Saponins from Panax japonicus alleviate HFD-induced impaired behaviors through inhibiting NLRP3 inflammasome to upregulate AMPA receptors.

Obesity is characterized by a condition of low-grade chronic inflammation that facilitates development of numerous comorbidities and dysregulation of brain homeostasis. It is reported that obesity can lead to behavioral alterations such as cognitive decline and depression-like behaviors both in humans and rodents. Saponins from panax japonicus (SPJ) have been reported to exhibit anti-inflammatory action in mouse model of diet-induced obesity. We evaluated the neuroprotection of SPJ on high fat diet (HFD) induced impaired behaviors such as memory deficit and depressive-like behaviors, and explored the underlying mechanisms. 6-week male Balb/c mice were divided into normal control group (NC, 17% total calories from fat), HFD group (60% total calories from fat), and HFD treated with SPJ groups (orally gavaged with dosages of 15 mg/kg and 45 mg/kg), respectively. After treatment for 16 weeks, behavioral tests were performed to evaluate the cognition and depression-like behaviors of the mice. The underling mechanisms of SPJ on HFD-induced impaired behaviors were investigated through histopathological observation, Western blot analysis and immunofluorescence. Our results showed that HFD-fed mice caused behavioral disorders, neuronal degeneration as well as elevated neuroinflammation, which was partly involved in NLRP3 inflammasome that finally resulted in decreased protein levels of AMPA receptors and down-regulated phosphorylated levels of CaMKII and CREB in cortex and hippocampus. All the above changes in cortex and hippocampus induced by HFD were mitigated by SPJ treatment. SPJ treatment alleviated HFD-induced recognitive impairment and depression-like behaviors of mice, which could be partly due to the capacity of SPJ to mitigate neuroinflammation through inhibition of NLRP3 inflammasome and upregulation of AMPA receptors signaling pathway.

The Roles of Superoxide on At-Level Spinal Cord Injury Pain in Rats.

BACKGROUND: In the present study, we examined superoxide-mediated excitatory nociceptive transmission on at-level neuropathic pain following spinal thoracic 10 contusion injury (SCI) in male Sprague Dawley rats. METHODS: Mechanical sensitivity at body trunk, neuronal firing activity, and expression of superoxide marker/ionotropic glutamate receptors (iGluRs)/CamKII were measured in the T7/8 dorsal horn, respectively. RESULTS: Topical treatment of superoxide donor t-BOOH (0.4 mg/kg) increased neuronal firing rates and pCamKII expression in the naïve group, whereas superoxide scavenger Tempol (1 mg/kg) and non-specific ROS scavenger PBN (3 mg/kg) decreased firing rates in the SCI group (* p < 0.05). SCI showed increases of iGluRs-mediated neuronal firing rates and pCamKII expression (* p < 0.05); however, t-BOOH treatment did not show significant changes in the naïve group. The mechanical sensitivity at the body trunk in the SCI group (6.2 ± 0.5) was attenuated by CamKII inhibitor KN-93 (50 µg, 3.9 ± 0.4) or Tempol (1 mg, 4 ± 0.4) treatment (* p < 0.05). In addition, the level of superoxide marker Dhet showed significant increase in SCI rats compared to the sham group (11.7 ± 1.7 vs. 6.6 ± 1.5, * p < 0.05). CONCLUSIONS: Superoxide and the pCamKII pathway contribute to chronic at-level neuropathic pain without involvement of iGluRs following SCI.

Treadmill exercise enhances synaptic plasticity in the ischemic penumbra of MCAO mice by inducing the expression of Camk2a via CYFIP1 upregulation.

