Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5+/-6.3% and 92. 5+/-11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1+/-0.3% and 8.6+/-2. 1%, respectively. However, the effect of KN-93 was attenuated (47. 8+/-3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.
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PMID:Phosphorylation of phospholamban at threonine-17 in the absence and presence of beta-adrenergic stimulation in neonatal rat cardiomyocytes. 1111 93

To decipher the mechanism(s) underlying glucocorticoid action on cardiac contractile function, this study investigated the effects of adrenalectomy and dexamethasone treatment on the contents of sarcoplasmic reticulum (SR) Ca(2+)-cycling proteins, their phosphorylation by endogenous Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), and SR Ca(2+) sequestration in the rat myocardium. Cardiac SR vesicles from adrenalectomized rats displayed significantly diminished rates of ATP-energized Ca(2+) uptake in vitro compared with cardiac SR vesicles from control rats; in vivo administration of dexamethasone to adrenalectomized rats prevented the decline in SR function. Western immunoblotting analysis showed that the relative protein amounts of ryanodine receptor/Ca(2+)-release channel, Ca(2+)-ATPase, calsequestrin, and phospholamban were neither diminished significantly by adrenalectomy nor elevated by dexamethasone treatment. However, the relative amount of SR-associated CaM kinase II protein was increased 2.5- to 4-fold in dexamethasone-treated rats compared with control and adrenalectomized rats. Endogenous CaM kinase II activity, as judged from phosphorylation of ryanodine receptor, Ca(2+)-ATPase, and phospholamban protein, was also significantly higher (50--80% increase) in the dexamethasone-treated rats. The stimulatory effect of CaM kinase II activation on Ca(2+) uptake activity of SR was significantly depressed after adrenalectomy and greatly enhanced after dexamethasone treatment. These findings identify the SR as a major target for glucocorticoid actions in the heart and implicate modification of the SR CaM kinase II system as a component of the mechanisms by which dexamethasone influences SR Ca(2+)-cycling and myocardial contraction.
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PMID:Glucocorticoid modulation of protein phosphorylation and sarcoplasmic reticulum function in rat myocardium. 1140

Sarcoplasmic reticulum (SR) dysfunction is one of the multiple alterations that occurs in ischemia-reperfused hearts. Because SR function is regulated by phosphorylation of phospholamban (PLB), a SR protein phosphorylated by cAMP-dependent protein kinase (PKA) at Ser(16)and Ca(2+)-calmodulin-dependent protein kinase (CaMKII) at Thr(17), the phosphorylation of these residues during ischemia and reperfusion was examined in Langendorff-perfused rat hearts. Ser(16)phosphorylation increased significantly after 20 min of ischemia from 2.5+/-0.6% to 99.8+/-25.5% of maximal isoproterenol-induced site-specific phosphorylation and decreased to control values immediately after reperfusion. Thr(17)phosphorylation transiently increased at 2-5 min of ischemia and at 1 min of reperfusion (R1, 166.2+/-28.2%). The ischemia-induced increase in Ser(16)phosphorylation was significantly diminished in hearts from catecholamine-depleted animals and/or after beta-blockade and abolished in the presence of the PKA-inhibitor, H-89. Thr(17)phosphorylation at the beginning of ischemia was blunted by nifedipine, whereas at R1 it was significantly diminished by perfusion with 0 m m Ca(2+)in the presence of EGTA and by the Na(+)/Ca(2+)exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit CaMKII, decreased Thr(17)phosphorylation at R1 and significantly prolonged half relaxation time. The results demonstrated a dissociation between the phosphorylation of PLB sites, being phosphorylation of Ser(16)dependent on the beta-adrenergic cascade during ischemia and phosphorylation of Thr(17)on Ca(2+)influx both, at the beginning of ischemia and reperfusion. Phosphorylation of Thr(17)at the onset of reflow may provide the cell a mechanism to cope with Ca(2+)overload, transiently favoring the recovery of relaxation during early reperfusion.
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PMID:Time course and mechanisms of phosphorylation of phospholamban residues in ischemia-reperfused rat hearts. Dissociation of phospholamban phosphorylation pathways. 1181 63

