Using lipid bilayer reconstitution technique, we investigated the oxidation effect of t-butyl hydrogen peroxide (tBHP) on the single channel activity of the sarcoplasmic reticulum (SR) calcium release channels isolated from canine latissimus dorsi muscles. When 0.7% tBHP was added in the cytosolic side, the channel activity became suppressed (n = 7), and it was recovered by changing the solution to the control solution. The suppression was due to the change in the gating mode of the channel: before tBHP the channel opened to four sub-conductance levels, but it opened to only one level after tBHP. These effects by tBHP were different from the previous finding using hydrogen peroxide (H2O2), which may be explained by different oxidation patterns between the two oxidants.
Animal ; Calcium Channels/drug effects* ; Dogs ; Hydrogen Peroxide/pharmacology ; Peroxides/pharmacology* ; Sarcoplasmic Reticulum/metabolism ; Sarcoplasmic Reticulum/drug effects* ; tert-Butylhydroperoxide

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

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

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

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

OBJECTIVETo investigate the effects of power frequency magnetic field on the Ca2+ transport dynamics of isolated sarcoplasmic reticulum vesicles.

METHODSThe assays of Ca2+ uptake time course and the Ca2+-ATPase activity of sarcoplasmic reticulum vesicles were investigated by using dynamic mode of spectrometry with a Ca2+ dye; Ca2+ release channel activation was examined by 3H-ryanodine binding and Ca2+ release assays; membrane fluidity of sarcoplasmic reticulum vesicles was examined by fluorescence polarization, without or with exposure to the vesicles at a 0.4 mT, 50 Hz sinusoidal magnetic field.

RESULTS0.4 mT, 50 Hz sinusoidal magnetic field exposure caused about a 16% decline of the initial Ca2+ uptake rate from a (29.18 +/- 3.90) pmol.mg(-1).s(-1) to a (24.60 +/- 3.81) pmol.mg(-1).s(-1) and a 26% decline of the Ca2+-ATPase activity from (0.93 +/- 0.05) micromol.mg(-1).min(-1) to (0.69 +/- 0.07) micromol.mg(-1).min(-1) of sarcoplasmic reticulum vesicles, whereas caused a 15% increase of the initial Ca2+ release rate from (4.83 +/- 0.82) pmol.mg(-1).s(-1) to (5.65 +/- 0.43) pmol.mg(-1).s(-1) and a 5% increase in 3H-ryanodine binding to the receptor from (1.10 +/- 0.12) pmol/mg to (1.16 +/- 0.13) pmol/mg, respectively.

CONCLUSIONThe decline of Ca2+-ATPase activity and the increase of Ca2+ release channel activity should result in a down-regulation of Ca2+ dynamic uptake and an up-regulation of Ca2+ release induced by exposing the sarcoplasmic reticulum to a 0.4 mT, 50 Hz power frequency magnetic field.

Animals ; Calcium ; metabolism ; Calcium Signaling ; Electromagnetic Fields ; Muscle, Skeletal ; metabolism ; Rabbits ; Sarcoplasmic Reticulum ; metabolism ; radiation effects

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

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

OBJECTIVETo study the changes in diaphragmatic function and gene expressions of calcium regulatory proteins in diabetic rats and explore the mechanism of diaphragm dysfunction in diabetes mellitus.

METHODSSD rats were randomly divided into normal control group and diabetic (induced by intraperitoneal STZ injection) group. After 4 and 8 weeks, the body weight and diaphragm to body weight ratio were measured, and the activities of succinic dehydrogenase (SDH) in the diaphragm and blood glucose were assayed. The diaphragm contractility was assessed and the alterations of diaphragm ultrastructure were observed. RT-PCR was used to detect the changes in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLB) mRNA expressions in the diaphragm.

RESULTSThe diabetic rats showed a significant weight loss with a lowered diaphragm to body weight ratio (P<0.01) and SDH activity (P<0.01). The peak twitch tension and maximum tetanic tension of the diaphragm were significantly lowered and the time to peak contraction and half relaxation time significantly prolonged (P<0.01) in the diabetic rats, which also exhibited a lowered tetanic force in response to stimulus (P<0.01). Transmission electron microscopy revealed obvious ultrastructural changes of the diaphragm in diabetic rats. RT-PCR showed significantly decreased SERCA and increased PLB mRNA expressions in diabetic rat diaphragm (P<0.01), and these changes intensified with time (P<0.01).

CONCLUSIONDiabetes can cause impairment of diaphragmatic ultrastructure, mitochondrial injuries, and lowered SDH activity and ATP production. Decreased SERCA and increased PLB mRNA expressions in diabetes result in reduced Ca(2+) uptake by the diaphragm sarcoplasmic reticulum to induce diaphragm dysfunction.

Animals ; Body Weight ; Calcium ; metabolism ; Calcium-Binding Proteins ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; physiopathology ; Diaphragm ; metabolism ; physiopathology ; Glucose ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Succinate Dehydrogenase ; metabolism

AIMTo investigate the biological mechanism of the dynamic changes of living myocardial nuclear calcium which was isolated from aerobic exercised rats.

METHODSAcutely isolated myocardial cells were incubated with STDBT and Fluo-3Ca2+ fluorescent indicator. The changes of the fluorescence signal of free calcium were measured by laser scanning confocal microscope (LSCM).

RESULTSThe fluorescence of the ventricular myocardium cell was weak which was incubated with nucleus specific Ca2+ fluorescent probe STDBT-AM, transverse striation and transverse tubules was looked ambiguous. The most significant fluorescence intensity was distributed in the place of nucleus, bottle green was observed and the borderline of nuclear membrane. It was showed that STDBT could though cytoplasm and specifically clustered in nucleus and characterized by targeting to the distribution of nucleus. Compared with control group, aerobic exercise group had the same characteristic of the change of the basic and peak value of [Ca2+]n. When isoproterenol was added, the [Ca2+]n of aerobic exercise group was increased significantly, its change rate was 33.3412% (P < 0.01). Before and after added isoproterenol, myocardial cell's [Ca2+]n changed significantly. After added isoproterenol, the [Ca2+]n of aerobic exercise group was increased significantly and the change rate was 33.224%.

CONCLUSIONThis article firstly studied the changes of living myocardial nuclear calcium and discussed the transport of the [Ca2+]n of myocardial nuclear which was isolated from aerobic exercised rats; proved that aerobic exercise could increase its level of [Ca2+]n significantly, isoproterenol could increase the level of [Ca2+]n and decrease the level of [Ca2+]i. Myocardial nuclear is one of calcium stores of myocardial cell, aerobic exercise could impact on the modulation of myocardial cell.

Animals ; Calcium ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; Nuclear Envelope ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; metabolism