Calcium/calmodulin-dependent Protein Kinase II Phosphorylation Research Articles

Costunolide covalently targets and inhibits CaMKII phosphorylation to reduce ischemia-associated brain damage

BackgroundChronic cerebral hypoperfusion (CCH) is a leading cause of disability and mortality worldwide. Restoring cerebral blood flow (CBF) through vasodilatation is particularly important in the treatment of CCH. Costunolide (Cos) is a natural sesquiterpenoid compound with vasodilatory effect, but its mechanism is unclear. PurposeThis study aimed to investigate the vasodilatory mechanism of Cos and provide a new therapeutic regimen for treating CCH. MethodsThe therapeutic effect of Cos on CCH was assessed in a rat model of permanent common carotid artery occlusion. The direct target protein for improving CBF was identified by drug affinity responsive target stability combined with quantitative differential proteomics analysis. The molecular mechanism of Cos acting on its target protein was analyzed by multidisciplinary approaches. The signalling involved was assessed using site-directed pharmacological intervention. ResultsCos has a significant therapeutic effect on ischemic brain injury by restoring CBF. Multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) was identified as a direct target of the natural small molecule Cos with a therapeutic effect on CCH. Mechanistic studies revealed that the α,β-unsaturated-γ-lactone ring of Cos covalently binds to the Cys116 residue of CaMKII. It then inhibits the phosphorylation of CaMKII and reduces the calcium concentration in vascular smooth muscle cells, thus playing a role in vasodilation and increasing CBF. Notably, this covalent binding between Cos and CaMKII can exert a long-term vasodilator activity. ConclusionWe reported for the first time that Cos reduced ischemia-associated brain damage by covalently binding to the Cys116 residue of CaMKII, inhibiting CaMKII phosphorylation, and exerting long-term vasodilatory activity. This study not only found a new covalent inhibitor against the phosphorylation of CaMKII but also suggested that pharmacologically targeting CaMKII is a promising therapeutic strategy for CCH.

Alpha-Asarone Ameliorates Neurological Dysfunction of Subarachnoid Hemorrhagic Rats in Both Acute and Recovery Phases via Regulating the CaMKII-Dependent Pathways.

Early brain injury (EBI) is the leading cause of poor prognosis for patients suffering from subarachnoid hemorrhage (SAH), particularly learning and memory deficits in the repair phase. A recent report has involved calcium/calmodulin-dependent protein kinase II (CaMKII) in the pathophysiological process underlying SAH-induced EBI. Alpha-asarone (ASA), a major compound isolated from the Chinese medicinal herb Acorus tatarinowii Schott, was proven to reduce secondary brain injury by decreasing CaMKII over-phosphorylation in rats' model of intracerebral hemorrhage in our previous report. However, the effect of ASA on SAH remains unclear, and the role of CaMKII in both acute and recovery stages of SAH needs further investigation. In this work, we first established a classic SAH rat model by endovascular perforation and intraperitoneally administrated different ASA doses (10, 20, and 40mg/kg) 2h after successful modeling. Then, the short- and long-term neurobehavioral performances were blindly evaluated to confirm ASA's efficacy against SAH. Subsequently, we explored ASA's therapeutic mechanism in both acute and recovery stages using histopathological examination, TUNEL staining, flow cytometry, Western-blot, double-immunofluorescence staining, and transmission electron microscopy (TEM) observation. Finally, KN93, a selective CaMKII inhibitor, was applied in oxyhemoglobin-damaged HT22 cells to explore the role of CaMKII in ASA's neuroprotective effect. The results demonstrated that ASA alleviated short- and long-term neurological dysfunction, reduced mortality and seizure rate within 24h, and prolonged 14-day survival in SAH rats. Histopathological examination showed a reduction of neuronal damage and a restoration of the hippocampal structure after ASA treatment in both acute and recovery phases of SAH. In the acute stage, the Western-blot and flow cytometer analyses showed that ASA restored E/I balance, reduced calcium overload and CaMKII phosphorylation, and inhibited mitochondrion-involved apoptosis, thus preventing neuronal damage and apoptosis underlying EBI post-SAH. In the recovery stage, the TEM observation, double-immunofluorescence staining, and Western-blot analyses indicated that ASA increased the numbers of synapses and enhanced synaptic plasticity in the ipsilateral hippocampi, probably by promoting NR2B/CaMKII interaction and activating subsequent CREB/BDNF/TrkB signaling pathways. Furthermore, KN93 notably reversed ASA's neuroprotective effect on oxyhemoglobin-damaged HT22 cells, confirming CaMKII a potential target for ASA's efficacy against SAH. Our study confirmed for the first time that ASA ameliorated the SAH rats' neurobehavioral deterioration, possibly via modulating CaMKII-involved pathways. These findings provided a promising candidate for the clinical treatment of SAH and shed light on future drug discovery against SAH.

