The ATP2C1 gene mutation in one case of familial benign chronic pemphigus was investigated. One patient was diagnosed as familial benign chronic pemphigus by pathology, ultrastructral examination and clinical features. Genomic DNA was extracted from blood samples. Mutation of ATP2C1 gene was detected by polymerase chain reaction (PCR) and DNA sequencing. The results showed that deletion mutation was detected in ATP2C1 gene in this patient, which was 2374delTTTG. No mutation was found in the family members and normal individuals. It was concluded that the 2374delTTTG mutation in ATP2C1 gene was the specific mutation for the clinical phenotype for this patient and was a de novo mutation.
Calcium-Transporting ATPases/*genetics ; DNA Mutational Analysis ; Pemphigus, Benign Familial/*genetics ; Sequence Deletion

OBJECTIVETo study a Chinese pedigree with Hailey-Hailey disease (HHD) and examine the ATP2C1 gene mutation in this family.

METHODAll exons of ATP2C1 gene were analyzed with polymerase chain reaction and DNA sequencing in all patients of this family and 100 unrelated population-match controls.

RESULTSWe identified a novel heterozygous nucleotide A --> G transition at position 235 - 2 in intron 3 of ATP2C1 gene. This splice site mutation was not found in the healthy members of this pedigree and in the controls.

CONCLUSIONThe splicing mutation can affect the result of transcription and translation, and it is a specific novel mutation of ATP2C1 gene.

Asian Continental Ancestry Group ; Calcium-Transporting ATPases ; genetics ; Humans ; Mutation ; Pedigree ; Pemphigus, Benign Familial ; 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 study a Chinese pedigree with Hailey-Hailey disease (HHD) and identify the ATP2C1 gene mutation in this family.

METHODSAll exons of the ATP2C1 gene were analyzed with polymerase chain reaction and DNA sequencing in all patients of this family and 80 unrelated population-matched controls.

RESULTSWe identified a nonsense mutation 163C to T, resulting in a premature termination codon in ATP2C1 gene. The mutation was not found in normal individuals of the family and controls.

CONCLUSIONThe mutation can affect the result of transcription and translation of ATP2C1 gene, and it is firstly reported in the Chinese pedigree with HHD.

Asian Continental Ancestry Group ; genetics ; Calcium-Transporting ATPases ; genetics ; DNA Mutational Analysis ; Humans ; Pedigree ; Pemphigus, Benign Familial ; genetics

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*

OBJECTIVETo investigate the effects of curcumin on sarcoplasmic reticulum Ca2+-ATPase in heart failure rabbits.

METHODSRabbit heart failure model was made with aortic regurgitation and abdominal aorta constriction and 40 rabbits were randomly divided into 4 groups including: (1) heart failure treated with curcumin; (2) heart failure treated with placebo; (3) healthy control treated with curcumin and (4) healthy control treated with placebo. All rabbits were administrated with curcumin capsules or placebo capsules 100 mg x kg(-1) x d(-1), respectively. All groups were sacrificed after eight weeks. Myocardial ultrastructural organization was detected by transmission electron microscope. RT-PCR and Western blot were used to measure the expression of sarcoplasmic reticulum Ca2+-ATPase in mRNA and protein levels, respectively. Malachite green colorimetric assay was used to evaluate the activity of sarcoplasmic reticulum Ca2+-ATPase.

RESULTSAll detected parameters were similar between control curcumin group and control placebo group. Compared with the control groups (Groups 3 and 4), the heart/body weight ratio was significantly increased in the heart failure-curcumin group (Group 1) and the heart failure-placebo group (Group 2, all P < 0.05), but the ratio was significantly lower in heart failure-curcumin group than in heart failure-placebo group (P < 0.05). The degree of heart failure was decreased by curcumin. Activity and mRNA and protein expression for sarcoplasmic reticulum Ca2+-ATPase were significantly reduced in the heart failure-placebo group and which could be significantly attenuated by curcumin (all P < 0.05).

CONCLUSIONCurcumin could improve cardiac function via upregulating the expression of sarcoplasmic reticulum Ca2+-ATPase in this model.

