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

Cardiovascular Diseases ; Heart Failure ; Humans ; Protein Processing, Post-Translational ; Sarcoplasmic Reticulum Calcium-Transporting ATPases

OBJECTIVETo investigate the abnormal abundances of calcium regulatory proteins in rabbit myocytes with failing hearts.

METHODSSixteen rabbits were divided into two groups: 8 rabbits with heart failure induced by volume plus pressure overload and 8 sham-operated animals. The hemodynamic parameters and cardiac structure and function were detected via catheterization and echocardiography respectively. L-type calcium channel (LTCC), Ryanodine receptor 2 (RyR2), Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), and Na(+)-Ca(2+) exchanger (NCX) protein abundances were determined by Western blot analysis.

RESULTSThe ratio of left ventricular mass to body weight, heart rate and left ventricular end diastolic pressure in heart failure rabbits were significantly increased compared with sham-operated rabbits (P < 0.01), but their left ventricular shorten fraction [(21.3 +/- 4.00)% vs. (36.5 +/- 1.36)%] and ejection fraction (0.45 +/- 0.07 vs. 0.70 +/- 0.02) were decreased (P < 0.01). In heart failure rabbits, the abundances of LTCC and RyR2 were significantly decreased (R(LTCC/actin): 0.287 +/- 0.029 vs. 0.624 +/- 0.009; R(RyR2/actin): 0.106 +/- 0.001 vs. 0.203 +/- 0.011; P < 0.01), whereas the expressions of SERCA2a and NCX were markedly increased (R(NCX/actin): 0.497 +/- 0.015 vs. 0.221 +/- 0.014; R(SERCA2a/actin): 0.611 +/- 0.036 vs. 0.433 +/- 0.008; P < 0.01).

CONCLUSIONSReductions of LTCC and RyR2 might contribute to risk factors of systolic dysfunction in failing hearts. In early stage of heart failure, upregulated SERCA2a and NCX protein levels may be helpful for maintaining cardiac performance.

Animals ; Calcium ; metabolism ; Calcium-Binding Proteins ; biosynthesis ; Female ; Heart Failure ; metabolism ; Male ; Rabbits ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum ; chemistry ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

Ischemia-reperfusion injury (IRI) has been recognized as a serious problem for therapy of cardiovascular diseases. Calcium regulation appears to be an important issue in the study of IRI. This article reviews calcium regulation in myocardial and vascular IRI, including the calcium overload and calcium sensitivity in IRI. This review is focused on the key players in Ca(2+) handling in IRI, including membrane damage resulting in increase in Ca(2+) influx, reverse-mode of Na(+)-Ca(2+) exchangers leading to increased Ca(2+) entry, the decreased activity of sarcoplasmic reticulum (SR) Ca(2+)-ATPase causing SR Ca(2+) uptake dysfunction, and increased activity of Rho kinase. These key players in Ca(2+) homeostasis will provide promising strategies and potential targets for therapy of cardiovascular IRI.
Animals ; Calcium ; metabolism ; Heart ; physiopathology ; Homeostasis ; Humans ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism

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 aim of the present study is to investigate the differences in cardiac function, and the expression and activity of calcium regulatory proteins between rabbit systolic heart failure (SHF) and diastolic heart failure (DHF) models. New Zealand white rabbits were randomly divided into three groups: sham operation (SO) group, DHF group (receiving abdominal aortic constriction) and SHF group (receiving aortic valve destruction and abdominal aortic constriction). The cardiac function was detected by echocardiographic and hemodynamic assays. The mRNA expression levels of sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) and phospholamban (PLB) were evaluated by RT-PCR. The protein expression levels of SERCA2a, PLB, phosphoserine 16-PLB (pSer-16-PLB) and protein kinase A (PKA) were evaluated by Western blot, and the phosphorylation status of PLB was determined by the ratio of pSer-16-PLB protein level to that of PLB. The activity of SERCA2a was measured through inorganic phosphate. The activity of PKA was measured by gamma-(32)P ATP-binding assays. Compared with SO group, there were significantly increased ventricular wall thickness, raised left ventricular end diastolic pressure (LVEDP), reduced diastolic function in DHF group (P<0.05 or P<0.01), and significantly increased ventricular cavity size and LVEDP, reduced systolic function in SHF group (P<0.05 or P<0.01). The expression levels of SERCA2a in DHF and SHF groups were lower than that in SO group (P<0.05), while the expression and activity of PKA in DHF and SHF groups were higher than that in SO group (P<0.05 or P<0.01), and there was no significant difference between DHF and SHF groups. The expression levels of PLB and pSer-16-PLB as well as the phosphorylation status of PLB and activity of SERCA2a in SHF group were lower than those in DHF and SO groups respectively. Posing a contrast, the phosphorylation status of PLB and activity of SERCA2a in DHF group were higher than that in SO group (P<0.05). These results indicate that the SHF and DHF models were successfully established, and there are some differences in the expression and activity of calcium regulatory proteins between two models.
Animals ; Calcium-Binding Proteins ; metabolism ; Disease Models, Animal ; Heart Failure, Diastolic ; metabolism ; Heart Failure, Systolic ; metabolism ; Rabbits ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

