Calcium/calmodulin-dependent Protein Kinase Type II Research Articles

Enhanced capacity for CaMKII signaling mitigates calcium release related contractile fatigue with high intensity exercise

BackgroundWe tested whether enhancing the capacity for calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling would delay fatigue of excitation-induced calcium release and improve contractile characteristics of skeletal muscle during fatiguing exercise. MethodsFast and slow type muscle, gastrocnemius medialis (GM) and soleus (SOL), of rats and mouse interosseus (IO) muscle fibers, were transfected with pcDNA3-based plasmids for rat α and β CaMKII or empty controls. Levels of CaMKII, its T287-phosphorylation (pT287-CaMKII), and phosphorylation of components of calcium release and re-uptake, ryanodine receptor 1 (pS2843-RyR1) and phospholamban (pT17-PLN), were quantified biochemically. Sarcoplasmic calcium in transfected muscle fibers was monitored microscopically during trains of electrical excitation based on Fluo-4 FF fluorescence (n = 5–7). Effects of low- (n = 6) and high- (n = 8) intensity exercise on pT287-CaMKII and contractile characteristics were studied in situ. ResultsCo-transfection with αCaMKII-pcDNA3/βCaMKII-pcDNA3 increased α and βCaMKII levels in SOL (+45.8 %, +250.5 %) and GM (+40.4 %, +89.9 %) muscle fibers compared to control transfection. High-intensity exercise increased pT287-βCaMKII and pS2843-RyR1 levels in SOL (+269 %, +151 %) and GM (+354 %, +119 %), but decreased pT287-αCaMKII and p17-PLN levels in GM compared to SOL (−76 % vs. +166 %; 0 % vs. +128 %). α/β CaMKII overexpression attenuated the decline of calcium release in muscle fibers with repeated excitation, and mitigated exercise-induced deterioration of rates in force production, and passive force, in a muscle-dependent manner, in correlation with pS2843-RyR1 and pT17-PLN levels (|r| > 0.7). ConclusionEnhanced capacity for α/β CaMKII signaling improves fatigue-resistance of active and passive contractile muscle properties in association with RyR1- and PLN-related improvements in sarcoplasmic calcium release.

Ser-Thr phosphatases in the rat brain that dephosphorylate phospho-Ser(1291)-GluN2A subunit of glutamate receptor

N-methyl-D-aspartate receptors (NMDARs), are one of the major ionotropic glutamate receptors found in excitatory synapses which play a key role in glutamatergic synaptic transmission. The receptors are regulated by post translational modifications such as phosphorylation. One of the major receptor subunits is GluN2A which is likely to get phosphorylated in vitro at a putative site Ser1291. However, the regulation of phosphorylation of this site by kinases and phosphatases is not yet completely understood. In the present study, we have used the fusion constructs of GluN2A tagged with glutathione S-transferase (GST) as substrate for phosphorylation, purified calcium/calmodulin dependent protein kinase type II (CaMKII) and radioactive P32. We demonstrated that the site phosphorylated by αCaMKII on GluN2A was Ser1291 and that protein phosphatases 1, 2A and 2C were able to dephosphorylate this phospho-GST-GluN2A-Ser1291 in vitro. In the rat brain tissue post synaptic density and cytosolic fraction the major phosphatase responsible for dephosphorylating phospho-GluN2A-Ser1291 was protein phosphatase 1. Keywords: CaMKII, cytosol, GluN2A-Ser1291 subunite, N-methyl-D-aspartate receptors, protein phosphatase 1, rat brain

Ginsenoside Rb3 upregulates sarcoplasmic reticulum Ca2+-ATPase expression and improves the contractility of cardiomyocytes by inhibiting the NF-κB pathway

