Calmodulin-dependent Protein Kinase II Inhibitor Research Articles

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.

Proline hydroxylase domain-containing enzymes regulate calcium levels in cardiomyocytes by TRPA1 ion channel

The proline hydroxylase domain-containing enzymes (PHDs) acts as cellular oxygen sensors, inducing a series of responses to hypoxia, especially during the regulation of metabolism and energy homeostasis. The increase of Ca2+ in cardiomyocytes, induced by the opening of PHD signaling pathway, is the key initiation signal necessary for the PHD-mediated regulation of the energy metabolism pathway, but the underlying molecular mechanism remains incompletely understood. This study used PHD inhibitors (PHIs) and PHD2-specific RNA interference (PHD2shRNA) to inhibit PHD signals in cardiomyocytes to explore whether transient receptor potential ankyrin 1 (TRPA1) is involved in the regulation of calcium ion influx in the PHD activation pathway associated with to AMP-activated protein kinase (AMPK). The Fluo-3AM probe was used to measure changes in free intracellular calcium ion concentrations, and Western blot analysis was used to detect the levels of phosphorylated (P)-AMPK, TRPA1, and P–Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) levels. The PHI-mediated inhibition of PHD resulted in an increase in free Ca2+ fluorescence in cardiomyocytes, which activated AMPK, TRPA1, and CaMKⅡ. The TRPA1 inhibitor HC030031, the CaMKII inhibitor KN93, and a ryanodine inhibitor (Ryanodine) were all able to inhibit the PHI-induced increase in intracellular Ca2+ and AMPK activation. Both PHIs and PHD2shRNA were able to effectively activate CaMKII and TRPA1. However, an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor and the protein kinase A (PKA) inhibitor H89 did not significantly inhibit the PHI-induced increase in intracellular Ca2+ and AMPK activation. These results indicated that PHD might activate the CaMKⅡ pathway through the TRPA1 ion channel, inducing the release of calcium from the sarcoplasmic reticulum through ryanodine receptor 2 (RyR2), activating AMPK to initiate the protective effects of hypoxia in cardiomyocytes.

Latent Sex Differences in CaMKII-nNOS Signaling That Underlie Antidepressant-Like Effects of Yueju-Ganmaidazao Decoction in the Hippocampus.

Previous studies have demonstrated that Yueju-Ganmaidazao (YG) decoction induces rapid antidepressant-like effects, and the antidepressant response is mostly dependent on the suppression of nitric oxide-cyclic guanosine monophosphate signaling in male mice. This study aimed to investigate the sex difference mediated by calcium/calmodulin-dependent protein kinase II (CaMKII)-neuronal nitric oxide synthase (nNOS) signaling involved in the antidepressant-like effect of YG in mice. We found that the immobility times in the tail suspension test (TST) were found to be decreased after the single injection of YG in male and female mice with the same dosage. Additionally, chronic administration for 4 days of subthreshold dosage of YG and escitalopram (ES) also significantly decreased the immobility time in mice of both sexes. Chronic subthreshold dosage of YG and ES in LPS-treated mice and in chronic unpredictable stress (CUS) mice both decreased the immobility time, which was increased by stress. Meanwhile, in CUS-treated mice, sucrose preference test, forced swimming test, and open field test were applied to further confirm the antidepressant-like effects of YG and ES. Moreover, CUS significantly decreased the expression of nNOS and CaMKII, and both YG and ES could enhance the expression in the hippocampus of female mice, which was opposite to that in male mice, while endothelial nitric oxide synthase expression was not affected by stress or drug treatment neither in male mice nor in female mice. Finally, subthreshold dosage of YG combined with 7-nitroindazole (nNOS inhibitor) induced the antidepressant-like effects both in female and in male mice, while the single use of YG or 7-NI did not display any effect. However, pretreatment with KN-93 (CaMKII inhibitor) only blocked the antidepressant-like effect of high-dosage YG in female mice. Meanwhile, in CUS mice, chronic stress caused NR1 overexpression and inhibited cAMP response element binding protein action, which were both reversed by YG and ES in male and female mice, implying that YG and ES produced the same antidepressant-like effect in mice of both sexes. The study revealed that chronic treatment with a subthreshold dose of YG also produced antidepressant-like effects in female mice, and these effects depended on the regulation of the CaMKII-nNOS signaling pathway.

