Singlet oxygen interaction with Ca(2+)-ATPase of cardiac sarcoplasmic reticulum.

We investigated the role of singlet oxygen (generated from photoactivation of rose bengal) on the calcium transport and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum (SR). Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at 560 nm resulted in significant inhibition of Ca2+ uptake (from 2.27 +/- 0.05 to 0.62 +/- 0.05 mumol Ca2+/mg.min [mean +/- SEM], p less than 0.01) and Ca(2+)-ATPase activity (from 2.08 +/- 0.05 to 0.28 +/- 0.04 mumol Pi/min.mg [mean +/- SEM], p less than 0.01). The inhibition of calcium uptake and Ca(2+)-ATPase activity by rose bengal-derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. Singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca(2+)-ATPase against rose bengal-derived activated oxygen species, but superoxide dismutase and catalase did not attenuate the inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal for up to 14 minutes demonstrated complete loss of the Ca(2+)-ATPase monomer band, which was significantly protected by histidine. The addition of dithiothreitol (5 mM) had a slight protective effect, showing that new disulfide bond formation was not a major cause of aggregation. The results were also confirmed by high-performance liquid chromatography of the SR exposed to irradiated rose bengal. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide radical (generated from xanthine oxidase action on xanthine) and hydroxyl radical (in the presence of Fe(3+)-EDTA or 0.5 mM H2O2 plus Fe(2+)-EDTA) as well as H2O2 (0.25-12 mM) were without any effect on the 97,000-d Ca(2+)-ATPase band of SR. Generation of radical species (superoxide and hydroxyl radical) from rose bengal was studied by electron paramagnetic resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results showed that irradiation of rose bengal formed a 1:2:2:1 quartet, characteristic of the DMPO-OH adduct, which was scavenged by ethanol but not by superoxide dismutase, catalase, or histidine. No radical species could be detected from irradiated rose bengal or irradiated DMPO under the assay conditions used. Peroxy adducts of DMPO might be produced but would be observed only at very low temperatures. Similarly, we could not detect any measurable.O2- anion from irradiation of rose bengal as indicated by either cytochrome c reduction at 550 nm or nitro blue tetrazolium reduction at 560 nm. These results show that SR is damaged most likely by singlet oxygen derived from rose bengal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Overexpression of sarcoplasmic reticulum Ca(2+)-ATPase improves cardiac contractile function in hypothyroid mice.

OBJECTIVE: Prolonged cardiac contraction and relaxation in hypothyroidism are in part related to diminished expression of the gene coding for the calcium pump of the sarcoplasmic reticulum (SERCA2a). Therefore, we examined whether or not transgenic SERCA2a gene expression in mice may compensate for the cardiac effects of hypothyroidism. METHODS: SERCA2a mRNA and protein were analyzed from hearts of euthyroid and hypothyroid mice of wild-type or SERCA2a transgene status. Contractile function was studied in isolated left ventricular papillary muscles. RESULTS: We found significant decreases of SERCA2a mRNA and protein levels in hearts of hypothyroid wild-type mice in comparison with euthyroid wild-type mice (controls). Papillary muscles from hypothyroid wild-type mice showed significant increases in time to peak contraction and relaxation times compared with controls. In contrast, SERCA2a mRNA and protein levels were significantly higher in hypothyroid SERCA2a transgenic mice than in hypothyroid wild-type mice. The transgene led to a functional improvement by compensating for the prolonged contraction and relaxation of papillary muscles. CONCLUSIONS: Our murine model of hypothyroidism revealed decreases in SERCA2a gene expression accompanied by prolonged contraction and relaxation of papillary muscles, and an improvement of the contractile phenotype due to compensated SERCA2a gene expression in SERCA2a transgenic mice.     

Diminished expression of sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine sensitive Ca(2+)Channel mRNA in streptozotocin-induced diabetic rat heart

The diabetic heart has an abnormal intracellular calcium ([Ca(2+)]i) metabolism. However, the responsible molecular mechanisms are unclear. The present study aimed to investigate mRNAs expressed in the proteins which regulate heart [Ca(2+)]i metabolism in streptozotocin (STZ)-induced diabetic rats. Expression of sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (SR Ca(2+)-ATPase) mRNA was significantly less in the heart 3 weeks after STZ injection than that in the age-matched controls. Together with the down-regulation of SR Ca(2+)-ATPase, expression of ryanodine sensitive Ca(2+)channel (RYR) mRNA was also decreased 12 weeks after STZ injection. Insulin supplementation fully restored the decreased mRNAs expression of SR Ca(2+)-ATPase and RYR. The diminished expression and restoration with insulin supplementation of SR Ca(2+)-ATPase was further confirmed at the protein level. In contrast, expression of mRNAs coding the L-type Ca(2+)channel, Na(+)-Ca(2+)exchanger, or phospholamban were not affected 3 or 12 weeks after STZ injection. These results can be taken to indicate that the down-regulation of SR Ca(2+)-ATPase and RYR mRNAs is a possible underlying cause of cardiac dysfunction in STZ-induced diabetic rats.     

Function of the sarcoplasmic reticulum and expression of its Ca2(+)-ATPase gene in pressure overload-induced cardiac hypertrophy in the rat

The reduction in Ca2+ concentration during diastole and relaxation occurs differently in normal hearts and in hypertrophied hearts secondary to pressure overload. We have studied some possible molecular mechanisms underlying these differences by examining the function of the sarcoplasmic reticulum and the expression of the gene encoding its Ca2(+)-ATPase in rat hearts with mild and severe compensatory hypertrophy induced by abdominal aortic constriction. Twelve sham-operated rats and 31 operated rats were studied 1 month after surgery. Eighteen animals exhibited mild hypertrophy (left ventricular wt/body wt less than 2.6) and 13 animals severe hypertrophy (left ventricular wt/body wt greater than 2.6). During hypertrophy we observed a decline in the function of the sarcoplasmic reticulum as assessed by the oxalate-stimulated Ca2+ uptake of homogenates of the left ventricle. Values decreased from 12.1 +/- 1.2 nmol Ca2+/mg protein/min in sham-operated rats to 9.1 +/- 1.5 and 6.7 +/- 1.1 in rats with mild and severe hypertrophy, respectively (p less than 0.001 and p less than 0.001, respectively, vs. shams). This decrease was accompanied by a parallel reduction in the number of functionally active CA2(+)-ATPase molecules, as determined by the level of Ca2(+)-dependent phosphorylated intermediate: 58.8 +/- 7.4 and 48.1 +/- 13.5 pmol P/mg protein in mild and severe hypertrophy, respectively, compared with 69.7 +/- 8.2 in shams (p less than 0.05 and p less than 0.01, respectively, vs. shams). Using S1 nuclease mapping, we observed that the Ca2(+)-ATPase messenger RNA (mRNA) from sham-operated and hypertrophied hearts was identical. Finally, the relative level of expression of the Ca2(+)-ATPase gene was studied by dot blot analysis at both the mRNA and protein levels using complementary DNA clones and a monoclonal antibody specific to the sarcoplasmic reticulum Ca2(+)-ATPase. In mild hypertrophy, the concentrations of Ca2(+)-ATPase mRNA and protein in the left ventricle were unchanged when compared with shams (mRNA, 93.8 +/- 10.6% vs. sham, NS; protein, 105.5 +/- 14% vs. sham, NS). in severe hypertrophy, the concentration of Ca2(+)-ATPase mRNA decreased to 68.7 +/- 12.9% and that of protein to 80.1 +/- 15.5% (p less than 0.001 and p less than 0.05, respectively), whereas the total amount of mRNA and enzyme per left ventricle was either unchanged or slightly increased. The slow velocity of relaxation of severely hypertrophied heart can be at least partially explained by the absence of an increase in the expression of the Ca2(+)-ATPase gene and by the relative diminution in the density of the Ca2+ pumps.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduced sarcoplasmic reticulum Ca(2+)-uptake and expression of phospholamban in left ventricular myocardium of dogs with heart failure.

The purpose of this study was to examine the sarcoplasmic reticulum (SR) Ca(2+)-uptake and the expression of phospholamban (PLB) and Ca(2+)-ATPase (CAA) in left ventricular (LV) and right ventricular (RV) myocardium of 6 normal (NL) dogs and 6 dogs with chronic heart failure (HF). In addition, gene expression of PLB and CAA was also examined in LV myocardium of NL and HF dogs. HF (LV ejection fraction 23+/-2%) was produced by multiple sequential intracoronary microembolizations. Oxalate-dependent Ca(2+)-uptake was measured in isolated membrane vesicles. Using specific dog myocardial monoclonal antibody, the expression of CAA, PLB and calsequestrin (CSQ) were measured in sodium dodecyl sulfate extract prepared from LV and RV tissue. Steady-state mRNA levels were determined by Northern hybridization using specific cDNA clones of PLB, CAA, CSQ, and glyceraldehyde-3-phosphate dehydrogenase (GADPH), a house keeping gene. SR Ca(2+)-uptake of NL and HF dogs increased with increasing Ca(2+)concentrations and reached a plateau at 3 microm in both LV and RV. Total capacity (134+/-9 v 224+/-10 nmol(45)Ca/mg protein/10 min, P<0.05) and maximal velocity (15+/-2 v 2 nmol(45)Ca/mg protein/min, P<0.05) of the SR to sequester Ca(2+)was significantly lower in LV myocardium of HF dogs compared to NL, whereas the Hill coefficient and the affinity of the Ca(2+)-pump for Ca(2+)were unchanged. LV tissue levels of the PLB and CAA, normalized to noncollagen protein or to CSQ and the PLB and CAA mRNA levels, normalized to CSQ or GADPH mRNA, were also significantly lower in HF dogs compared to NL. In RV myocardial tissue, no significant differences in total capacity of SR to sequester Ca(2+), maximal velocity of SR Ca(2+)-uptake, the affinity and Hill Coefficient of the Ca(2+)-pump for Ca(2+), or tissue levels of PLB and CAA were observed between NL dogs compared to HF dogs. We conclude that SR Ca(2+)-uptake and SR PLB and CAA protein and gene expression levels are reduced in LV myocardium of dogs with chronic HF. These abnormalities can lead to Ca(2+)-overload and subsequent global LV dysfunction.     

Transgenic expression of sarcoplasmic reticulum Ca(2+) atpase modifies the transition from hypertrophy to early heart failure

To examine the contribution of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) to early heart failure, we subjected transgenic (TG) mice expressing SERCA2a gene and wild-type (WT) mice to aortic stenosis (AS) for 7 weeks. At an early stage of hypertrophy (4-week AS), in vivo hemodynamic and echocardiographic indices were similar in TG and WT mice. By 7 weeks of AS, which is the stage of early failure in this model, TG mice with AS had lower mortality than WT mice with AS (6.7% versus 29%). The magnitude of left ventricular (LV) hypertrophy was similar in WT and TG 7-week AS mice. In vivo LV systolic function was higher in TG than in WT 7-week AS mice. In LV myocytes loaded with fluo-3, fractional cell shortening and the amplitude of the [Ca(2+)](i) transients were higher in TG than in WT 7-week AS mice under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C). The rates of relengthening and decay in [Ca(2+)](i) were faster in TG than in WT 7-week AS myocytes. In myocytes from WT 7-week AS compared with sham-operated WT mice, contractile reserve in response to rapid pacing was depressed with impaired augmentation of both peak-systolic [Ca(2+)](i) and the SR Ca(2+) load. In contrast, contractile reserve and the capacity to augment SR Ca(2+) load were maintained in TG 7-week AS mice. SERCA2a protein levels were depressed in WT 7-week AS mice, but were preserved in TG 7-week AS mice. These data suggest that defective SR Ca(2+) loading contributes to the onset of contractile failure in animals with chronic pressure overload.     

微电极阵列技术研究肌质网钙ATP酶过表达对大鼠衰竭心肌电活动的改善作用

目的 探索重组腺病毒(rAd)介导的肌质网Ca2+-ATP酶(SERCA2a)过表达对大鼠心肌梗死后心力衰竭心肌电活动节律和传导的改善作用,并探讨可能的电活动机制.方法 将26只成年雄性SD大鼠随机分为3组:假手术组(n=l0),空病毒对照组(rAd.β-gal组,n=8)和肌质网Ca2+-ATP酶(SERCA2a)转染组(rAd.SERCA2a组,n=8).假手术组仅开胸不结扎动脉,rAd.β-gal组和rAd.SERCA2a组分别进行左冠状动脉前降支结扎建立大鼠心肌梗死后心力衰竭动物模型,同时分别将携带β-gal和SERCA2a基因的重组腺病毒(rAd)导入衰竭心脏,术后2周超声心电图检测心脏舒张功能和收缩功能,心电图监测体表心电活动以及微电极阵列(MEA)技术监测离体心脏组织电活动情况.结果 rAd携带SERCA2a与β-gal基因均成功转入大鼠衰竭心脏.rAd.SERCA2a组可改善心功能,与假手术组相比心室舒张末期容积与心室收缩末期容积轻微增加[(0.41±0.13)cm2对(0.39±0.02)cm2,(0.08±0.02)cm2对(0.06±0.01)cm2,P＞0.05],左心室射血分数[(0.82±0.05)对(0.86±0.01),P＞0.05]和短轴缩短率[(46.6±2.32)％对(49.58±1.71)％,P＞0.05]无明显改变.与假手术组相比,rAd.β-gal组体表心电图QT间期延长[(111.02±7.42) ms对(94.7±1.55) ms,n=6,P＜0.05],室性早搏发生率达71.5％ (5/7),而rAd.SERCA2a组QT间期缩短[(81.45±4.97)ms对(94.7±1.55)ms,n=6,P＜0.05],室性早搏发生率达14.3％(1/7).MEA记录可发现rAd.SERCA2a组心率与假手术组相比差异无统计学意义[(435±31)次/min对(442 ±22)次/min,n=6,P＞0.05],与rAd.β-gal组相比,rAd.SERCA2a组最大场电位[(0.82±0.39)mV对(0.64±0.13) mV,n=6,P＜0.05]、最小场电位[(1.88±0.57) mV对(1.35±0.12) mV n=6,P＜0.05]、场电位时限[(124.17±21.08)ms对(113.23±12.02) ms n=6,P＜0.05]均延长;rAd.β-gal组梗死区与梗死对侧区心肌组织场电位时限差异有统计学意义[(60.36±2.08)ms对(103.24±7.35) ms,n=5,P＜0.05],并且60通道记录梗死区心肌组织场电位时限离散度大于rAd.SERCA2a组[(38.5ms±4.62)ms对(26.88±5.09) ms,n=5];rAd.SERCA2a组传导基本一致,使心肌梗死面心室肌组织电活动呈均一性传导.结论 SERCA2a转基因治疗可以显著改善心力衰竭大鼠的左心室收缩功能、舒张功能,同时可以降低心肌梗死后心力衰竭伴发心律失常的发生,改善心脏电活动的均一传导.MEA技术是一项检测心血管疾病动物模型心脏组织电生理节律和频率以及传导活动的理想技术.     

rAAV-asPLB transfer attenuates abnormal sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac dysfunction in rats with myocardial infarction

BACKGROUND: Diminished myocardial sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) activity and upregulated phospholamban (PLB) level during cardiac dysfunction, had been reported in many studies. AIMS: The current study was designed to examine the effects of rAAV-antisense phospholamban (asPLB) gene transfer on cardiac function, SERCA expression and activity, as well as PLB expression and phosphorylation (Pser16-PLB), in a rat myocardial infarction (MI) model. METHODS AND RESULTS: Rat MI model was generated by ligating the left anterior descending coronary artery. Four weeks later, left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), the maximal rates of increase and decrease in intraventricular pressure (+/-dp/dt(max)) were significantly depressed, and left ventricular end diastolic pressure (LVEDP) was increased. Myocardial PLB was markedly increased while both SERCA activity and Pser16-PLB level were decreased. In rAAV-asPLB transfected rats, rAAV-asPLB, which was injected into the myocardium around the infarction area immediately after the coronary artery ligation, effectively attenuated the depression of cardiac function, significantly inhibited the expression of PLB, restored Pser16-PLB level and enhanced myocardium SERCA activity. CONCLUSION: rAAV-asPLB transfer in rats with MI effectively prevented the progression of heart failure.     

Reduced sarcoplasmic reticulum Ca(2+) load mediates impaired contractile reserve in right ventricular pressure overload

Myocardial contractile reserve is significantly attenuated in patients with advanced heart failure. The aim of this study was to identify mechanisms of impaired contractile reserve in a large animal model that closely mimics human myocardial failure. Progressive right ventricular hypertrophy and failure were induced by banding the pulmonary artery in kittens. Isometric contractile force was measured in right ventricular trabeculae (n=115) from age-matched Control and Banded feline hearts. Rapid cooling contractures (RCC) were used to determine sarcoplasmic reticulum (SR) Ca(2+) load while assessing the ability of changes in rate, adrenergic stimulation and bath Ca(2+) to augment contractility. The positive force-frequency relationship and robust pre- and post-receptor adrenergic responses observed in Control trabeculae were closely paralleled by increases in RCC amplitude and the RCC2/RCC1 ratio. Conversely, the severely blunted force-frequency and adrenergic responses in Banded trabeculae were paralleled by an unchanged RCC amplitude and RCC2/RCC1 ratio. Likewise, supraphysiologic levels of bath Ca(2+) were associated with severely reduced contractility and RCC amplitude in Banded trabeculae compared to Controls. There were no differences in myofilament Ca(2+) sensitivity or length-dependent increases in contractility between Control and Banded trabeculae. There was a significant decrease in SR Ca(2+)-ATPase pump abundance and phosphorylation of phospholamban and ryanodine receptor in Banded trabeculae compared with Controls. A reduced ability to increase SR Ca(2+) load is the primary mechanism of reduced contractile reserve in failing feline myocardium. The similarity of impaired contractile reserve phenomenology in this feline model and transplanted hearts suggests mechanistic relevance to human myocardial failure.     

Alteration of myocardial sarcoplasmic reticulum Ca2+-ATPase and Na+-Ca2+ exchanger expression in human left ventricular volume overload

BACKGROUND: Reduced myocardial contractility is often attributed to altered Ca(2+) transients and expression of Ca(2+)-ATPase of the SR (SERCA) and Na+/Ca(2+)exchanger (NCX) genes. AIMS: To assess myocardial expression of SERCA and NCX protein levels in left ventricular (LV) remodelling due to chronic severe mitral regurgitation (MR). METHODS: Myocardial expression of SERCA/NCX in biopsy specimens obtained during mitral surgery was assessed in 36 MR patients with LV remodelling and plasma neurohumoral/cytokine activation and in four non-failing hearts (NFH). RESULTS: Myocardial protein levels of SERCA were significantly (20%) lower in the MR group than in NFH group (p=0.016). No significant changes in NCX were observed. However, a lack of homogeneity with regard to SERCA/NCX proteins was observed. Moreover, SERCA was negatively correlated with BNP (r=-0.49, p=0.02), TNFalpha (r=-0.68, p=0.0005) and IL-6 (r=-0.52, p=0.02), whereas NCX was only negatively correlated with TNFalpha (r=-0.62, p=0.002). CONCLUSIONS: MR patients showed wide variations in SERCA/NCX protein expression. Myocardial protein levels of SERCA were significantly lower in the MR population. Moreover, a correlation between BNP, cytokines (IL-6, TNFalpha) and the expression of SERCA/NCX proteins was observed.     

Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.

Intramolecular crosslinking of the active site of the sarcoplasmic reticulum Ca(2+)-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive E1 approximately P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked E1 approximately P(Ca) (approximately 5 nmol/min per mg of protein at 25 degrees C) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2+ release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca2+ is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen.     

Differences in Ca(2+)-handling and sarcoplasmic reticulum Ca(2+)-content in isolated rat and rabbit myocardium

We made novel measurements of the influence of rest intervals and stimulation frequency on twitch contractions and on sarcoplasmic reticulum (SR) Ca(2+)-content (using rapid cooling contractures, RCCs) in isolated ventricular muscle strips from rat and rabbit hearts at a physiological temperature of 37 degrees C. In addition, the frequency-dependent relative contribution of SR Ca(2+)-uptake and Na(+)/Ca(2+)-exchange for cytosolic Ca(2+)-removal was assessed by paired RCCs. With increasing rest intervals (1-240 s) post-rest twitch force and RCC amplitude decreased monotonically in rabbit myocardium (after 240 s by 45+/-10% and 61+/-11%, respectively P<0. 05, n=14). In contrast, rat myocardium (n=11) exhibited a parallel increase in post-rest twitch force (by 67+/-16% at 240 s P<0.05) and RCC amplitude (by 20+/-14%P<0.05). In rabbit myocardium (n=11), increasing stimulation frequency from 0.25 to 3 Hz increased twitch force by 295+/-50% (P<0.05) and RCC amplitude by 305+/-80% (P<0.05). In contrast, in rat myocardium (n=6), twitch force declined by 43+/-7% (P<0.05), while RCC amplitude decreased only insignificantly (by 16+/-7%). The SR Ca(2+)-uptake relative to Na(+)/Ca(2+)-exchange (based on paired RCCs) increased progressively with frequency in rabbit, but not in rat myocardium (;66+/-2% at all frequencies). We conclude that increased SR Ca(2+)-load contributes to the positive force-frequency relationship in rabbits and post-rest potentiation of twitch force in rats. Decreased SR Ca(2+)-load contributes to post-rest decay of twitch force in rabbits, but may play only a minor role in the negative force-frequency relationship in rats. SR Ca(2+)-release channel refractoriness may contribute importantly to the negative force-frequency relationship in rat and recovery from refractoriness may contribute to post-rest potentiation.