A causal relationship between ginsenoside Rb3 (G-Rb3) and improved inflammation and cardiac function has not been established. To determine which specific signaling pathways were involved in G-Rb3 improvement of inflammation and myocardial function. In vivo, we found that G-Rb3 decreased the levels of both nuclear factor κB (NF-κB p65) and CD45, an inflammatory marker. G-Rb3 also enhanced key proteins of the contraction unit (cardiac troponin protein I (cTnI) and α-actinin) to improve cardiac function. G-Rb3 inhibited NF-κB p65 nuclear translocation in vitro, as verified by western blot and IF. When NF-κB p65 was overexpressed, a decrease in cyclic nucleotide phosphodiesterase 3B (PDE3B) and SERCA2a expression, while no statistical significance was observed in the expressions of cAMP, PKA, and calcium/calmodulin-dependent protein kinase type II (CaMKⅡ) in each group. The NF-κB p65 plasmid blocked the SERCA2a promoter, as verified by the luciferase reporter system, and G-Rb3 truncated the NF-κB p65 block on the SERCA2a promoter. qPCR was also used to confirm that G-Rb3 increased the mRNA of SERCA2a. In conclusion, we confirmed that the mechanisms of G-Rb3 on ventricular systolic dysfunction causing inflammation are not via the cAMP/PKA pathway, but via suppressing the blockage of NF-κB p65 on the SERCA2a promoter and increasing the SERCA2a expression.

The Protective Effect of Qishen Granule on Heart Failure after Myocardial Infarction through Regulation of Calcium Homeostasis.

Qishen granule (QSG) is a frequently prescribed traditional Chinese medicine formula, which improves heart function in patients with heart failure (HF). However, the cardioprotective mechanisms of QSG have not been fully understood. The current study aimed to elucidate whether the effect of QSG is mediated by ameliorating cytoplasmic calcium (Ca2+) overload in cardiomyocytes. The HF rat model was induced by left anterior descending (LAD) artery ligation surgery. Rats were randomly divided into sham, model, QSG-low dosage (QSG-L) treatment, QSG-high dosage (QSG-H) treatment, and positive drug (diltiazem) treatment groups. 28 days after surgery, cardiac functions were assessed by echocardiography. Levels of norepinephrine (NE) and angiotensin II (AngII) in the plasma were evaluated. Expressions of critical proteins in the calcium signaling pathway, including cell membrane calcium channel CaV1.2, sarcoendoplasmic reticulum ATPase 2a (SERCA2a), calcium/calmodulin-dependent protein kinase type II (CaMKII), and protein phosphatase calcineurin (CaN), were measured by Western blotting (WB) and immunohistochemistry (IHC). Echocardiography showed that left ventricular ejection fraction (EF) and fractional shortening (FS) value significantly decreased in the model group compared to the sham group, and illustrating heart function was severely impaired. Furthermore, levels of NE and AngII in the plasma were dramatically increased. Expressions of CaV1.2, CaMKII, and CaN in the cardiomyocytes were upregulated, and expressions of SERCA2a were downregulated in the model group. After treatment with QSG, both EF and FS values were increased. QSG significantly reduced levels of NE and AngII in the plasma. In particular, QSG prevented cytoplasmic Ca2+ overload by downregulating expression of CaV1.2 and upregulating expression of SERCA2a. Meanwhile, expressions of CaMKII and CaN were inhibited by QSG treatment. In conclusion, QSG could effectively promote heart function in HF rats by restoring cardiac Ca2+ homeostasis. These findings revealed novel therapeutic mechanisms of QSG and provided potential targets in the treatment of HF.

Alpha1A-adrenoceptor is involved in norepinephrine-induced proliferation of pulmonary artery smooth muscle cells via CaMKII signaling.

Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). Some studies have demonstrated the sympathetic nervous system is activated in PAH and norepinephrine (NE) released is closely linked with its activation. However, the subtypes of adrenoreceptor (AR) and the downstream molecular cascades which are involved in the proliferation of PASMCs are still unclear. In this study, adult male Wistar rats were exposed to chronic hypoxia and PASMCs were cultured in hypoxic condition. Significant upregulation of α1A -AR was identified by Western blot analysis or immunofluorescence in all of the pulmonary arteries, lung tissues, and cell hypoxic models. Western blot analysis, flow cytometry, and immunofluorescence were applied to detect the roles of α1A -AR in NE mediated proliferation of PASMCs. We revealed 5-methylurapidil (5-MU) reversed NE-induced upregulation of PCNA, CyclinA and CyclinE, more cells from G0 /G1 phase to G2 /M+S phase, enhancement of the microtubule formation. In addition, we found calcium/calmodulin(CaM)-dependent protein kinase type II (CaMKII) pathway was involved in α1A -AR-mediated cell proliferation. [Ca2+ ]i measurements showed that an increase of [Ca2+ ]i caused by NE or/and hypoxia could be blocked by 5-MU in PASMCs. Western blot analysis results demonstrated the augmentation of CaMKII phosphorylation level was caused by hypoxia or NE in pulmonary arteries, lung tissues, and PASMCs. KN62 attenuated NE-induced proliferation of PASMCs under normoxia and hypoxia. In conclusion, those results suggested NE which stimulated α1A -AR-mediated the proliferation of PASMCs, which may be via the CaMKII pathway, and it could be used as a novel treatment strategy in PAH.

Genotype-Dependent and -Independent Calcium Signaling Dysregulation in Human Hypertrophic Cardiomyopathy.

Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03). 45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.

Lipopolysaccharide-induced serotonin transporter up-regulation involves PKG-I and p38MAPK activation partially through A3 adenosine receptor.

Serotonin transporter (SERT) is a critical determinant of synaptic serotonin (5-hydroxytryptamine, 5-HT) inactivation which plays a critical role in the pathology of depression and other mood disorders. Lipopolysaccharide (LPS), a potent activator of the inflammatory system, has been reported to cause depression symptoms by the modulation of SERT in vivo and in vitro. This study is aimed to investigate the underlying mechanism of LPS-induced SERT modulation. The 4-(4-(dimethylamino) styryl)-N-methylpyridinium iodide (ASP) assay was used to detect dynamic 5-HT uptake as read out of SERT activities in RBL-2H3 cells, and cytosol Ca(2+) concentrations ([Ca(2+)]i) and nitric oxide (NO) were examined. Using specific cyclic GMP-dependent protein kinase type I (PKG-I), p38 mitogen-activated protein kinases (p38MAPK) and A3 adenosine receptor (A3AR) inhibitors, SERT expression was evaluated by western blot and immunofluorescence analysis. Results showed that 24 h treatment with LPS stimulated 5-HT transport and up-regulate plasma membrane distribution of SERT in RBL-2H3 cells. LPS treatment increased NO and [Ca(2+)]i, and led to significant increases in levels of phosphorylated calcium/calmodulin-dependent protein kinase type II (CaMK-II), inducible NOS (iNOS) and PKG-I as well as active p38 MAPK. Moreover, PKG-I inhibitor KT5823 or p38MAPK inhibitor SB203580 respectively impaired SERT activation and transposition to plasma membrane by LPS. Notably, A3 adenosine receptor inhibitor MRS1191 also hindered SERT stimulation by LPS. In conclusion, LPS-induced 5-HT uptake and transposition to plasma membrane of SERT in RBL-2H3 cells involves CaMK-II/iNOS/PKG-I and p38 MAPK activation, which may be partially mediated by A3 adenosine receptor activation. This finding provides a novel insight into the interrelationship between LPS and depression.