AIMS: Physical exercise is beneficial to the recovery of patients with ischemic stroke. However, the underlying mechanism by which exercise promotes dendritic remodeling and synaptic plasticity is still obscure. This study explored the mechanism by which treadmill exercise enhances synaptic plasticity and dendritic remodeling in the ischemic penumbra. MAIN METHODS: A middle cerebral artery occlusion (MCAO) model was generated in C57BL/6 mice, and lentivirus-mediated cytoplasmic FMRP-associated protein 1 (CYFIP1) shRNA expression was utilized to confirm the role of CYFIP1 in the exercise-induced increase in synaptic plasticity and dendritic remodeling. Neurological deficits were measured using the Zea Longa scale. Hematoxylin-eosin (H&E) staining and Nissl staining were performed to assess cerebral ischemic injury. Golgi-Cox staining was used to observe changes in dendritic remodeling and synaptic plasticity. Transmission electron microscopy (TEM) was performed to observe the synaptic ultrastructure. Molecular mechanisms were explored using immunofluorescence staining and western blotting. KEY FINDINGS: Treadmill training enhanced synaptic plasticity in the penumbra. Additionally, we observed significant increases in the expression of CYFIP1 and calcium/calmodulin-dependent kinase 2a (Camk2a); enhanced neurological recovery and a decreased infarct volume. However, the injection of a lentivirus containing CYFIP1 shRNA into the lateral ventricle exerted negative effects on synaptic plasticity. Moreover, the exercise-induced neuroprotective effects were abolished by lentivirus-mediated CYFIP1 shRNA expression, consistent with the downregulation of Camk2a expression and the deterioration of neurological function. SIGNIFICANCE: Treadmill training enhances synaptic plasticity and dendritic remodeling in the ischemic penumbra by inducing the expression of Camk2a via upregulation of CYFIP1.

Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIß, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca2+/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca2+/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo-electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca2+/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.

Role of ion channels in chronic intermittent hypoxia-induced atrial remodeling in rats.

AIMS: Atrial remodeling, including structural and electrical remodeling, is considered as the substrate in the development of atrial fibrillation (AF). Structural remodeling mainly involves atrial fibrosis, and electrical remodeling is closely related to the changes of ion channels in atrial myocytes. In this study, we aimed to investigate the changes of ion channels in atrial remodeling induced by CIH in rats, which provide the explication for the mechanisms of AF. MATERIALS AND METHODS: 80 male Sprague-Dawley rats were randomized into two groups: Control and CIH group (n = 40). CIH rats were subjected to CIH 8 h/d for 30 days. Atrial epicardial conduction velocity, conduction inhomogeneity and AF inducibility were examined. Masson's trichrome staining was used to evaluate the extent of atrial fibrosis, and the expression levels of ion channel subunits were measured by RT-qPCR, Western blot, and IHC, respectively. The remaining 40 rats were used for whole-cell patch clamp experiments. Action potential, INa, ICa-L, Ito were recorded and compared between two groups. KEY FINDINGS: CIH rats showed increased AF inducibility, atrial interstitial collagen deposition, APD, expression levels of RyR2, p-RyR2, CaMKII, p-CaMKII, and decreased atrial epicardial conduction velocity, expression levels of Nav1.5, Cav1.2, Kv1.5, Kv4.2, Kv4.3 compared to the Control rats, and the current density of INa, ICa-L, Ito were significantly decreased in CIH group. SIGNIFICANCE: We observed significant atrial remodeling induced by CIH in our rat model, which was characterized by changes in ion channels. These changes may be the mechanisms of CIH promoting AF.

Inhibitor 1 of Protein Phosphatase 1 Regulates Ca2+/Calmodulin-Dependent Protein Kinase II to Alleviate Oxidative Stress in Hypoxia-Reoxygenation Injury of Cardiomyocytes.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), regulated by inhibitor 1 of protein phosphatase 1 (I1PP1), is vital for maintaining cardiovascular homeostasis. However, the role and mechanism of I1PP1 against hypoxia-reoxygenation (H/R) injury in cardiomyocytes remain a question. In our study, after I1PP1 overexpression by adenovirus infection in the neonatal cardiomyocytes followed by hypoxia for 4 h and reoxygenation for 12 h, the CaMKIIδ alternative splicing subtype, ATP content, and lactate dehydrogenase (LDH) release were determined. CaMKII activity was evaluated by phosphoprotein phosphorylation at Thr17 (p-PLB Thr17), CaMKII phosphorylation (p-CaMKII), and CaMKII oxidation (ox-CaMKII). Reactive oxygen species (ROS), mitochondrial membrane potential, dynamin-related protein 1 (DRP1), and optic atrophy 1 (OPA1) expressions were assessed. Our study verified that I1PP1 overexpression attenuated the CaMKIIδ alternative splicing disorder; suppressed PLB phosphorylation at Thr17, p-CaMKII, and ox-CaMKII; decreased cell LDH release; increased ATP content; attenuated ROS production; increased mitochondrial membrane potential; and decreased DRP1 expression but increased OPA1 expression in the cardiomyocytes after H/R. Contrarily, CaMKIIδ alternative splicing disorder, LDH release, ATP reduction, and ROS accumulation were aggravated after H/R injury with the I1PP1 knockdown. Collectively, I1PP1 overexpression corrected disorders of CaMKIIδ alternative splicing, inhibited CaMKII phosphorylation, repressed CaMKII oxidation, suppressed ROS production, and attenuated cardiomyocyte H/R injury.