CaMKII Inhibition Attenuates Distinct Gain-of-Function Effects Produced by Mutant Nav1.6 Channels and Reduces Neuronal Excitability.

Aberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the SCN8A gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.

UVR Promotes Keratinocyte Phagocytosis and Skin Pigmentation Through TRPA1 Channels.

PurposeUltraviolet radiation (UVR) enhances skin pigmentation, which involves the production of melanin by melanocytes and subsequent transfer to keratinocytes. In the epidermis, keratinocyte phagocytosis plays a pivotal role in the process of melanosome transfer to protect DNA of epidermal cells against damage from UVR. Previous research suggested that transient receptor potential channels ankyrin 1 (TRPA1) was required for UVR-induced early melanin synthesis in melanocytes. Currently, there is no evidence that supports the detailed mechanism of TRPA1 for UVR-induced phagocytosis by keratinocytes. Here, we investigated the effect and the possible mechanisms of TRPA1 on keratinocyte phagocytosis and skin pigmentation after UVR exposure.MethodsFlow cytometry was applied to investigate the effect of TRPA1 on intracellular calcium concentration ([Ca2+]ic) and fluorescent microspheres uptake was carried out to analyze phagocytosis in HaCaT cells (human immortalized keratinocytes). Western blotting was applied to measure the protein expression of calcium/calmodulin-dependent protein kinase II (CaMKII), phosphorylated CaMKII and β-catenin after UVA/UVB exposure. Masson-Fontana staining was applied to observe the effect of XAV-939 (decreasing the expression of β-catenin) on UVB-induced skin pigmentation in guinea pigs.ResultsTRPA1 channels activated by UVR increased the [ca2+]ic and phosphorylation of CaMKII in HaCaT cells. The UVR-induced phagocytosis was regulated by TRPA1 in HaCaT cells. TRPA1 promoted the protein expression of β-catenin after UVR exposure in HaCaT cells. XAV-939, inhibiting β-catenin expression, decreased the UVB-induced skin pigmentation on in vivo guinea pig models.ConclusionTaken together, TRPA1 activated by UVR led to the increase of intracellular calcium, which promoted the phosphorylation of CaMKII, enhancing keratinocyte phagocytosis. Moreover, TRPA1 regulated the protein expression of β-catenin to exert a lightening effect on skin pigmentation. Our findings suggest that TRPA1 may be a potential therapeutic target for UVR-induced skin pigmentary diseases.

Trans-cinnamaldehyde protects against phenylephrine-induced cardiomyocyte hypertrophy through the CaMKII/ERK pathway

BackgroundTrans-cinnamaldehyde (TCA) is one of the main pharmaceutical ingredients of Cinnamomum cassia Presl, which has been shown to have therapeutic effects on a variety of cardiovascular diseases. This study was carried out to characterize and reveal the underlying mechanisms of the protective effects of TCA against cardiac hypertrophy.MethodsWe used phenylephrine (PE) to induce cardiac hypertrophy and treated with TCA in vivo and in vitro. In neonatal rat cardiomyocytes (NRCMs), RNA sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out to identify potential pathways of TCA. Then, the phosphorylation and nuclear localization of calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-related kinase (ERK) were detected. In adult mouse cardiomyocytes (AMCMs), calcium transients, calcium sparks, sarcomere shortening and the phosphorylation of several key proteins for calcium handling were evaluated. For mouse in vivo experiments, cardiac hypertrophy was evaluated by assessing morphological changes, echocardiographic parameters, and the expression of hypertrophic genes and proteins.ResultsTCA suppressed PE-induced cardiac hypertrophy and the phosphorylation and nuclear localization of CaMKII and ERK in NRCMs. Our data also demonstrate that TCA blocked the hyperphosphorylation of ryanodine receptor type 2 (RyR2) and phospholamban (PLN) and restored Ca2+ handling and sarcomere shortening in AMCMs. Moreover, our data revealed that TCA alleviated PE-induced cardiac hypertrophy in adult mice and downregulated the phosphorylation of CaMKII and ERK.ConclusionTCA has a protective effect against PE-induced cardiac hypertrophy that may be associated with the inhibition of the CaMKII/ERK pathway.