Animals ; Calcium ; metabolism ; Curcumin ; pharmacology ; Heart Failure ; metabolism ; RNA, Messenger ; genetics ; Rabbits ; Sarcoplasmic Reticulum ; drug effects ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

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

BACKGROUNDChronic heart failure (CHF) is associated with calcium transients and calcium handling proteins. Angiotensin converting enzyme (ACE) inhibitor has been demonstrated to have beneficial effect on CHF. Yet studies addressed to the relationship between ACE inhibitor and calcium transients in CHF are rare. The aim of this study was to investigate the influence of ACE inhibitor (perindopril) on the contractility and calcium transients and calcium handling proteins in ventricular myocytes from rats with experimental heart failure.

METHODSMale Wistar rats were randomized to heart failure group treated with perindopril [CHF-T, 3 mg.kg(-1).d(-1)], heart failure group without treatment (CHF-C) and sham-operated group (PS). Heart failure was induced by abdominal aortic constriction. All groups were further followed up for 12 weeks. Left ventricular myocytes were then isolated. Single cell shortening fraction and [Ca(2+)]i were simultaneously measured by laser scanning confocal microscope under the field stimulation (1.0 Hz). Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate the changes of mRNA and protein of Na(+)-Ca(2+) exchanger (NCX1), sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) and phospholamban (PLB).

RESULTSThe fraction of cell shortening (FS%) and [Ca(2+)]imax (nmol/L) were significantly reduced in group CHF-C compared with group PS (FS%: 7.51 +/- 1.15 vs 13.21 +/- 1.49; [Ca(2+)]i max: 330.85 +/- 50.05 vs 498.16 +/- 14.07; both P < 0.01), and restored at least partially in CHF-T group. In CHF-C group, the left ventricular mRNA of NCX1 and PLB were significantly upregulated in comparing with PS group (RNCX1/beta-Actin: 0.51 +/- 0.12 vs 0.19 +/- 0.06, P < 0.01; RPLB/beta-Actin: 0.26 +/- 0.12 vs 0.20 +/- 0.08, P < 0.05), while SERCA2 mRNA was downregulated (0.48 +/- 0.10 vs 0.80 +/- 0.11, P < 0.01). The mRNA levels of NCX1 and SERCA2 in CHF-T group were between the CHF-C and PS group, and the differences of the latter two groups were significant (all P < 0.05). In CHF-C and CHF-T groups, the protein expression of NCX1 were 1.141 +/- 0.047 and 1.074 +/- 0.081 times of that in PS group respectively (both P < 0.05), and SERCA2 protein levels were 0.803 +/- 0.100 and 0.893 +/- 0.084 times of that in PS group respectively (both P < 0.05). The protein expression of NCX1 and SERCA2 in the CHF-C and CHF-T groups is significantly different (both P < 0.05).

CONCLUSIONACE inhibitor could improve cardiac function of failing heart through directly enhancing the contractility of single cardiomyocyte, and these effects are probably mediated by its roles in preventing the deleterious changes of calcium transients and calcium handling proteins in CHF.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium-Binding Proteins ; genetics ; Calcium-Transporting ATPases ; genetics ; Heart Failure ; drug therapy ; metabolism ; Heart Ventricles ; drug effects ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; Perindopril ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; Sodium-Calcium Exchanger ; genetics

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

OBJECTIVETo assess the effect of RNA interference-mediated gene silencing of plasma membrane-related Ca(2+)-ATPase-1 (PMR1) gene on the insulin secretion in islet beta cells NIT-1 in vitro.

METHODSA small interfering RNA duplex (siPMR1) corresponding to the nucleotides 337-357 of mouse PMR1 cDNA was introduced into NIT-1 cells via liposomes. The gene silencing effect was assessed by RT-PCR, and the total insulin level in the transfected cells was measured by radioimmunoassay.

RESULTSTransfection with siPMR1 resulted in obviously reduced PMR1 expression and increased insulin secretion in NIT-1 cells.

CONCLUSIONThe synthesized siPMR1 can significantly silence the expression of PMR1 and promote the secretion of insulin in the islet cells in vitro, which shed light on further studies of RNAi-based therapy of diabetes.

Animals ; Calcium-Transporting ATPases ; deficiency ; genetics ; Cell Line ; Gene Expression Regulation ; Insulin ; secretion ; Insulin-Secreting Cells ; metabolism ; secretion ; Mice ; RNA Interference ; RNA, Messenger ; genetics ; metabolism