MicroRNA Expression Profiling Screen miR-3557/324-Targeted CaMK/mTOR in the Rat Striatum of Parkinson's Disease in Regular Aerobic Exercise.

This study aimed to screen the target miRNAs and to investigate the differential miR-3557/324-targeted signal mechanisms in the rats' model of Parkinson's disease (PD) with regular aerobic exercise. Rats were divided into sedentary control PD group (SED-PD, n = 18) and aerobic exercise PD group (EX-PD, n = 22). After 8 weeks of regular aerobic exercise, a 6-hydroxydopamine- (6-OHDA-) induced PD lesion model was constructed. Preregular aerobic exercises enhanced the injury resistance of rats with 6-OHDA-induced PD. The rotational behavior after injection of apomorphine hydrochloride was alleviated. Under the scanning electron microscopy, we found the neurons, axons, and villi of the striatum were clearly and tightly arranged, and neurons and axons significantly becoming larger. Tyrosine hydroxylase (TH) was increased significantly and α-synuclein protein expression was reduced in the EX-PD group compared to the SED-PD group. Screening from miRNA microarray chip, we further found upregulation of miR-3557 and downregulation of miR-324 were closely related to the calcium-modulating signaling pathway, remitting the progress of Parkinson's disease on aerobic exercise. Compared to the SED-PD group, Ca2+/calmodulin dependent protein kinase II (CaMK2α) was upregulated, but CaMKV and voltage-dependent anion-selective channel protein 1 (Vdac1) were significantly downregulated in the EX-PD group. Additionally, phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) expression were activated, and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) expression was upregulated in the EX-PD group. Conclusions: the adaptive mechanism of regular aerobic exercise delaying neurodegenerative diseases and lesions was that miR-3557/324 was activated to regulate one of its targets CaMKs signaling pathways. CaMKs, coordinated with mTOR pathway-related gene expression, improved UCH-L1 level to favor for delaying neurodegeneration or improving the pathogenesis of PD lesions.

Blockade of calcium-permeable AMPA receptors in the lateral habenula produces increased antidepressant-like effects in unilateral 6-hydroxydopamine-lesioned rats compared to sham-lesioned rats.

At present, role of the lateral habenula (LHb) calcium-permeable AMPA receptors (CP-AMPARs) in depression is not understood, particularly in Parkinson's disease-related depression. Here we found that lesions of the substantia nigra pars compacta (SNc) in rats induced depressive-like behaviors, and intra-LHb injection of CP-AMPAR antagonist Naspm produced antidepressant-like effects in SNc sham-lesioned and SNc-lesioned rats, however, the doses inducing these effects in SNc-lesioned rats were lower than that of SNc sham-lesioned rats. Blockade of LHb CP-AMPARs decreased the firing rate of the neurons and increased release of dopamine and serotonin in the medial prefrontal cortex (mPFC) in both groups, but the duration of Naspm action on the firing rate and release of the transmitters were prolonged in SNc-lesioned rats. These changes in SNc-lesioned rats were involved in increased expression of ßCaMKII and p-GluR1-S831 in the LHb. Intra-LHb injection of Naspm inhibited dopaminergic neurons in the anterior ventral tegmental area and serotonergic neurons in the dorsal raphe nucleus and excited dopaminergic neurons in the posterior ventral tegmental area (pVTA) and serotonergic neurons in the median raphe nucleus (MRN), and lesioning the GABAergic rostromedial tegmental nucleus (RMTg) decreased the percentages of excited pVTA dopaminergic neurons and MRN serotonergic neurons. Our findings indicate that blockade of LHb CP-AMPARs produces antidepressant-like effects, which attribute to decreased firing activity of LHb neurons and increased levels of dopamine and serotonin in the mPFC, and provide further evidence that LHb CP-AMPARs regulate the firing activity of pVTA dopaminergic neurons and MRN serotonergic neurons indirectly via the RMTg.