MLKL and CaMKII Are Involved in RIPK3-Mediated Smooth Muscle Cell Necroptosis.

Receptor interacting protein kinase 3 (RIPK3)-mediated smooth muscle cell (SMC) necroptosis has been shown to contribute to the pathogenesis of abdominal aortic aneurysms (AAAs). However, the signaling steps downstream from RIPK3 during SMC necroptosis remain unknown. In this study, the roles of mixed lineage kinase domain-like pseudokinase (MLKL) and calcium/calmodulin-dependent protein kinase II (CaMKII) in SMC necroptosis were investigated. We found that both MLKL and CaMKII were phosphorylated in SMCs in a murine CaCl2-driven model of AAA and that Ripk3 deficiency reduced the phosphorylation of MLKL and CaMKII. In vitro, mouse aortic SMCs were treated with tumor necrosis factor α (TNFα) plus Z-VAD-FMK (zVAD) to induce necroptosis. Our data showed that both MLKL and CaMKII were phosphorylated after TNFα plus zVAD treatment in a time-dependent manner. SiRNA silencing of Mlkl-diminished cell death and administration of the CaMKII inhibitor myristoylated autocamtide-2-related inhibitory peptide (Myr-AIP) or siRNAs against Camk2d partially inhibited necroptosis. Moreover, knocking down Mlkl decreased CaMKII phosphorylation, but silencing Camk2d did not affect phosphorylation, oligomerization, or trafficking of MLKL. Together, our results indicate that both MLKL and CaMKII are involved in RIPK3-mediated SMC necroptosis, and that MLKL is likely upstream of CaMKII in this process.

CaMKII is activated in opioid induced conditioned place preference, but αCaMKII Thr286 autophosphorylation is not necessary for its establishment

Activation of calcium/calmodulin-dependent protein kinase II (CaMKII), particularly its α isoform, is known to be important for neuronal processes central for learning and memory and has also been implicated in the maladaptive learning involved in drug addiction.Thr286 autophosphorylation of αCaMKII has been shown to be indispensable for establishment of cocaine-induced CPP (Easton et al., 2014). To study the contribution of CaMKII in opioid induced conditioned learning, we examined how establishment of conditioned place preference (CPP) induced by 10 or 30 μmol/kg morphine or its active metabolite morphine-6-glucuronide (M6G) affects the levels and Thr286 autophosphorylation of the α- and β-isoforms of CaMKII, as well as β-actin levels, in dorsal and ventral striatum and in hippocampus of mice. An acute and a sub-chronic treatment were used as controls. Whereas an acute single administration of morphine or M6G caused increases in CaMKII levels and phosphorylation at Thr286 and β-actin in striatal areas, CPP induced by these opioids was accompanied primarily by an increase in the protein levels of both CaMKII isoforms and β-actin in dorsal striatum and hippocampus. Decreases in CaMKII Thr286 phosphorylation were observed in dorsal striatum after the sub-chronic pharmacological treatment. Despite the changes observed in αCaMKII activity in wild type mice, morphine-induced CPP was not affected in αCaMKIIT286A autophosphorylation-deficient mice. These results indicate that opioid-induced CPP is accompanied by activation of α- and βCaMKII in striatum and hippocampus, but, in opposition to what has been observed with cocaine, αCaMKII autophosphorylation is not essential for establishment of opioid-induced CPP.

Mechanism of deoxynivalenol-induced neurotoxicity in weaned piglets is linked to lipid peroxidation, dampened neurotransmitter levels, and interference with calcium signaling.