Nociceptin/orphanin FQ-induced inhibition of delayed rectifier potassium currents by calcium/calmodulin-dependent protein kinase type II.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been shown to inhibit delayed rectifier potassium current (IK) in acutely dissociated rat parietal cortical neurons. However, the detailed mechanism of N/OFQ-induced inhibition on IK is not clear. This study is the first to explore an involvement of calcium/calmodulin (CaM)-dependent protein kinase type II (CaMKII) in mediating N/OFQ-induced responses. Utilizing pharmacological inhibitors of CaM and CaMKII, we have investigated the contribution of CaMKII in N/OFQ-induced effects using the whole-cell patch-clamp technique. In whole-cell voltage clamp, W-7 (100 μM), an antagonist of CaM, as well as KN-62, an inhibitor of CaMKII activity, attenuated the inhibitory effects of N/OFQ on IK. Activation and inactivation analysis indicated that the kinetics of IK were altered by N/OFQ, with decreased activation and promoted inactivation of IK. W-7 and KN-62 (10 μM) partly abolished the activation and inactivation curves shift of IK induced by N/OFQ. These findings show that CaMKII plays a critical role in N/OFQ-induced inhibition of IK in acutely dissociated rat parietal cortical neurons.

Disrupted cortical function underlies behavior dysfunction due to social isolation

Stressful events during early childhood can have a profound lifelong influence on emotional and cognitive behaviors. However, the mechanisms by which stress affects neonatal brain circuit formation are poorly understood. Here, we show that neonatal social isolation disrupts molecular, cellular, and circuit developmental processes, leading to behavioral dysfunction. Neonatal isolation prevented long-term potentiation and experience-dependent synaptic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors normally occurring during circuit formation in the rodent barrel cortex. This inhibition of AMPA receptor trafficking was mediated by an increase of the stress glucocorticoid hormone and was associated with reduced calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling, resulting in attenuated whisker sensitivity at the cortex. These effects led to defects in whisker-dependent behavior in juvenile animals. These results indicate that neonatal social isolation alters neuronal plasticity mechanisms and perturbs the initial establishment of a normal cortical circuit, which potentially explains the long-lasting behavioral effects of neonatal stress.

Neuronal excitability and calcium/calmodulin‐dependent protein kinase type II: Location, location, location

Calcium/calmodulin-dependent protein kinase type II (CaMKII) is a highly abundant serine/threonine kinase comprising a significant fraction of total protein in mammalian forebrain and forming a major component of the postsynaptic density. CaMKII is essential for certain forms of synaptic plasticity and memory consolidation and this is mediated through substrate binding and intramolecular phosphorylation of holoenzyme subunits. CaMKII is multifunctional; it targets a variety of cellular substrates, and this diversity depends on holoenzyme subunit composition. CaMKII comprises homooligomeric and heterooligomeric complexes generated from four subunits (α, β, δ, and γ) encoded by separate genes that are further expanded by extensive alternative splicing to more than 30 different isoforms. Much attention has been paid to understanding the regulation of CaMKII function through its structural diversity and/or substrate specificity. However, given the importance of subunit composition to holoenzyme activity, it is likely that specificity of cellular expression of CaMKII isoforms also plays a major role in regulation of enzyme function. Herein we review the cellular colocalization of CaMKII isoforms with special regard to the cell-type specificity of isoform expression in brain. In addition, we highlight the remarkable specificity of subcellular localization by the CaMKIIα isoform. In addition, we discuss the role that this cellular specificity of expression might play in propagating the type of recurrent neuronal activity associated with disorders such as temporal lobe epilepsy.

Identification of Pin1-Binding Phosphorylated Proteins in the Mouse Brain

To determine the role of Pin1 in the neurotransmission pathway, Pin1-binding proteins in mouse brain extract were identified. The Pin1-binding proteins were extracted from mouse brain homogenate, and the trypsin-digested peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins that involve the neurotransmission pathway, such as synapsin I, synapsin II, and calcium/calmodulin-dependent protein kinase type II (CaMKII), were identified in a Mascot search. Pull-down and immunoprecipitation assay indicated that Pin1 binds CaMKII in a phosphorylation-specific manner. It was assumed that Pin1 participates in the neurotransmission pathway involving the phosphorylation signal by CaMKII.

The C-terminus of CaMKII is truncated when expressed in E. coli.