Upregulation of MMP-9 and CaMKII prompts cardiac electrophysiological changes that predispose denervated transplanted hearts to arrhythmogenesis after prolonged cold ischemic storage.

BACKGROUND: Although the importance of the potential mechanisms of arrhythmogenesis, including cardiac electrophysiology dysfunction and gap junction electrical uncoupling by Cx43 degradation under pathological conditions, has been widely and thoroughly established, the correlations between these mechanisms and biomarkers of cold ischemic insult have not yet been confirmed. Limited evidence suggests that these biomarkers are significantly associated with cardiac dysrhythmia. Thus, in this study, we analyzed such correlations in denervated transplanted hearts. METHODS AND RESULTS: Rat hearts were divided into two groups according to a random number table. The hearts in the first group were stored in histidine-tryptophan-ketoglutarate (HTK) solution for 6 h to produce cold ischemic insult (insult group, n = 8), while the hearts in the second group were left untreated (control group, n = 8). Ventricular arrhythmias (VAs) were successively observed in 8/8 hearts stored in HTK solution and 0/8 untreated hearts (P < 0.05). The analysis of the electrophysiological parameters by programmed electrical stimulation (PES) and monophasic action potential (MAP) recording demonstrated that compared with the control hearts, the insult hearts had increased transmural dispersion of repolarization (TDR) and effective refractory periods (ERPs) (P < 0.001), decreased conduction velocities (CVs) and excitation wavelengths (CV × ERP, λ) (P < 0.05), and longer times between peak MAP values and 90% repolarization (MAPD90) in the epicardium (P < 0.05). However, no significant difference in endocardial MAPD90 was observed between the two groups of hearts. The investigation of biomarkers and Cx43 proteins by a western blot analysis showed that the matrix metalloproteinase-9 (MMP-9) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) levels were strongly elevated and that the Cx43 protein levels were markedly reduced in the insult hearts. CONCLUSION: MMP-9 overexpression mediates gap junction (GJ) electrical uncoupling induced by Cx43 decreases, while CaMKII upregulation induces transmural dispersion of repolarization (TDR) increase, both of which increase the risk of ventricular arrhythmias (VAs) in denervated transplanted hearts after prolonged cold ischemic storage.

Prostaglandin E2 inhibits profibrotic function of human pulmonary fibroblasts by disrupting Ca2+ signaling.

We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-ß (TGF-ß) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-ß in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-ß-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-ß-stimulated expression of collagen A1, fibronectin, and α-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-ß. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.

Frankincense upregulates the hippocampal calcium/calmodulin kinase II-α during development of the rat brain and improves memory performance.

BACKGROUND: Frankincense is an oleo gum resin derived from trees of genus Boswellia. It has favorable effects on memory formation. However, the probable underlying molecular mechanisms have not been assessed. Frankincense exerts some of its effects via activation of protein kinases. Calcium/calmodulin kinaseII (CaMKII) and CaMKIV are crucial mediators of learning and memory. We studied the effect of maternal injection of the aqueous extract of frankincense during gestation and lactation periods on spatial memory performance and the mRNA expression levels of the hippocampal CaMKIIand CaMKIV in the offspring rats. METHODS: Aqueous extract of Frankincense (50 and 100 mg/kg) or tap water was gavaged to distinct female rats during gestation and lactation periods. Memory performance was assessed in groups of male offspring using Morris water maze. In other groups of the offspring (with no memory test), the hippocampi of the juvenile rats were removed 30 days after labor. A real-time PCR method was used to measure the mRNA levels of CaMKII and CaMKIV. RESULTS: Frankincense improved spatial memory retrieval in the offspring rats in a dose-dependent manner. The mRNA expression of hippocampal CaMKIV was unchanged between groups. However, the mRNA expression of hippocampal CaMKII was dose-dependently upregulated in the rats, whose mothers had received frankincense. CONCLUSIONS: Due to the crucial role of the CaMKII in memory formation, the results provide a molecular basis for the effect of administration of frankincense to mother rats on improvement of the memory in the offspring.