The purpose of the present study was to investigate whether interleukin-2 (IL-2) changes the activity of sarcoplasmic reticulum (SR) Ca(2+) ATPase, sarcolemmal Ca(2+)ATPase and Na(+)/K(+) ATPase by measuring the Pi liberated from ATP hydrolysis with colorimetrical methods. It was shown that the activity of Ca(2+)ATPase in SR from IL-2-perfused (10, 40, 200, 800 U/ml) rat heart increased dose-dependently. After incubation of the SR with ATP (0.1 approximately 4 mmol/L), the activity of SR Ca(2+)ATPase increased dose-dependently in the control group. In the SR from 200 U/ml IL-2-perfused hearts, the activity of Ca(2+)ATPase was much higher than that in the control group. On the other hand, incubation of the SR with Ca(2+) (1 approximately 40 micromol/L) increased the activity of SR Ca(2+) ATPase in the control group. The activity of SR Ca(2+)ATPase of IL-2-perfused hearts was inhibited as the function to Ca(2+). Pretreatment with specific kappa-opioid receptor antagonist nor-BNI (10 nmol/L) for 5 min attenuated the effect of IL-2 (200 U/ml) on the activity of SR Ca(2+) ATPase. After pretreatment with pertussis toxin (PTX, 5 mg/L) or U73122 (5 micromol/L), IL-2 failed to increase SR Ca(2+)ATPase activity. The activity of SR Ca(2+)ATPase was not changed by incubation of SR isolated from normal hearts with IL-2. Perfusion of rat heart with IL-2 did not affect the activity of sarcolemmal Ca(2+)ATPase and Na(+)/K(+)ATPase. It is concluded that perfusion of rat heart with IL-2 increases the activity of SR Ca(2+)ATPase dose-dependently, which is mainly mediated by cardiac kappa-opioid receptor pathway including a PTX sensitive Gi-protein and phospholipase C. IL-2 increases the activity of SR Ca(2+)ATPase as the function to ATP, but inhibits the activity of SR Ca(2+)ATPase as the function to Ca(2+). IL-2 has no effect on the activity of sarcolemmal Ca(2+)ATPase and Na(+)/K(+)ATPase.
Animals ; Interleukin-2 ; pharmacology ; Male ; Myocardium ; enzymology ; Rats ; Rats, Sprague-Dawley ; Sarcolemma ; enzymology ; Sarcoplasmic Reticulum ; enzymology ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Potassium-Exchanging ATPase ; metabolism

Sarco/endoplasmic reticulum Ca(2+)-ATPase (Serca) is responsible for transporting Ca2+ into the endoplasmic reticulum and maintaining a suitable calcium environment in cells. The suitable calcium environment created by BmSerca is vital for the growth and development of silkworm. With a large molecular weight and 10 transmembrane domains, Serca is very difficult to express in Escherichia coli expression system. In order to obtain recombinant Serca with biological activity, pFastBac Dual vector was used to construct a binary baculovirus expression vector for expressing egfp and serca in cells. After transfection and infection, EGFP and Serca were expressed successfully in BmN-SWU1 cell line. Fluorescent observation revealed that the expression patterns of EGFP and Serca in infected cells were the same. Western blotting analysis showed that the recombinant proteins were about to express in cells 48 h post infection and highly expressed 96 h post infection. Ca(2+)-ATPase activities assays were used to evaluate the enzyme activities of recombinant Serca and found that the enzyme activities increased significantly after infection. The obtained data showed that this binary baculovirus expression system can be successfully used to express Serca with biological activity. The expression of Serca protein with this system is useful for further research on the function of Serca.
Animals ; Baculoviridae ; Bombyx ; enzymology ; Cell Line ; Genetic Vectors ; Recombinant Proteins ; biosynthesis ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; biosynthesis ; Transfection

OBJECTIVEThe explore the mechanism responsible for diaphragmatic contractile and relaxation dysfunction in a rat model of sepsis.

METHODSThirty-six adult male Sprague-Dawley rats were randomized equally into a sham-operated group and two model groups of sepsis induced by cecal ligation and puncture (CLP) for examination at 6 and 12 h following CLP (CLP-6 h and CLP-12 h groups). The parameters of diaphragm contractile and relaxation were measured, and the calcium uptake and release rates of the diaphragmatic sarcoendoplasmic reticulum (SR) and the protein expressions of SERCA1, SERCA2 and RyR in the diaphragmatic muscles were determined.

RESULTSThe half-relaxation time of the diaphragm was extended in both the CLP-6 h and CLP-12 h groups with significantly reduced maximum tension declinerate and the peek uptake rate of SERCA (P<0.01). Diaphragmatic maximum twitch force development rate, the maximal twitch, tetanus tensions and the peek release rate of SR decreased only at 12h after CLP (P<0.01). The expression levels of SERCA1 protein decreased significantly in the diaphragmatic muscles at 12h following CLP (P<0.01) while SERCA2 expression level and SERCA activity showed no significant changes.

CONCLUSIONIn the acute stage of sepsis, both the contractile and relaxation functions of the diaphragm are impaired. Diaphragmatic relaxation dysfunction may result from reduced calcium uptake in the SR and a decreased level of SERCA1 in the diaphragmatic muscles.

Animals ; Calcium ; metabolism ; Cecum ; Diaphragm ; drug effects ; metabolism ; Endoplasmic Reticulum ; metabolism ; Ligation ; Male ; Muscle Contraction ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sepsis

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*