Studies on the status of multifunctional Ca(2+)-calmodulin (CaM)-dependent protein kinase-II (CaMKII) in failing hearts are limited and controversial. The study was performed in the left ventricular (LV) myocardium of six dogs with heart failure (HF) (LV ejection fraction, 23 +/- 2%) and six normal (NL) dogs. In the LV homogenate, CaMKII activity and its protein level were determined by using the CaMKII peptide and antibody, respectively. Furthermore, the protein level of CaM and phosphorylated phospholamban (PLB) at threonine-17 (PLB-Thr(17)) and serine-16 (PLB-Ser(16)) were also determined in the LV homogenate using a specific antibody. In addition, the level of zinc, which inhibits protein kinase A activity, was determined in the LV tissue by inductively coupled plasma mass spectrometry. CaMKII activity and phosphorylated PLB-Thr(17) and PLB-Ser(16) levels, but not CaM and Zn levels, were significantly reduced in the LV homogenate of dogs with HF compared with NL dogs. These results suggest that CaMKII activity is reduced in the failing LV myocardium, and this abnormality is associated with reduced protein expression level of the enzyme but not due to changes in CaM and zinc levels. In conclusion, reduced CaMKII activity and phosphorylated PLB level may be partly responsible for impaired sarcoplasmic reticulum function in HF.
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PMID:Reduced Ca2+-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure. 1242 92

Dephosphorylated phospholamban (PLB) is an inhibitor of the affinity of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA2) for Ca2+. Phosphorylation of PLB relieves its inhibitory effects on SERCA2, with subsequent acceleration of Ca2+ transport into the SR lumen, which has been suggested to underlie the positive inotropic and lusitropic actions of beta-adrenergic agonists in the mammalian heart. PLB can be phosphorylated at Ser16 by cAMP-dependent protein kinase (PKA) and Thr17 by Ca2+-calmodulin-dependent protein kinase (CaMKII) during beta-agonist stimulation. However, the interrelationship and relative contribution of dual site phosphorylation to the cardiac stimulatory effects are not clear. The recent availability of the PLB knockout mouse, in combination with mutagenesis and transgenic technologies, have provided excellent model systems for expression of each of the phosphorylation site-specific PLB mutants in the heart and elucidation of the functional interplay between PKA- and CaMKII-dependent pathways of PLB phosphorylation. Transgenic mice expressing similar levels of the wild-type, S16A, or T17A mutant PLB in the null background were generated and they were characterized in parallel. Our results indicate that 1) reinsertion of PLB into the knockout mouse heart reverses the hyperdynamic cardiac function associated with PLB deficiency, 2) phosphorylation of Ser16 in PLB is sufficient to mediate its maximal cardiac contractile responses to beta-adrenergic stimulation, and 3) Ser16 phosphorylation is a prerequisite for Thr17 phosphorylation in vivo during beta-agonist stimulation, but Thr17 can be phosphorylated independently of Ser16 in vitro. Thus, these studies revealed novel insights into the interdependence and physiological significance of PKA (Ser16) and CaMKII (Thr17) pathways of PLB phosphorylation during beta-adrenergic stimulation in the mammalian heart.
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PMID:Functional interplay between dual site phospholambam phosphorylation: insights from genetically altered mouse models. 1247 33

Recent studies have demonstrated that transgenic (TG) expression of either Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIdeltaB, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIdeltaB but also a cytoplasmic isoform, CaMKIIdeltaC. In the present study, we demonstrate that expression of the deltaC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic deltaC isoform of CaMKII can lead to phosphorylation of Ca2+ regulatory proteins and induce hypertrophy, we generated TG mice that expressed the deltaC isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2+ handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIdeltaC can mediate phosphorylation of Ca2+ regulatory proteins in vivo and provide evidence for the involvement of CaMKIIdeltaC activation in the pathogenesis of dilated cardiomyopathy and heart failure.
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PMID:The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. 1267 14