Agmatine attenuates methamphetamine-induced passive avoidance learning and memory and CaMKII-α gene expression deteriorations in hippocampus of rat.

Methamphetamine (METH) abuse is one the most worldwide problems with wide-ranging effects on the central nervous system (CNS). Chronic METH abuse can associate with cognitive abnormalities and neurodegenerative changes in the brain. Agmatine, a cationic polyamine, has been proposed as a neuromodulator that modulates many effects of abused drugs. The aim of this study was to determine if agmatine can decrease the impairment effect of METH on memory and hippocampal CaMKII-α gene expression, a gene that plays a major role in memory. Male wistar rats (200-220 g) were allocated into 7 groups, including 5 groups of saline, METH (1, 2 mg/kg), Agmatine (5, 10 mg/kg) and 2 groups of agmatine (5, 10 mg/kg) with higher doses of METH (2 mg/kg) for 5 consecutive days (n = 8 in each group). All injections were done intraperitoneally and agmatine was administrated 10 min before METH treatment. Furthermore, Passive avoidance learning (PAL) test was assessed on the 5th day. Retention test was done 24 h after training and the rats were sacrificed immediately. Hippocampi were removed and stored at -80 °C. Finally, hippocampal CaMKII-α gene expression was measured using Quantitative Real-time PCR. Our data showed that chronic METH dose-dependently impaired PAL retrieval, as it decreased step-through latency (STL) and increased time spent in the dark compartment (TDC). While Agmatine with a higher dose (10 mg/kg) significantly decreased impairment effect of METH (2 mg/kg) on PAL and memory. Also, molecular results revealed that METH (2 mg/kg) markedly decreased hippocampal CaMKII-α gene expression while agmatine (10 mg/kg) co-adminstration prevented it. Taken together, the results propose that agmatine may provide a potential therapy for learning and memory deficits induced by METH.

Impaired Hippocampal Synaptic Plasticity and Enhanced Excitatory Transmission in a Novel Animal Model of Autism Spectrum Disorders with Telomerase Reverse Transcriptase Overexpression.

Recently, we have reported that animals with telomerase reverse transcriptase (TERT) overexpression exhibit reduced social interaction, decreased preference for novel social interaction and poor nest-building behaviors symptoms that mirror those observed in human autism spectrum disorders (ASD). Overexpression of TERT also alters the excitatory/inhibitory (E/I) ratio in the medial prefrontal cortex. However, the effects of TERT overexpression on hippocampal-dependent learning and synaptic efficacy have not been investigated. In the present study, we employed electrophysiological approaches in combination with behavioral analysis to examine hippocampal function of TERT transgenic (TERT-tg) mice and FVB controls. We found that TERT overexpression results in enhanced hippocampal excitation with no changes in inhibition and significantly impairs long-term synaptic plasticity. Interestingly, the expression levels of phosphorylated CREB and phosphory-lated CaMKIIα were significantly decreased while the expression level of CaMKIIα was slightly increased in the hippocampus of TERT-overexpressing mice. Our observations highlight the importance of TERT in normal synaptic function and behavior and provide additional information on a novel animal model of ASD associated with TERT overexpression.

Inhibition of miR-219 Alleviates Arsenic-Induced Learning and Memory Impairments and Synaptic Damage Through Up-regulating CaMKII in the Hippocampus.

Epidemiological investigations and experimental studies indicate that chronic arsenic exposure can reduce learning and memory function. However, the underlying mechanism of this effect remains largely unknown. Emerging evidence suggests that microRNA (miRNA) play an important role in toxicant exposure and a regulatory role in cognitive function. In this study, we observed that subchronic arsenic exposure induced impairment of learning and memory and significantly up-regulated miRNA-219 (miR-219) expression in the mouse hippocampus. Furthermore, the expression of CaMKII, an experimentally validated target of miR-219, was decreased in the mice exposed to arsenic. Suppression of miR-219 by adeno-associated viral (AAV)-delivered anti-miR-219 prevented the arsenic-induced impairment of learning and memory and relieved the pathological changes in the synaptic structure of the hippocampus. Furthermore, we observed that the NMDA receptor subunit 2 (NR2) and the memory-related proteins c-Fos and c-Jun were up-regulated by inhibition of miR-219 in the mouse hippocampus. Taken together, the results of this study indicate that inhibition of miR-219 regulates arsenic-induced damage in the structure of the hippocampus and impairment of learning and memory, possibly by targeting CaMKII. Suppression of miR-219 may be a potential strategy to ameliorate arsenic-induced neurotoxicity.