Calcium Regulation on the Atrial Regional Difference of Collagen Production Activity in Atrial Fibrogenesis.

Background: Atrial fibrosis plays an important role in the genesis of heart failure and atrial fibrillation. The left atrium (LA) exhibits a higher level of fibrosis than the right atrium (RA) in heart failure and atrial arrhythmia. However, the mechanism for the high fibrogenic potential of the LA fibroblasts remains unclear. Calcium (Ca2+) signaling contributes to the pro-fibrotic activities of fibroblasts. This study investigated whether differences in Ca2+ homeostasis contribute to differential fibrogenesis in LA and RA fibroblasts. Methods: Ca2+ imaging, a patch clamp assay and Western blotting were performed in isolated rat LA and RA fibroblasts. Results: The LA fibroblasts exhibited a higher Ca2+ entry and gadolinium-sensitive current compared with the RA fibroblasts. The LA fibroblasts exhibited greater pro-collagen type I, type III, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), phosphorylated phospholipase C (PLC), stromal interaction molecule 1 (STIM1) and transient receptor potential canonical (TRPC) 3 protein expression compared with RA fibroblasts. In the presence of 1 mmol/L ethylene glycol tetra-acetic acid (EGTA, Ca2+ chelator), the LA fibroblasts had similar pro-collagen type I, type III and phosphorylated CaMKII expression compared with RA fibroblasts. Moreover, in the presence of KN93 (a CaMKII inhibitor, 10 μmol/L), the LA fibroblasts had similar pro-collagen type I and type III compared with RA fibroblasts. Conclusion: The discrepancy of phosphorylated PLC signaling and gadolinium-sensitive Ca2+ channels in LA and RA fibroblasts induces different levels of Ca2+ influx, phosphorylated CaMKII expression and collagen production.

CaMKII as a therapeutic target in catecholaminergic polymorphic ventricular tachycardia type 1

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority Background In catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) adrenergic activation of the cardiac ryanodine receptor (RyR2) causes spontaneous calcium release and triggers arrhythmias. Calcium/calmodulin-dependent protein kinase II (CaMKII) can contribute to such arrhythmogenic calcium release and has been proposed as a therapeutic target in CPVT1. To predict the efficacy and safety of this strategy, it is necessary to know whether the mechanism for CaMKII activation is important for its arrhythmogenic effects, and if inhibition has proarrhythmic effects. We tested (1) if oxidation of CaMKII contributes to spontaneous calcium release in CPVT1 and (2) if inhibition of CaMKII in this condition can induce calcium alternans. Methods Mice with the CPVT1-causative mutation RyR2-R2474S (RyR2-RS) were crossed with mice with the CaMKII M281/282V (MMVV) mutation that prevents CaMKII M281/282 oxidation, to create double mutants (RyR2-RSxMMVV). Telemetric ECG surveillance was used to study in vivo arrhythmias following an adrenergic challenge by i.p. administration of the beta-adrenoceptor agonist isoprenaline. Confocal line-scan imaging and whole-cell calcium imaging were used to study arrhythmogenic calcium release in isolated left ventricular cardiomyocytes during stimulation with isoprenaline. Results As expected, RyR2-RS mice exhibited more arrhythmic events and spontaneous calcium release (i.e. calcium sparks and calcium waves) compared to wild-type mice. Treatment of RyR2-RS cardiomyocytes with either the CaMKII inhibitor KN-93 or the antioxidant n-acetyl-cysteine reduced spontaneous calcium release (i.e. calcium sparks and calcium waves, for KN-93 and n-acetyl-cysteine, respectively). Interestingly, CaMKII inhibition by KN-93 also increased both incidence and degree of arrhythmogenic calcium alternans in RyR2-RS cardiomyocytes. This adverse effect was a result of prolonged refractoriness of calcium release. Furthermore, to test whether the protective effect of antioxidant treatment in RyR2-RS was mediated via CaMKII oxidation, we compared arrhythmias and spontaneous calcium release (i.e. calcium waves) in RyR2-RSxMMVV with RyR2-RS. However, these two genotypes did not differ in either incidence or severity of arrhythmias, and showed similar degree of spontaneous calcium release. Conclusions Inhibition of CaMKII protects against spontaneous calcium release in CPVT1, and is a promising therapeutic strategy. However, the fact that such inhibition also induces calcium alternans needs further exploration. Antioxidative agents also attenuate arrhythmogenic calcium release in CPVT1 cardiomyocytes, but this effect does not seem to involve the M281/282 CaMKII oxidation site. Future studies should explore other oxidation sites.