Deoxynivalenol（DON） has broad toxicity in livestock, but we know little about its neurotoxic mechanisms. We investigated DON neurotoxicity in the cerebral cortex, cerebellum, and hippocampus of "Duroc×Landrace×Yokshire" piglets. Control piglets were fed a basal diet, while those in low- and high-treatment groups were fed diets with 1.3mg/kg and 2.2mg/kg DON, respectively. After a 60d trial, scanning electron microscopy revealed the destruction of hippocampal cell ultrastructure. As DON concentrations increased, oxidative damage also increased in the cerebral cortex, cerebellum, and hippocampus. Norepinephrine and 5-hydroxytryptamine concentrations tended to increase, whereas dopamine and γ-aminobutyric acid concentrations decreased. We also observed an increase in calcium concentration, relative mRNA expression of calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII phosphorylation. However, calmodulin (CaM) mRNA and protein content decreased. Overall, our results suggest that DON acts through the Ca2+/CaM/CaMKII signaling pathway to influence cerebral lipid peroxidation and neurotransmitter levels.

Tocopherol suppresses 24(S)-hydroxycholesterol-induced cell death via inhibition of CaMKII phosphorylation

Although 24(S)-hydroxycholesterol (24S-OHC) plays an important role to maintain homeostasis of cholesterol in the brain, it induces neuronal cell death at high concentrations. 24S-OHC-induced cell death was suppressed by γ-tocopherol (γ-Toc) but not by γ-tocotrienol (γ-Toc3) in a similar way to α-tocopherol (α-Toc) and α-tocotrienol (α-Toc3) in human neuroblastoma SH-SY5Y cells. Both γ-Toc and γ-Toc3 significantly inhibited cumene hydroperoxide-induced cell death, as previously shown in the case of α-Toc and α-Toc3. Lipid droplet-like structure formation induced by 24S-OHC was suppressed by neither γ-Toc nor γ-Toc3. The phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) was induced by 24S-OHC, which was suppressed by CaMKII phosphorylation-site inhibitor mM3 but not by calmodulin-binding-site inhibitor KN62. A calcium chelator, BAPTA-AM, inhibited calcium ionophore A23187-induced CaMKII phosphorylation but not 24S-OHC-induced CaMKII phosphorylation. Receptor-interacting protein kinase 1 (RIPK1) phosphorylation induced by 24S-OHC was not inhibited by either mM3 or KN62, suggesting that CaMKII activation does not affect RIPK1 phosphorylation. Knockdown of RIPK1 using siRNA induced not only inhibition of CaMKII phosphorylation but also reduction of total CaMKII protein levels, suggesting that RIPK1 may regulate CaMKII signalling. 24S-OHC-induced RIPK1 phosphorylation was inhibited by neither α-Toc nor α-Toc3. In contrast, CaMKII phosphorylation induced by 24S-OHC was significantly suppressed by α-Toc but not by α-Toc3. These results suggest that CaMKII activation is involved in the mechanism of 24S-OHC-induced cell death and that Toc inhibits the cell death via inhibition of CaMKII activation through a RIPK1 phosphorylation-independent pathway.

Abstract 444: Role of CaMKII in Atherosclerotic Plaque Progression in ApoE-Deficient Mice

Non-obstructive mixed atherosclerotic plaques are an independent risk factor in predicting mortality in coronary artery disease patients. The multifactorial process of plaque progression and complication is a result of proliferation and migration of VSMCs and macrophages, apoptosis and calcification. A number of osteogenic regulators and inflammatory stimuli driving these processes in plaque advancement have been identified. Yet rupture, a result of complex mixed plaques, remains the leading cause of fatal cardiovascular events. Further understanding of the mechanisms underlying plaque progression is required to identify potential targets for therapies. Key signalling factors that modulate cell proliferation and migration, which potentially underlie plaque complication include the nodal multifunctional calcium/calmodulin dependent protein kinase II (CaMKII). Thus we hypothesized that, as plaques progress in a mouse model of atherosclerosis (ApoE -/- ), CaMKII activity would increase. We also hypothesized that systemic inhibition of CaMKII would impede plaque progression. Our data show a trend towards increased CaMKII phosphorylation, an indirect measure of kinase activity, in plaques as mice advance from 26 to 30 weeks. Pharmacological inhibition of CaMKII with KN-93, on alternate days for 4 weeks, in 30 week old mice reduced the incidence of calcification from 50% (KN-92 control: 4 out of 8) to 25% (KN-93: 2 out of 8). This was associated with a trend towards reduced calcification area (KN-92 control: 0.43mm 2 vs. KN-93: 0.06mm 2 ). These results suggest that CaMKII is activated during atherosclerosis and may be involved with plaque progression through calcification.

Biophysical attributes that affect CaMKII activation deduced with a novel spatial stochastic simulation approach.