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

BACKGROUND: Sustained ß-adrenergic receptor (ß-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. OBJECTIVE: The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained ß-AR stimulation and associated functional implications on IKs. METHODS: KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained ß-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. RESULTS: Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained ß-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained ß-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained ß-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). CONCLUSION: CaMKII regulates KCNQ1 at S484 during sustained ß-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

Selective cephalic upregulation of p-ERK, CamKII and p-CREB in response to glyceryl trinitrate infusion.

Background A common characteristic of migraine-inducing substances is that they cause headache and no pain in other areas of the body. Few studies have compared pain mechanisms in the trigeminal and spinal systems and, so far, no major differences have been noted. We compared signalling molecules in the trigeminal and spinothalamic system after infusion of the migraine-provoking substance glyceryltrinitrate. Method A catheter was placed in the femoral vein of rats and one week later glyceryltrinitrate 4 µg/kg/min was infused for 20 min. Protein expression in the dura mater, trigeminal ganglion, nucleus caudalis, dorsal root ganglion and the dorsal horn of the thoracic spinal cord was analysed at different time points using western blotting and immunohistochemistry. Results Glyceryltrinitrate caused a threefold increase in expression of phosphorylated extracellular signal-regulated kinases at 30 min in the dura mater and nucleus caudalis ( P < 0.05) and at 2 h in the trigeminal ganglion with very few expressions in the dorsal root ganglion. In the nucleus caudalis, expression of phosphorylated extracellular signal-regulated kinases and Cam KII increased 2.6-fold and 3.2-fold, respectively, at 2 h after glycerytrinitrate infusion ( P < 0.01). p-CREB/ATF-1 upregulation was observed only at 30 min ( P < 0.05) in the nucleus caudalis. None of these markers showed increased expression in the regions of thoracic spinal cord dorsal horn. Conclusion The dura, trigeminal ganglion and nucleus caudalis are activated shortly after glycerytrinitrate infusion with long-lasting expression of phosphorylated extracellular signal-regulated kinases observed in the nucleus caudalis. These activations were not observed at the spinal level.

In vitro neurotoxicity by ropivacaine is reduced by silencing Cav3.3 T-type calcium subunits in neonatal rat sensory neurons.

Neurotoxicity of local anaesthetics has been alerted by more and more peoples. Cav3.1 and Cav3.2 T-type calcium channels were closely related with local anaesthetics toxicity. However, the role of Cav3.3, another subtype of the T-type calcium channel, on the neurotoxicity induced by local anaesthetics remains unclear. CaMKIIγ is a kind of multifunctional kinase and associated with a variety of physiological and pathological process. T-type calcium channel is closely related with CaMKIIγ. Up-regulation CaMKIIγ can increase T-type currents at the dorsal root ganglia (DRG). On the contrary, down-regulation results in the T-type currents decrease. Is the relation between Cav3.3 T-type channel calcium and CaMKIIγ involved with the ropivacaine hydrochloride neurotoxicity? In this study, we generated pAd-Cav3.3 and pAd-shRNA adenovirus vector to up-regulate and down-regulate Cav3.3 mRNA expression of the DRG. The cells treated or untreated with ropivacaine hydrochloride (3 mM) for 4 h were used to evaluate the neurotoxicity. Cell viability, cell death rate and apoptosis rate, Cav3.3 and CaMKIIγ expression were detected with MTT method, Hoechst-PI, flow cytometry, qRT-PCR and western blotting. Results showed that the cell viability of the DRG treated with ropivacaine hydrochloride markedly decreased, death rate and apoptosis rate, Cav3.3 and CaMKIIγ mRNA and protein expression significantly increased. Cav3.3 overexpression aggravated DRG injury induced by ropivacaine hydrochloride and inhibition of Cav3.3 expression improved the cell damages. Cav3.3 can regulate CaMKIIγ mRNA and protein expression. In conclusion, Cav3.3 regulated CaMKIIγ in DRG, which was involved with the cell injury induced by ropivacaine hydrochloride.