To investigate the role of Ca2+/calmodulin-dependent kinase II in cardiac sarcoplasmic reticulum function, transgenic mice were designed and generated to target the expression of a Ca2+/calmodulin-dependent kinase II inhibitory peptide in cardiac longitudinal sarcoplasmic reticulum using a truncated phospholamban transmembrane domain. The expressed inhibitory peptide was highly concentrated in cardiac sarcoplasmic reticulum. This resulted in a 59.7 and 73.6% decrease in phospholamban phosphorylation at threonine 17 under basal and beta-adrenergic stimulated conditions without changing phospholamban phosphorylation at serine 16. Sarcoplasmic reticulum Ca2+ uptake assays showed that the Vmax was decreased by approximately 30% although the apparent affinity for Ca2+ was unchanged in heterozygous hearts. The in vivo measurement of cardiac function showed no significant reductions in positive and negative dP/dt, but a moderate 18% decrease in dP/dt40, indicative of isovolumic contractility, and a 26.1% increase in the time constant of relaxation (tau) under basal conditions. The changes in these parameters indicate a moderate cardiac dysfunction in transgenic mice. Although the 3 and 4-month-old transgenic mice displayed no overt signs of cardiac disease, when stressed by gestation and parturition, the 7-month-old female mice develop dilated heart failure, suggesting the important role of Ca2+/calmodulin-dependent kinase II pathway in the development of cardiac disease.
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PMID:Targeted inhibition of Ca2+/calmodulin-dependent protein kinase II in cardiac longitudinal sarcoplasmic reticulum results in decreased phospholamban phosphorylation at threonine 17. 1269 24

The status of phospholamban (PLB) phosphorylation in the ischemia-reperfused hearts remains controversial. Although a decrease in the phosphorylation of both PLB residues (Ser16, PKA site, and Thr17, CaMKII site) was previously reported, experiments from our laboratory failed to detect this decrease. In an attempt to elucidate the cause for this discrepancy, experiments were performed in Langendorff-perfused rat hearts with two main goals: (1) To determine whether keeping pacing during ischemia, a protocol followed in other ischemia-reperfusion models, decreases the phosphorylation of PLB residues, below pre-ischemic values; (2) To investigate whether a maximal beta-adrenergic challenge allows to detect a decrease in the ability of PLB to be phosphorylated in ischemia-reperfused hearts. Hearts were submitted to a global ischemia/reperfusion protocol (20/30 min) with (P) or without (NP) pacing during ischemia, and phosphorylation of PLB residues was assessed by immunodetection. The recovery of contractility upon reperfusion was lower in P vs. NP hearts. Ser16 of PLB, was phosphorylated at the end of ischemia in NP hearts. This increase appeared earlier in P hearts and was significantly diminished by catecholamine depletion and beta-blockade. Thr17 site was phosphorylated at the beginning of ischemia and the onset of reperfusion. The ischemia-induced phosphorylation of Thr17 was higher and more sustained in P vs. NP hearts, and inhibited by the calcium channel blocker, nifedipine, whereas the reperfusion-induced increase in Thr17 phosphorylation was similar in P and NP hearts and was significantly diminished by the Na+/Ca2+ exchanger inhibitor KB-R7943. Phosphorylation of PLB residues did not decrease below basal levels at any time during ischemia and reperfusion. However, the phosphorylation, inotropic and lusitropic response to beta-adrenergic stimulation was significantly decreased both in P and NP hearts.
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PMID:Phospholamban phosphorylation in ischemia-reperfused heart. Effect of pacing during ischemia and response to a beta-adrenergic challenge. 1457 98