Calcium/calmodulin-dependent protein kinase II (CaMKII) holoenzymes play a critical role in decoding Ca2+ signals in neurons. Understanding how this occurs has been the focus of numerous studies including many that use models. However, CaMKII is notoriously difficult to simulate in detail because of its multi-subunit nature, which causes a combinatorial explosion in the number of species that must be modeled. To study the Ca2+-calmodulin-CaMKII reaction network with detailed kinetics while including the effect of diffusion, we have customized an existing stochastic particle-based simulator, Smoldyn, to manage the problem of combinatorial explosion. With this new method, spatial and temporal aspects of the signaling network can be studied without compromising biochemical details. We used this new method to examine how calmodulin molecules, both partially loaded and fully loaded with Ca2+, choose pathways to interact with and activate CaMKII under various Ca2+ input conditions. We found that the dependence of CaMKII phosphorylation on Ca2+ signal frequency is intrinsic to the network kinetics and the activation pattern can be modulated by the relative amount of Ca2+ to calmodulin and by the rate of Ca2+ diffusion. Depending on whether Ca2+ influx is saturating or not, calmodulin molecules could choose different routes within the network to activate CaMKII subunits, resulting in different frequency dependence patterns. In addition, the size of the holoenzyme produces a subtle effect on CaMKII activation. The more extended the subunits are organized, the easier for calmodulin molecules to access and activate the subunits. The findings suggest that particular intracellular environmental factors such as crowding and calmodulin availability can play an important role in decoding Ca2+ signals and can give rise to distinct CaMKII activation patterns in dendritic spines, Ca2+ channel nanodomains and cytoplasm.

Calcium/calmodulin-dependent protein kinase II regulation of IKs during sustained β-adrenergic receptor stimulation

Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P=.04) but not at T482 (P = .17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P < .01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P < .01) but not in the dephosphorylated S484 mimic (P = .99). CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Urotensin II induction of neonatal cardiomyocyte hypertrophy involves the CaMKII/PLN/SERCA 2a signaling pathway

Although studies have shown that Urotensin II (UII) can induce cardiomyocyte hypertrophy and UII-induced cardiomyocyte hypertrophy model has been widely used for hypertrophy research, but its precise mechanism remains unknown. Recent researches have demonstrated that UII-induced cardiomyocyte hypertrophy has a relationship with the changes of intracellular Ca2+ concentration. Therefore, the aim of this study was to investigate the mechanisms of cardiomyocyte hypertrophy induced by UII and to explore whether the calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulating of phospholamban (PLN) Thr17–phosphorylation signaling pathway contributed to UII-induced cardiomyocyte hypertrophy. Primary cultures of neonatal rat cardiomyocytes were stimulated for 48h with UII. Cell size, protein/DNA contents and intracellular Ca2+ were determined. Phosphorylated and total forms of CaMKII, PLN and the total amount of serco/endo-plasmic reticulum ATPases (SERCA 2a) were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the CaMKII inhibitor, KN-93. These results showed that UII increased cell size, protein/DNA ratio and intracellular Ca2+, consistent with a hypertrophic response. Furthermore, the phosphorylation of CaMKII and its downstream target PLN (Thr17), SERCA 2a levels were up-regulated by UII treatment. Conversely, treatment with KN-93 reversed all those effects of UII. Taken together, the results suggest that UII can induce cardiomyocyte hypertrophy through CaMKII-mediated up-regulating of PLN Thr17–phosphorylation signaling pathway.

Ischemic preconditioning protects the brain against injury via inhibiting CaMKII–nNOS signaling pathway

Although studies have shown that cerebral ischemic preconditioning (IPC) can ameliorate ischemia/reperfusion (I/R) induced brain damage, but its precise mechanisms remain unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of IPC against ischemic brain damage induced by cerebral I/R and to explore whether the Calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway contributed to the protection provided by IPC. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with 3min of IPC alone or KN62 (selective antagonist of CaMKII) treatment before IPC, after reperfusion for 3 days, 6min ischemia was induced. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of CaMKII, nNOS, c-Jun and the expression of FasL. Immunoprecipitation was used to examine the binding between PSD95 and nNOS. The results showed that IPC could significantly protect neurons against cerebral I/R injury, furthermore, the combination of PSD95 and nNOS was increased, coinstantaneously the phosphorylation of CaMKII and nNOS (ser847) were up-regulated, however the activation of c-Jun and FasL were reduced. Conversely, KN62 treatment before IPC reversed all these effects of IPC. Taken together, the results suggest that IPC could diminish ischemic brain injury through CaMKII-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway.