The neuronal enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key molecule in biochemical events necessary for learning and memory. The alpha-subunit of CaMKII expressed in E. coli as well as in insect cells shows similar catalytic behavior [Praseeda, M., Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J. In Press]. The association domain of the enzyme has been crystallized in its native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product accompanies the full-length protein when expressed in E. coli. We show by epitope labeling and immunoblotting that the truncation occurs at the C-terminal half of the protein so that the N-terminal catalytic domain is complete in the truncated product. This supports the use of the preparation of alpha-CaMKII expressed in E. coli for studies on functions of the catalytic site. Our data will also be helpful in designing modified prokaryotic expression systems for CaMKII devoid of the trun-cation product, which are easier to use compared to the insect cell system.

Phosphorylation of Ca 2+/calmodulin-dependent protein kinase type ii and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet

The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and responds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c- fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related increase in neuronal excitability, but previous studies have shown effects on intracellular Ca 2+ in deficient APC slices in vitro. In the present study we hypothesized that indispensable amino acid deficiency increases intraneuronal Ca 2+, resulting in autophosphorylation of calcium/calmodulin-dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine-devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit returned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex. These findings support the notion that calcium and glutamate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII.

Sequence determinants on the NR2A and NR2B subunits of NMDA receptor responsible for specificity of phosphorylation by CaMKII

Calcium/calmodulin-dependent protein kinase type II (CaMKII) and NMDA-type glutamate receptor (NMDAR) are neuronal proteins involved in learning and memory. CaMKII binds to the NR2B subunit of NMDAR in more than one mode, a stable association involving a noncatalytic site on CaMKII and an enzyme–substrate mode of interaction by its catalytic site. The latter binding results in phosphorylation of serine-1303 on NR2B. We have investigated this binding by studying the kinetics of phosphorylation of synthetic peptides harboring nested sequences of the phosphorylation site motif. We find that residues 1292–1297 of NR2B enhance the affinity of the catalytic site-mediated binding of CaMKII to the minimal phosphorylation site motif, 1298–1308 of NR2B, as evident from measurements of K m values for phosphorylation. However, CaMKII shows decreased affinity towards the closely related NR2A subunit due to an -Ile-Asn- motif present as a natural insertion in the analogous sequence on NR2A.

PKC and CaMKII dependent synaptic potentiation in cultured cerebral neurons

We have reported that the long-lasting potentiation of spontaneous excitatory postsynaptic currents (SEPSCs) was induced by a Mg 2+-free treatment in cultured chick cerebral neurons and a factor(s) extracellularly released during the treatment could induce the potentiation by itself. In this paper, protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase type II (CaMKII) but not protein kinase A (PKA) were reported to contribute to the potentiation mechanism during a step between the activation of the N-methyl- d-aspartate receptors by the Mg 2+-free treatment and the secretion of the protein factor(s).

Decreased expression of the alpha subunit of Ca2+/ calmodulin-dependent protein kinase type II mRNA in the adult rat CNS following recurrent limbic seizures.

Calcium/calmodulin-dependent protein kinase type II (CamKII) is a ubiquitous brain enzyme implicated in a wide variety of neuronal processes. Understanding CamKII has become increasingly complicated with the recent identification of multiple gene transcripts coding for separate subunits. Previous studies have shown that mRNA for the alpha subunit of CamKII can be increased by reduction of afferent input. In this study we have examined the regulation of alpha CamKII mRNA following increased activity due to seizures. Using in situ hybridization with a cRNA probe against the rat alpha CamKII sequence we found reduced levels of hybridization following limbic seizures induced by lesions of the hilus of the dentate gyrus. Hybridization was most dramatically reduced in the granule cells of the dentate gyrus and the pyramidal cells of hippocampal region CA1. There were also significant reductions in hybridization in the superficial layers of neocortex and piriform cortex. In each of these region hybridization was decreased in the molecular layers which is consistent with the reported dendritic localization of alpha CamKII mRNA. All changes in mRNA content were transient, with maximal reductions at 24 h following lesion placement and a return to control levels by 96 h. These findings demonstrate the negative regulation of alpha CamKII mRNA by seizure activity and raise the possibility that synthesis of this kinase may be regulated by normal physiological activity.