Taurine Protects Primary Neonatal Cardiomyocytes Against Apoptosis Induced by Hydrogen Peroxide.

The aim of this study was to investigate the effects of taurine (Tau) on primary cultured neonatal myocardial cells treated with hydrogen peroxide (H2O2) and the underlying mechanism. Primary cardiac myocytes from neonatal Wistar rats were pre-incubated with Tau, and its effects on cell viability and expression of CaM, CaMKII, p53, Bcl-2, and Bax were examined. Tau enhanced the viability of myocardial cells, decreased apoptosis, and alleviated the intracellular calcium overload, especially at dosages of 40 or 80 mM (P < 0.01 or P < 0.001, respectively). Moreover, Tau could inhibit the H2O2-induced decrease in CamKII and CaM expression at both the mRNA and protein levels. The pattern of CaMKII expression was consistent with that of the anti-apoptotic protein Bcl-2, but contrasted the pattern of the pro-apoptotic proteins p53 and Bax. Thus, our results show that Tau protects myocardial cells against damage caused by H2O2 exposure, suggesting that it might play a role in the mitochondrial apoptotic pathway by upregulating the expression of CaMKII to rescue myocardial cells. However, the underlying mechanism still needs to be investigated. In addition, we tested the protective effect of taurine on cardiac myocytes, and the effect of taurine on another model, specifically an animal model.

CaMKIIα Expression Defines Two Functionally Distinct Populations of Granule Cells Involved in Different Types of Odor Behavior.

Granule cells (GCs) in the olfactory bulb (OB) play an important role in odor information processing. Although they have been classified into various neurochemical subtypes, the functional roles of these subtypes remain unknown. We used in vivo two-photon Ca2+ imaging combined with cell-type-specific identification of GCs in the mouse OB to examine whether functionally distinct GC subtypes exist in the bulbar network. We showed that half of GCs express Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα+) and that these neurons are preferentially activated by olfactory stimulation. The higher activity of CaMKIIα+ neurons is due to the weaker inhibitory input that they receive compared to their CaMKIIα-immunonegative (CaMKIIα-) counterparts. In line with these functional data, immunohistochemical analyses showed that 75%-90% of GCs expressing the immediate early gene cFos are CaMKIIα+ in naive animals and in mice that have been exposed to a novel odor and go/no-go operant conditioning, or that have been subjected to long-term associative memory and spontaneous habituation/dishabituation odor discrimination tasks. On the other hand, a perceptual learning task resulted in increased activation of CaMKIIα- cells. Pharmacogenetic inhibition of CaMKIIα+ GCs revealed that this subtype is involved in habituation/dishabituation and go/no-go odor discrimination, but not in perceptual learning. In contrast, pharmacogenetic inhibition of GCs in a subtype-independent manner affected perceptual learning. Our results indicate that functionally distinct populations of GCs exist in the OB and that they play distinct roles during different odor tasks.

Memory Erasure Experiments Indicate a Critical Role of CaMKII in Memory Storage.

The abundant synaptic protein CaMKII is necessary for long-term potentiation (LTP) and memory. However, whether CaMKII is required only during initial processes or whether it also mediates memory storage remains unclear. The most direct test of a storage role is the erasure test. In this test, a putative memory molecule is inhibited after learning. The key prediction is that this should produce persistent memory erasure even after the inhibitory agent is removed. We conducted this test using transient viral (HSV) expression of dominant-negative CaMKII-alpha (K42M) in the hippocampus. This produced persistent erasure of conditioned place avoidance. As an additional test, we found that expression of activated CaMKII (T286D/T305A/T306A) impaired place avoidance, a result not expected if a process other than CaMKII stores memory. Our behavioral results, taken together with prior experiments on LTP, strongly support a critical role of CaMKII in LTP maintenance and memory storage.