Regulation of serotonin transport by geranylgeranyl transferase and Ca 2+ /calmodulin‐dependent protein kinase II

The serotonin transporter (SERT) transports extracellular serotonin (5HT) into neuron terminals and serves as the primary mechanism by which 5HT is regulated in the brain. Recently, it has been shown that SERT activity is increased after treatment with cholesterol-lowering statin drugs in a manner independent of cholesterol but dependent on the cholesterol biosynthetic intermediate geranylgeranyl pyrophosphate (GGPP). GGPP and its corresponding enzyme geranylgeranyl transferase (GGT) lipidate proteins for targeting to the plasma membrane. Although statins are known to increase constitutive activity of the canonical targets of GGPP including Rac1, CDC42, and RhoA, the mechanism by which GGPP regulates SERT activity is unknown. To investigate the role of GGPP on neuronal SERT function, serotonin neurons were modeled in serotonergic RN46A-B14 cells with stably overexpressed SERT. Simvastatin or a GGT inhibitor that specifically blocks geranylgeranylation by GGPP were applied to cells for 24hr and 5HT transport was assayed by measuring 5HT uptake in cells after incubation with exogenous 5HT. Simvastatin and GGT inhibition increased 5HT uptake by 58% and 82% (Mean (SEM): Veh=2.09 (0.30), Sim=4.99 (0.53), GGT=11.84 (2.63), p<0.05), respectively, in a manner blocked by fluoxetine. Pharmacological inhibition of CDC42 or knockdown of the mRNA for RhoA did not block statin-increased 5HT uptake. Protein kinases p38 mitogen-activated protein kinase (P38MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) were also investigated based on their regulation downstream of GGPP. While phosphorylation of P38MAPK was significantly increased by 37% after 24hr simvastatin treatment (Mean (SEM): Veh=100 (6.7), Sim=159 (19.0), p<0.02), inhibition of P38MAPK did not block statin-increased uptake. However, inhibition of CaMKII with CaMKII inhibitor Tat-CN21 dose-dependently blocked statin-increased 5HT uptake (Mean (SEM): Veh=100 (0), Sim=263 (25.8), Tat-CN21=119.48 (12.2), p<0.001). Ongoing studies of the inhibition of GGT on 5HT uptake are being conducted using a panel of CaMKII inhibitors including Tat-CN21, myristoylated autocamtide-2-related inhibitory peptide, and small molecule inhibitors with corresponding control agents. Our results indicate that SERT is regulated through the isoprenylation pathway in a GGPP, GGT and CaMKII-dependent manner.

Loss of Camk2n1 aggravates cardiac remodeling and malignant ventricular arrhythmia after myocardial infarction in mice via NLRP3 inflammasome activation

AimsInflammation response and subsequent ventricular remodeling are critically involved in the development of ventricular arrhythmia post myocardial infarction (MI). However, as the vital endogenous inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), the effects of CaMKII inhibitor 1 (Camk2n1) on the process of arrhythmia substrate generation following MI remains unclear. In this study, we investigated the role of Camk2n1 in ventricular arrhythmia post-MI and the underlying mechanisms. Methods and resultsCamk2n1 was mainly expressed in cardiomyocytes and inhibited the phosphorylation of CaMKIIδ in the infarcted border zone. Compared to wild type (WT) littermates mice, Camk2n1 knockout mice (Camk2n1−/−) manifested exacerbated cardiac dysfunction, larger fibrosis area, higher incidence of premature ventricular contractions (PVCs) and higher vulnerability to ventricular tachycardia (VT) or ventricular fibrillation (VF) after MI. The results of RNA sequencing (RNA-seq) identified that excessive activation of NLRP3 inflammasome was responsible for aggravated inflammation response which led to adverse cardiac remodeling in Camk2n1−/− mice subjected to MI. More importantly, both in vivo and in vitro experiments verified that aggravated NLRP3 inflammasome activation occurred via CaMKIIδ-p38/JNK pathway in Camk2n1−/− mice. ConclusionsCollectively, our results highlight the importance of Camk2n1 in alleviating ventricular remodeling and malignant ventricular arrhythmia post-MI by reducing cardiomyocytes inflammation activation via CaMKIIδ-p38/JNK-NLRP3 inflammasome pathway, targeting Camk2n1 might serve as a novel therapeutic strategy after MI.