Initially during acidosis, Ca transient amplitude (Delta[Ca]i) and the rate constant of [Ca]i decline (k(Ca)) are decreased, but later during acidosis Delta[Ca]i and k(Ca) partially recover. This recovery in rat myocytes could be inhibited by KN-93 suggesting that CaMKII-dependent protein phosphorylation (and enhanced SR Ca uptake) may be responsible. To test whether phospholamban (PLB) is required for the Delta[Ca]i and k(Ca) recovery during acidosis, we used isolated myocytes from PLB knockout (PLB-KO) vs. wild-type (WT) mice. [Ca]i was measured using fluo-3. During the initial phase of acidosis (1-4 min), Delta[Ca]i decreased in WT myocytes (n = 8) from 1.75 +/- 0.19 to 1.10 +/- 0.13 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 3.20 +/- 0.22 to 2.38 +/- 0.18 s(-1) (P < 0.05). Later during acidosis (6-12 min), Delta[Ca]i partially recovered to 1.41 +/- 0.18 DeltaF/F0 and k(Ca) to 2.78 +/- 0.22 s(-1) (i.e. both recovered by approximately 50%). CaMKII inhibition using KN-93 completely prevented this recovery of Delta[Ca]i and k(Ca) during late acidosis in WT myocytes. In PLB-KO myocytes (n = 11) Delta[Ca]i decreased during early acidosis from 2.92 +/- 0.31 to 1.33 +/- 0.17 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 10.45 +/- 0.56 to 7.58 +/- 0.68 s(-1) (P < 0.05). However, Delta[Ca]i did not recover during late acidosis and k(Ca) decreased even more (6.59 +/- 0.65 s(-1)). Parallel results were seen for contractile parameters. We conclude that PLB is crucial to the recovery of Delta[Ca]i and k(Ca) during acidosis. Moreover, PLB phosphorylation by CaMKII plays an important role in limiting the decline in Ca transients (and contraction) during acidosis.
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PMID:Phospholamban is required for CaMKII-dependent recovery of Ca transients and SR Ca reuptake during acidosis in cardiac myocytes. 1473 43

beta-adrenergic stimulation helps to synchronize Ca release in myocytes from failing hearts. Transverse (t-) tubules, which synchronize Ca release in normal cells and contain many of the elements of the beta-adrenergic pathway, may be depleted in such cells. The objective of the present study was to determine whether beta-adrenergic stimulation could reverse the desynchronization of Ca release observed in detubulated ventricular myocytes. The effect of isoprenaline (0.5 microM) on control and detubulated rat ventricular myocytes was investigated. Ca transients were monitored using whole-cell fluorescence and confocal microscopy, and Ca current recorded using the patch-clamp technique. Immunocytochemistry was used to investigate phospholamban (PLB) phosphorylation. Detubulation reduces and slows the Ca transient; these effects were reversed by isoprenaline. This restoration was associated with partial reversal of the desynchronization of Ca release that occurs in detubulated cells. Sarcoplasmic reticulum Ca load increased by the same amount in normal and detubulated cells, but Ca current increased less in detubulated cells (64%) than in control cells (124%) in response to isoprenaline. The pattern and extent of cAMP-dependent protein kinase and CaMKII-induced phosphorylation of PLB in response to isoprenaline was the same in both cell types. Thus, the beta-adrenergic pathway is functional in the absence of t-tubules; such stimulation appears to increase the speed of propagation of Ca via Ca-induced Ca release between adjacent clusters of ryanodine receptors, which may be relevant in pathological conditions, such as heart failure, in which t-tubules are depleted. The data also suggest that the Ca current responds to local signaling pathways, which are better coupled to the channel in the t-tubules than at the surface membrane, whereas PLB responds to whole-cell signaling.
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PMID:beta-adrenergic stimulation restores the Ca transient of ventricular myocytes lacking t-tubules. 1487 54


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