Exercise-induced metabolic fluctuations influence AMPK, p38-MAPK and CaMKII phosphorylation in human skeletal muscle.

During transition from rest to exercise, metabolic reaction rates increase substantially to sustain intracellular ATP use. These metabolic demands activate several kinases that initiate signal transduction pathways which modulate transcriptional regulation of mitochondrial biogenesis. The purpose of this study was to determine whether metabolic fluctuations per se affect the signaling cascades known to regulate peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). On two separate occasions, nine men performed a continuous (30-min) and an intermittent exercise (30 × 1-min intervals separated by 1-min of recovery) at 70% of . Skeletal muscle biopsies from the vastus lateralis were taken at rest and at +0 h and +3 h after each exercise. Metabolic fluctuations that correspond to exercise-induced variation in metabolic rates were determined by analysis of VO2 responses. During intermittent exercise metabolic fluctuations were 2.8-fold higher despite identical total work done to continuous exercise (317 ± 41 vs. 312 ± 56 kJ after intermittent and continuous exercise, respectively). Increased phosphorylation of AMP-activated protein kinase (AMPK) (˜2.9-fold, P < 0.01), calcium/calmodulin-dependent protein kinase II (CaMKII) (˜2.7-fold, P < 0.01) and p38-mitogen-activated protein kinase (MAPK) (˜4.2-fold, P < 0.01) occurred immediately in both exercises and to a greater extent after the intermittent exercise (condition x time interaction, P < 0.05). A single bout of intermittent exercise induces a greater activation of these signaling pathways regulating PGC-1α when compared to a single bout of continuous exercise of matched work and intensity. Chronic adaptations to exercise on mitochondria biogenesis are yet to be investigated.

Intrathecal injection of KN93 attenuates paradoxical remifentanil-induced postoperative hyperalgesia by inhibiting spinal CaMKII phosphorylation in rats

AimsRemifentanil is a short-acting and highly selective mu opiate agonist that is used in many clinical surgical situations for intraoperative pain relief. Under certain conditions, remifentanil can produce “paradoxical” hyperalgesia. This study aims to investigate mechanisms of actions mediating this “paradoxical” effect. MethodsSprague–Dawley rats were divided into 6 groups including control and treatment groups. The paw withdrawal mechanical threshold and the paw withdrawal thermal latency of the rats were tested. The changes of rat behaviors were measured at 24h before intrathecal injection and at 2h, 6h, 24h, and 48h after operation. According to the changes in behavioral indicators of pain, the specimens of all groups were collected at 2h, 6h, 24h, and 48h after the operation. The level of calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in the spinal dorsal horn was analyzed by Western blotting. ResultsIntraoperative infusion of remifentanil induced postoperative hyperalgesia in the rats. Intrathecal KN93 injection increased nociceptive thresholds of paw withdrawal mechanical threshold and paw withdrawal thermal latency in a dose-dependent manner. Western blotting results showed that CaMKII phosphorylation in the spinal dorsal horn was increased significantly by remifentanil. Inhibition of CaMKII phosphorylation relieved the hyperalgesia pain state. ConclusionsIntrathecal injection of KN93 attenuates postoperative hyperalgesia induced by intraoperative infusion of remifentanil in rats through inhibiting spinal CaMKII phosphorylation.

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition ameliorates arrhythmias elicited by junctin ablation under stress conditions.

Aberrant calcium signaling is considered one of the key mechanisms contributing to arrhythmias, especially in the context of heart failure. In human heart failure, there is significant down-regulation of the sarcoplasmic reticulum (SR) protein junctin, and junctin deficiency in mice is associated with stress-induced arrhythmias. The purpose of this study was to determine whether the increased SR Ca(2+) leak and arrhythmias associated with junctin ablation may be associated with increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phosphorylation of the cardiac ryanodine receptor (RyR2) and whether pharmacologic inhibition of CaMKII activity may prevent these arrhythmias. Using a combination of biochemical, cellular, and in vivo approaches, we tested the ability of KN-93 to reverse aberrant CaMKII phosphorylation of RyR2. Specifically, we performed protein phosphorylation analysis, in vitro cardiomyocyte contractility and Ca(2+) kinetics, and in vivo ECG analysis in junctin-deficient mice. In the absence of junctin, RyR2 channels displayed CaMKII-dependent hyperphosphorylation. Notably, CaMKII inhibition by KN-93 reduced the in vivo incidence of stress-induced ventricular tachycardia by 65% in junctin null mice. At the cardiomyocyte level, KN-93 reduced the percentage of junctin null cells exhibiting spontaneous Ca(2+) aftertransients and aftercontractions under stress conditions by 35% and 37%, respectively. At the molecular level, KN-93 blunted the CaMKII-mediated hyperphosphorylation of RyR2 and phospholamban under stress conditions. Our data suggest that CaMKII inhibition is effective in preventing arrhythmogenesis in the setting of junctin ablation through modulation of both SR Ca(2+) release and uptake. Thus, it merits further investigation as promising molecular therapy.

HsBAFF promotes proliferation and survival in cultured B lymphocytes via calcium signaling activation of mTOR pathway

B-cell activating factor of the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) has revealed its critical function in B lymphocyte proliferation and survival, as well as the pathogenesis of autoimmune disease. However, the molecular mechanisms of excess BAFF-extended aggressive B lymphocytes have not been completely defined. Here we show that excessive hsBAFF-elevated [Ca2+]i activated mammalian target of rapamycin (mTOR) signaling pathway, leading to proliferation and survival in B lymphocytes. This is supported by the findings that intracellular Ca2+ chelator (BAPTA/AM) or mTOR inhibitor (rapamycin) abolished the events. Sequentially, we observed that preventing [Ca2+]i elevation using EGTA or 2-APB dramatically inhibited hsBAFF activation of mTOR signaling, as well as cell growth and survival, suggesting that hsBAFF-induced extracellular Ca2+ influx and ER Ca2+ release elevates [Ca2+]i contributing to B lymphocyte proliferation and survival via activation of mTOR signaling. Further, we noticed that pretreatment with BAPTA/AM, EGTA or 2-APB blocked hsBAFF-increased phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibiting CaMKII with KN93 attenuated hsBAFF-activated mTOR signaling, as well as cell growth and survival, revealing that the effects of hsBAFF-elevated [Ca2+]i on mTOR signaling as well as proliferation and survival in B lymphocytes is through stimulating phosphorylation of CaMKII. The results indicate that hsBAFF activates mTOR pathway triggering B lymphocyte proliferation and survival by calcium signaling. Our findings suggest that manipulation of intracellular Ca2+ level or CaMKII and mTOR activity may be exploited for the prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases.

Phosphorylated CaMKII levels increase in rat central nervous system after large-dose intravenous remifentanil

BackgroundPostoperative remifentanil-induced pain sensitization is common, but its molecular mechanism remains unclear. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been shown to have a critical role in morphine-induced hyperalgesia. This study was designed to determine how CaMKII phosphorylation and protein expression levels change in the central nervous system of rats with remifentanil-induced hyperalgesia.Material/MethodsMale Sprague-Dawley® rats were exposed to large-dose (bolus of 6.0 μg/kg and 2.5 μg/kg/min for 2 hours) intravenous remifentanil to induce post-transfusion hyperalgesia. Levels of phosphorylated CaMKII (P-CaMKII) and total protein of CaMKII (T-CaMKII) were determined at different post-transfusion times by Western blot and immunostaining and were compared with controls.ResultsP-CaMKII increased significantly (P<0.05) at 0, 0.5, and 2 hours. However, P-CaMKII at 5 to 24 hours and T-CaMKII at 0 to 24 hours post-transfusion did not change significantly in rats’ spinal dorsal horn, hippocampus, or primary somatosensory (S1) cortex (n=6 per group). Similarly, immunostaining showed stronger P-CaMKII immunoreactants (P<0.05) and more P-CaMKII- positive cells (P<0.05) in the spinal dorsal horn, CA1 region of the hippocampus, and S1 cortex of rats 0.5 hours post-transfusion compared with the control group treated with 0.9% sodium chloride (n=3 per group).ConclusionsThese results suggest that a temporary rise in the P-CaMKII level in the central nervous system may correlate with remifentanil-induced pain sensitization in the postoperative period.