Chronic Ca2+/Calmodulin-Dependent Protein Kinase II Inhibition Rescues Advanced Heart Failure.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is upregulated in congestive heart failure (CHF), contributing to electrical, structural, and functional remodeling. CaMKII inhibition is known to improve CHF, but its direct cardiac effects in CHF remain unclear. We hypothesized that CaMKII inhibition improves cardiomyocyte function, [Ca2+]i regulation, and β-adrenergic reserve, thus improving advanced CHF. In a 16-week study, we compared plasma neurohormonal levels and left ventricular (LV)- and myocyte-functional and calcium transient ([Ca2+]iT) responses in male Sprague-Dawley rats (10/group) with CHF induced by isoproterenol (170 mg/kg sq for 2 days). In rats with CHF, we studied the effects of the CaMKII inhibitor KN-93 or its inactive analog KN-92 (n = 4) (70 µg/kg per day, mini-pump) for 4 weeks. Compared with controls, isoproterenol-treated rats had severe CHF with 5-fold-increased plasma norepinephrine and about 50% decreases in ejection fraction (EF) and LV contractility [slope of LV end-systolic pressure-LV end-systolic volume relation (EES)] but increased time constant of LV relaxation (τ). They also showed significantly reduced myocyte contraction [maximum rate of myocyte shortening (dL/dtmax)], relaxation (dL/dtmax), and [Ca2+]iT Isoproterenol superfusion caused significantly fewer increases in dL/dtmax and [Ca2+]iT KN-93 treatment prevented plasma norepinephrine elevation, with increased basal and acute isoproterenol-stimulated increases in EF and EES and decreased τ in CHF. KN-93 treatment preserved normal myocyte contraction, relaxation, [Ca2+]iT, and β-adrenergic reserve, whereas KN-92 treatment failed to improve LV and myocyte function, and plasma norepinephrine remained high in CHF. Thus, chronic CaMKII inhibition prevented CHF-induced activation of the sympathetic nervous system, restoring normal LV and cardiomyocyte basal and β-adrenergic-stimulated contraction, relaxation, and [Ca2+]iT, thereby playing a rescue role in advanced CHF. SIGNIFICANCE STATEMENT: We investigated the therapeutic efficacy of late initiation of chronic Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition on progression of advanced congestive heart failure (CHF). Chronic CaMKII inhibition prevented CHF-induced activation of the sympathetic nervous system and restored normal intrinsic cardiomyocyte basal and β-adrenergic receptor-stimulated relaxation, contraction, and [Ca2+]i regulation, leading to reversal of CHF progression. These data provide new evidence that CaMKII inhibition is able and sufficient to rescue a failing heart, and thus cardiac CaMKII inhibition is a promising target for improving CHF treatment.

Pka and CaMKII Signaling Synergistically Promotes Atrial Arrhythmia in Populations of Human Atrial Tissues

