signaling proteins in the parotid gland acini, however, overall Ca(2+) signaling is unchanged.
Amylases/metabolism ; Animals ; Blotting, Western ; Calcium/metabolism ; Calcium Signaling/drug effects/genetics/*physiology ; Carbachol/pharmacology ; Immunohistochemistry ; Inositol 1,4,5-Trisphosphate Receptors/metabolism ; Mice ; Mice, Knockout ; Parotid Gland/*metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics/*metabolism ; Signal Transduction/drug effects/genetics/physiology

AIMTo investigate the changes in the expression of four kinds of calcium regulatory proteins mRNA on the isolated ischemia/ reperfusion (IR) hearts.

METHODSThe rat hearts were divided into two groups: control group and IR group which received 45 min exposure to Krebs-Henseleit solution after 15 min zero-flow global ischemia. The indexes of left ventricular function, such as LVDP, +dp/dt(max), -dp/dt(max), and an arrhythmia scoring system were compared between the two groups. The messenger ribonucleic acid (mRNA) amount of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA), phospholamban (PLB), inositol 1,4,5-trisphosphate receptor2 (IP3R2) and ryanodine receptor2 (RyR2) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and normalized to the mRNA levels of beta-actin.

RESULTSIn the IR group, LVDP, +dp/dt(max) and -dp/dt(min) of the isolated hearts were depressed and the high rate of arrhythmias occurred during reperfusion. The levels of SERCA, IP3R2, RyR2 mRNA were lower in the IR isolated hearts group than those in the control group, while there was no difference in the level of phospholamban.

CONCLUSIONThese data suggest that myocardial ischemia/reperfusion can induce the depression of cardiac performance and an increased risk of arrhythmias, concomitant with the decrease in SERCA, IP3R2, RyR2 mRNA steady state levels.

Animals ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism