Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myocardial function, intracellular calcium and levels of calcium cycling proteins were analyzed in failing and nonfailing human myocardium. Myocardial function was evaluated by the isometric force-frequency relation, and intracellular calcium was studied by aequorin light emission. When stimulation frequency was increased above 30 min-1, there was a continuous increase in isometric tension development in the nonfailing myocardium. In contrast, in failing myocardium, frequency potentiation of contractile force was blunted or inverse. As a consequence, at higher rates of stimulation, twitch tension was reduced significantly in failing compared to nonfailing human myocardium. Aequorin measurements indicated that the contractile deficit in the failing myocardium at higher rates of stimulation is associated with decreased free intracellular calcium concentration. Western blot analysis indicated that in the failing myocardium protein levels of SR-Ca(2+)- ATPase are significantly reduced and protein levels of Na(+)-Ca(2+)- exchanger are significantly increased. Levels of phospholamban are slightly reduced in the failing myocardium, and ryanodine receptor and calsequestrin protein levels are unchanged. There was a close positive correlation between the protein levels of SR-Ca(2+)-ATPase and frequency potentiation of contractile force. From these data, we conclude that in failing compared to nonfailing human myocardium 1) force-frequency relation is blunted or inverse. 2) Frequency-dependence of contractile force is closely correlated with frequency-dependence of intracellular calcium cycling. 3) Protein levels of SR-Ca(2+)-ATPase may determine frequency-dependence of sarcoplasmic reticulum calcium release. 4) Calcium elimination by an increased number of Na(+)-Ca2-exchanger molecules may be a compensatory mechanism to prevent diastolic calcium accumulation in failing myocardium with a reduced number of SR calcium pumps.
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PMID:Calcium cycling proteins and force-frequency relationship in heart failure. 895 39

Although systolic left ventricular (LV) function is normal in the elderly, aging is associated in rat papillary muscle with mechanical and sarcoplasmic reticulum Ca2+ ATPase alterations similar to those observed in the hypertrophied heart. However, alterations in the other calcium-regulating proteins implicated in contraction and relaxation are still unknown. To investigate alterations in LV function and calcium-regulating proteins, we measured hemodynamics and Na(+)-Ca2+ exchanger (NCx), ryanodine receptor (RyR2), and sarcoplasmic reticular Ca2+ ATPase (SERCA2) mRNA levels (expressed in densitometric scores normalized to that of poly(A+) mRNA) in left ventricle from 4-month-old (adult, n = 13) and 24-month-old (senescent, n = 15) rats. For ex vivo contractile function, active tension was measured during isolated heart perfusion in adult (n = 11) and senescent (n = 11) rats. For comparison of age-dependent effects of moderate hypertension on both hemodynamics and calcium proteins, renovascular hypertension was induced or a sham operation performed at 2 (n = 11 and n = 6) and 22 (n = 26 and n = 5) months of age. In senescent rats, LV systolic pressure and maximal rates of pressure development were unaltered, although active tension was depressed (4.7 +/- 0.4 versus 8.3 +/- 0.7 g/g heart weight in adults, P < .0001). SERCA2 mRNA levels were decreased in senescent left ventricle (0.98 +/- 0.05 versus 1.18 +/- 0.05 in adults, P < .01), without changes in NCx and RyR2 mRNA accumulation. Renovascular hypertension resulted in 100% mortality in aged rats; in adults, renovascular hypertension resulted, 2 months later, in an increase of LV systolic pressure (170 +/- 7 versus 145 +/- 3 mm Hg in sham-operated rats, P < .05) and in mild LV hypertrophy (+18%, P < .01) associated with a decrease in SERCA2 mRNA levels (1.02 +/- 0.03 versus 1.18 +/- 0.03 in sham-operated rats, P < .001). Contractile dysfunction in senescent isolated heart and decreased SERCA2 mRNA levels were associated with in vivo normal LV function at rest, indicating the existence of in vivo compensatory mechanisms. RyR2 and NCx gene expressions were not implicated in the observed contractile dysfunction. In aged rats, renovascular hypertension resulted in 100% mortality, probably related to elevated levels of circulating angiotensin II, whereas in adult rats, renovascular hypertension induced a mild LV hypertrophy associated with a selective alteration in SERCA2 gene expression.
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PMID:Senescent heart compared with pressure overload-induced hypertrophy. 903 74

The ry1(53) dyspedic mouse contains two disrupted alleles for ryanodine receptor type 1 (skeletal isoform of ryanodine receptor; Ry1R) resulting in perinatal death. In the present study, whole skeletal muscle homogenates and sucrose gradient-purified junctional sarcoplasmic reticulum from neonatal wild-type and dyspedic mice were assayed for biochemical and functional markers. Equilibrium binding experiments performed with 1-120 nM [3H]ryanodine reveal saturable high and low affinity binding to membrane preparations from wild-type mice, but not to preparations from dyspedic mice. Binding experiments performed with [3H]PN200 show a 2-fold reduction in [3H]PN200 binding capacity in dyspedic muscle, compared to age-matched wild-type muscle, with no change in receptor affinity. The presence or absence of proteins known to be critical for normal ryanodine receptor/Ca2+ channel complex function was assessed by Western blot analysis. Results indicate that FKBP-12, DHPRalpha1, triadin, calsequestrin, SERCA1 (sarco(endo)plasmic reticulum Ca2+ ATPase), and skeletal muscle myosin heavy chain are present in both dyspedic and wild-type muscle. Only wild-type membranes showed immunoreactivity toward Ry1R antibody. Neither dyspedic nor wild-type mouse muscle showed detectable immunoreactivity toward Ry2R or Ry3R antibodies, even after sucrose gradient purification of sarcoplasmic reticulum. These results indicate that proteins critical for ryanodine receptor function are expressed in dyspedic skeletal muscle in the absence of Ry1R. Ca2+ transport measurements show that membranes from wild-type controls, but not dyspedic mice, release Ca2+ upon exposure to ryanodine. Dyspedic mice and cells derived from them serve as excellent homologous expression systems in which to study how Ry1R structure relates to function.
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PMID:Dyspedic mouse skeletal muscle expresses major elements of the triadic junction but lacks detectable ryanodine receptor protein and function. 905 35

The endothelial-derived relaxing factor, nitric oxide (NO.) has been shown to depress force in smooth and cardiac muscles through the activation of guanylyl cyclase and an increase in cGMP. In fast skeletal muscle, NO (i.e. NO-related compounds) elicits a modest decrease in developed force, but in contracting muscles NO increases force by a mechanism independent of cGMP. We now demonstrate an alternative mechanism whereby NO triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum (SR). NO delivered in the form of NO gas, NONOates (a class of sulfur-free compounds capable of releasing NO), or S-nitrosothiols (R-SNO) oxidized or transnitrosylated regulatory thiols on the release channel (or ryanodine receptor, RyR), resulting in channel opening and Ca2+ release from skeletal and cardiac SR. The process was reversed by sulfhydryl reducing agents which promoted channel closure and Ca2+ reuptake by ATP-driven Ca2+ pumps. NO did not directly alter Ca(2+)-ATPase activity but increased the open probability of RyRs reconstituted in planar bilayers and inhibited [3H]-ryanodine binding to RyRs. The formation of peroxynitrite or thiyl radicals did not account for the reversible R-SNO-dependent activation of RyRs. Ca2+ release induced by nitric oxide free radicals (NO.) was potentiated by cysteine providing compelling evidence that NO. in the presence of O2 formed nitrosylated cysteine followed by the transnitrosation of regulatory thiols on the RyR to activate the channel. These findings demonstrate direct interactions of NO derivatives with RyRs and a new fundamental mechanism to regulate force in striated muscle.
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PMID:Nitric oxide activates skeletal and cardiac ryanodine receptors. 905 74

In cardiac muscle, a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates the Ca(2+)-pumping ATPase in addition to its previously characterized substrates, phospholamban and Ca(2+)-release channel (ryanodine receptor). The phosphorylated amino acid in the Ca(2+)-ATPase has been identified as serine. Posphorylation of the Ca(2+)-ATPase is rapid and is reversible by a membrane-associated protein phosphatase, Ca(2+)-ATPase purified from cardiac SR underwent phosphorylation by exogenous CaM kinase, and the phosphorylated enzyme displayed twofold greater catalytic activity without alteration in its Ca(2+)-sensitivity. The phosphorylation of the Ca(2+)-ATPase was found to be isoform-specific in that the cardiac and slow-twitch skeletal muscle isoform (SERCA 2), but not the fast-twitch skeletal muscle isoform (SERCA 1), underwent phosphorylation by CaM kinase. Studies using SERCA 1 and SERCA 2 isoforms and their mutants expressed in a heterelogous cell system have resulted in i) confirmation of the isoform specificity of Ca(2+)-ATPase phosphorylation by CaM kinase, ii) identification of Ser38 as the site in SERCA 2 phosphorylated by CaM kinase, and iii) demonstration of phosphorylation-induced increase in Vmax of Ca2+ transport by the SERCA 2 enzyme. These observations suggest that in cardiac and slow-twitch skeletal muscle direct phosphorylation of the SR Ca(2+)-ATPase by the membrane-bound CaM kinase may serve to stimulate Ca2+ sequestration and therefore, the speed of muscle relaxation.
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PMID:Phosphorylation and regulation of the Ca(2+)-pumping ATPase in cardiac sarcoplasmic reticulum by calcium/calmodulin-dependent protein kinase. 920 41

There is accumulating evidence that disturbed calcium homeostasis may play a key role in the pathophysiology of human heart failure. Because disturbed calcium handling could result from altered protein expression, levels of calcium handling proteins were quantitated by Western Blot analysis in failing and nonfailing human myocardium from hearts with endstage failing dilated or ischemic cardiomyopathy. Protein levels of the sarcoplasmic reticulum calcium release channel (ryanodine receptor) and of calcium storage proteins (calsequestrin and calreticulin) were similar in failing and nonfailing human myocardium. However, proteins involved in calcium removal from the cytosol were significantly altered in the failing human heart: 1) SR-Ca(2+)-ATPase, relevant for removal of calcium from the cytosol into the lumen of the sarcoplasmic reticulum, was decreased; 2) phospholamban, which inhibits the SR-Ca(2+)-ATPase in the basal unphosphorylated state, was slightly decreased; 3) the ratio of SR-Ca(2+)-ATPase to phospholamban was decreased; 4) the sarcolemmal Na(+)-Ca(2+)-exchanger, relevant for transsarcolemmal calcium extrusion was increased in the failing hearts. In summary, altered levels of proteins involved in calcium removal from the cytosol suggest an increase in transsarcolemmal calcium elimination relative to sarcoplasmic reticulum calcium removal. These findings support the concept that reduced function of the sarcoplasmic reticulum to accumulate calcium may reflect a major defect in excitation-contraction coupling in human heart failure.
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PMID:Calcium handling proteins in the failing human heart. 920 48

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.
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PMID:Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. 921 15

Phospholamban (PLB) is expressed in slow-twitch skeletal, cardiac, and smooth muscles. Several studies have indicated that it is an important regulator of basal contractility and the stimulatory responses to isoproterenol in the mammalian heart. To determine whether PLB is also a key modulator of slow-twitch skeletal muscle contractility, we examined isometric twitch contractions of isolated, intact soleus muscles from wild-type (WT) and PLB-deficient mice in parallel. Soleus muscles from PLB-deficient mice exhibited a significant (25%) decrease in the time to half relaxation, with no change in contraction time compared with WT soleus muscles. The observed enhancement of relaxation in the PLB-deficient soleus was not associated with alterations in the protein levels of either the sarcoplasmic reticular Ca(2+)-adenosinetriphosphatase or the ryanodine receptor. Examination of the effects of isoproterenol on the twitch kinetics of these muscles revealed 1) no effect on the contraction times of either WT or PLB-deficient muscles and 2) a significant decrease in the half relaxation time of the WT soleus, whereas this parameter remained unchanged in the PLB-deficient muscle. Furthermore, with maximal isoproterenol stimulation, the half relaxation time of the WT soleus was similar to that of the nonstimulated PLB-deficient soleus. These results suggest that PLB is a key determinant of relaxation in slow-twitch skeletal muscle under basal conditions and during isoproterenol stimulation.
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PMID:Phospholamban ablation enhances relaxation in the murine soleus. 925 36

To examine mechanisms underlying force reduction after the onset of chronic low-frequency (10 Hz) stimulation (CLFS), we exposed rabbit tibialis anterior muscles to various durations of CLFS. To follow changes in isometric contractile properties and electromyographic (EMG) activity, we studied stimulated and contralateral muscles during a terminal test at 10 Hz for 10 min. In addition, activities and protein amounts of the sarcoplasmic reticulum Ca(2+)-ATPase, content of Na(+)-K(+)-ATPase, and expression patterns of triad junction components were examined. Force output and EMG amplitude declined abruptly soon after the onset of stimulation, suggesting refractoriness of a large fiber population. Although twitch force and to a lesser extent EMG activity gradually recovered after stimulation for 6 days and longer, the muscles exhibited profoundly altered properties, i.e., enhanced fatigue resistance, absence of twitch potentiation, and prolonged contraction and relaxation times. These changes were associated with significant increases in Na(+)-K(+)-ATPase concentration and significant decreases in Ca(2+)-ATPase, ryanodine receptor, dihydropyridine receptor, and triadin concentrations over the course of the 20 days of stimulation. Alterations in excitability, Ca2+ handling, and excitation-contraction coupling prior to changes in myofibrillar protein isoforms may thus be responsible for early functional alterations.
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PMID:Early functional and biochemical adaptations to low-frequency stimulation of rabbit fast-twitch muscle. 925 68

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.
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PMID:Ryanodine receptor dysfunction in porcine stunned myocardium. 927 97


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