The β-adrenergic receptor (βAR)/cAMP/PKA and multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) signaling pathways are key mediators of cardiac excitation-contraction coupling (ECC) and share multiple downstream targets. Hyperactivation of both signaling pathways contributes to the initiation and maintenance of atrial fibrillation (AF), the world's most common arrhythmia. Using novel populations of computational models coupling atrial electrophysiology and Ca2+ handling with detailed descriptions of βAR-PKA and CaMKII pathways, we have previously shown that PKA and CaMKII effects on atrial ECC proteins synergistically promote a vicious cycle of Ca2+and membrane potential instabilities. Since both kinases are known to affect tissue-level modifiers of atrial electrophysiology, here we sought to test whether PKA and CaMKII signaling also synergize to mediate arrhythmias in tissue, and to determine the mechanisms underlying the increased vulnerability. Populations of 1D strand models revealed increased vulnerability to unidirectional block with hyperactive CaMKII, mediated by Na+ channel loss of function and cell-to-cell uncoupling, which was exacerbated by activation of βARs. Simulations of 2D heterogeneous tissues demonstrated synergistic enhancement of arrhythmia vulnerability by βAR stimulation and CaMKII hyperactivation through dysregulating conduction and modifying source-sink mismatch, as well as promoting tissue repolarization dispersion and invoking spontaneous Ca2+-overload-mediated action potentials. CaMKII inhibition substantially reduced the vulnerability. Collectively, our simulations reveal synergy in PKA and CaMKII effects on tissue-level outcomes, and depict a novel paradigm for Ca2+-CaMKII-dependent involvement in both enhanced triggered activity and conduction disturbances in AF. These findings suggest that interrupting the vicious cycle of ever-increasing CaMKII and Ca2+ (e.g., via CaMKII inhibition) may be a valuable pharmacotherapy approach to counteract both AF triggers and functional substrate.

Sunitinib and Imatinib Display Differential Cardiotoxicity in Adult Rat Cardiac Fibroblasts That Involves a Role for Calcium/Calmodulin Dependent Protein Kinase II.

Background: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodeling of the heart following insult or injury, yet their involvement in anti-cancer drug induced cardiotoxicity has been largely overlooked. Here, we examine the direct effects of sunitinib malate and imatinib mesylate on adult rat CF viability, Ca2+ handling and mitochondrial function that may contribute to TKI-induced cardiotoxicity. In particular, we investigate whether Ca2+/calmodulin dependent protein kinase II (CaMKII), may be a mediator of TKI-induced effects.Methods: CF viability in response to chronic treatment with both drugs was assessed using MTT assays and flow cytometry analysis. Calcium mobilization was assessed in CFs loaded with Fluo4-AM and CaMKII activation via oxidation was measured via quantitative immunoblotting. Effects of both drugs on mitochondrial function was determined by live mitochondrial imaging using MitoSOX red.Results: Treatment of CFs with sunitinib (0.1–10 μM) resulted in concentration-dependent alterations in CF phenotype, with progressively significant cell loss at higher concentrations. Flow cytometry analysis and MTT assays revealed increased cell apoptosis and necrosis with increasing concentrations of sunitinib. In contrast, equivalent concentrations of imatinib resulted in no significant change in cell viability. Both sunitinib and imatinib pre-treatment increased Angiotensin II-induced intracellular Ca2+ mobilization, with only sunitinib resulting in a significant effect and also causing increased CaMKII activation via oxidation. Live cell mitochondrial imaging using MitoSOX red revealed that both sunitinib and imatinib increased mitochondrial superoxide production in a concentration-dependent manner. This effect in response to both drugs was suppressed in the presence of the CaMKII inhibitor KN-93.Conclusions: Sunitinib and imatinib showed differential effects on CFs, with sunitinib causing marked changes in cell viability at concentrations where imatinib had no effect. Sunitinib caused a significant increase in Angiotensin II-induced intracellular Ca2+ mobilization and both TKIs caused increased mitochondrial superoxide production. Targeted CaMKII inhibition reversed the TKI-induced mitochondrial damage. These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in CFs, consistent with the differential selectivity of sunitinib and imatinib.

Electroacupuncture Attenuates CFA-Induced Inflammatory Pain by Regulating CaMKII.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine kinase that is ubiquitously distributed in the central and peripheral nervous systems. Moreover, its phosphorylated protein (P-CaMKII) is involved in memory, mood, and pain regulation in the anterior cingulate cortex (ACC). Electroacupuncture (EA) is a traditional Chinese therapeutic technique that can effectively treat chronic inflammatory pain. However, the CaMKII-GluA1 role in EA analgesia in the ACC remains unclear. This study investigated the role of P-CaMKII and P-GluA1 in a mouse model of inflammatory pain induced by complete Freund's adjuvant (CFA). There were increased P-CaMKII and P-GluA1 levels in the ACC. We found that intracerebroventricular injection of KN93, a CaMKII inhibitor, as well as EA stimulation, attenuated complete Freund's adjuvant-induced pain behavior. Further, EA increased pCaMKII-PICK1 complex (abbreviated as C-P complex) levels. Our findings demonstrate that EA inhibits inflammatory pain by inhibiting CaMKII-GluA1 phosphorylation. P-CaMKII is involved in EA analgesia as the pCaMKII-PICK1 complex.

Feeding habit transition induced by social learning through CaMKII signaling in Chinese perch (Siniperca chuatsi)

Social learning plays pivotal roles in gaining new foraging skills and food preferences. However, the molecular mechanism underlying the effects of social learning on feeding habits is unclear. Chinese perch, as a demersal piscivore, has a unique feeding habit. Once the fry of Chinese perch start feeding, they only accept live prey fish in the wild and refuse dead prey fish or artificial diets. In the present study, we used the inhibitor of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling pathway KN-93 in vivo and in vitro, to explore how social learning can affect the transition from live prey fish to dead prey fish in Chinese perch. Analysis of gene expression, protein phosphorylation, success rate, learning times, and food intake allowed us to gain insights into the molecular mechanism of social learning in acquiring new feeding habits in Chinese perch. Our results showed that the p-CaMKII, phospho-cAMP-response element binding (p-CREB) and proto-oncogenes c-fos (c-fos), and melanin-concentrating hormone (mch) expression levels were significantly lower with KN-93 treatment than levels in the control condition, whereas the expression of the anorexigenic gene proopiomelanocortin (pomc) was significantly higher in vitro. The increased success rate and food intake of Chinese perch induced by social learning, were diminished after the injection of the CaMKII inhibitor KN-93 in vivo. Inhibited signaling of CaMKII was observed in Chinese perch after KN-93 treatment. We also found that the expression levels of the c-fos, mch or pomc genes were increased or decreased in the learning group and then decreased or increased in the learning group with KN-93 injection, respectively. These results indicate that the higher expression of the orexigenic mch gene and the decreased expression of the anorexigenic pomc gene might contribute to the learning of eating dead fish prey in Chinese perch. Altogether, our results showed that social learning could promote the feeding habit transition from live prey fish to dead prey fish through the CaMKII - c-fos - pomc/mch signaling pathway in Chinese perch.

Over-expression of Kv4.3 gene reverses cardiac remodeling and transient-outward K+ current (Ito) reduction via CaMKII inhibition in myocardial infarction

ObjectivePrevious study has shown that Kv4.3, a main coding subunit generating cardiac transient-outward K+ current (Ito), can inhibit Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. Based on these observations, we speculate that over-expression of Kv4.3 gene could reverse not only Ito reduction but also cardiac remodeling in the rat myocardial infarction (MI) model. Methods and resultsHealthy male Sprague-Dawley (SD) rats were used to establish MI model by ligation of left anterior descending coronary artery, and adenovirus integrated with Kv4.3 gene (AD-Kv4.3) was delivered in infarct border zone by intramyocardial injection. The hearts were harvested for histological analysis (HE or Masson trichrome staining), western blot or patch clamp 4 weeks after MI. Our data showed that the application of AD-Kv4.3 could reduce myocardial infarct size and fibrosis, and its cardioprotective effects were similar with medicine therapy (combination of metoprolol and captopril). Moreover, Kv4.3 over-expression significantly improved MI-induced cardiac dysfunction and enhanced Ito density while decreasing corrected QT (QTc) intervals and cardiac electrophysiological instability. Western blot showed that Kv4.3 transfection reduced CaMKII, PLB-17 and ryanodine receptor2 (RyR2 Ser2814) phosphorylation level, at same time increased SERCA2 expression dramatically. ConclusionOver-expression of Kv4.3 can not only attenuate cardiac electrophysiological instability and cardiac performance, but also reduce myocardial infarct area and cardiac fibrosis. Like traditional anti-remodeling therapy-angiotensin converting enzyme inhibitor (ACEI) combined with β-adrenergic receptor blocker, over-expression of Kv4.3 seems to be an effective and safe therapy for both structural and electrical remodeling induced by MI via CaMKII inhibition.

Physiological testosterone attenuates profibrotic activities of rat cardiac fibroblasts through modulation of nitric oxide and calcium homeostasis.

Testosterone deficiency is associated with poor prognosis among patients with chronic heart failure (HF). Physiological testosterone improves the exercise capacity of patients with HF. In this study, we evaluated whether treatment with physiological testosterone contributes to anti-fibrogenesis by modifying calcium homeostasis in cardiac fibroblasts and we studied the underlying mechanisms. Nitric oxide (NO) analyses, calcium (Ca2+) fluorescence, and Western blotting were performed in primary isolated rat cardiac fibroblasts with or without (control cells) testosterone (10, 100, 1,000 nmol/L) treatment for 48 hours. Physiological testosterone (10 nmol/L) increased NO production and phosphorylation at the inhibitory site of the inositol trisphosphate (IP3) receptor, thereby reducing Ca2+ entry, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression, type I and type III pro-collagen production. Non-physiological testosterone-treated fibroblasts exhibited similar NO and collagen production capabilities as compared to control (testosterone deficient) fibroblasts. These effects were blocked by co-treatment with NO inhibitor (L-NG-nitro arginine methyl ester [L-NAME], 100 μmol/L). In the presence of the IP3 receptor inhibitor (2-aminoethyl diphenylborinate [2-APB], 50 μmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar phosphorylated CaMKII expression. When treated with 2-APB or CaMKII inhibitor (KN93, 10 μmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar type I, and type III collagen production. In conclusion, physiological testosterone activates NO production, and attenuates the IP3 receptor/Ca2+ entry/CaMKII signaling pathway, thereby inhibiting the collagen production capability of cardiac fibroblasts.

Coordination between Calcium/Calmodulin-Dependent Protein Kinase II and Neuronal Nitric Oxide Synthase in Neurons.

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is highly abundant in the brain and exhibits broad substrate specificity, thereby it is thought to participate in the regulation of neuronal death and survival. Nitric oxide (NO), produced by neuronal NO synthase (nNOS), is an important neurotransmitter and plays a role in neuronal activity including learning and memory processes. However, high levels of NO can contribute to excitotoxicity following a stroke and neurodegenerative disease. Aside from NO, nNOS also generates superoxide which is involved in both cell injury and signaling. CaMKII is known to activate and translocate from the cytoplasm to the post-synaptic density in response to neuronal activation where nNOS is predominantly located. Phosphorylation of nNOS at Ser847 by CaMKII decreases NO generation and increases superoxide generation. Conversely, NO-induced S-nitrosylation of CaMKII at Cys6 is a prominent determinant of the CaMKII inhibition in ATP competitive fashion. Thus, the “cross-talk” between CaMKII and NO/superoxide may represent important signal transduction pathways in brain. In this review, we introduce the molecular mechanism of and pathophysiological role of mutual regulation between CaMKII and nNOS in neurons.

Phosphorylation of the Retinal Ribbon Synapse Specific t-SNARE Protein Syntaxin3B Is Regulated by Light via a Ca2 +-Dependent Pathway.

Neurotransmitter release at retinal ribbon-style synapses utilizes a specialized t-SNARE protein called syntaxin3B (STX3B). In contrast to other syntaxins, STX3 proteins can be phosphorylated in vitro at T14 by Ca2+/calmodulin-dependent protein kinase II (CaMKII). This modification has the potential to modulate SNARE complex formation required for neurotransmitter release in an activity-dependent manner. To determine the extent to which T14 phosphorylation occurs in vivo in the mammalian retina and characterize the pathway responsible for the in vivo phosphorylation of T14, we utilized quantitative immunofluorescence to measure the levels of STX3 and STX3 phosphorylated at T14 (pSTX3) in the synaptic terminals of mouse retinal photoreceptors and rod bipolar cells (RBCs). Results demonstrate that STX3B phosphorylation at T14 is light-regulated and dependent upon the elevation of intraterminal Ca2+. In rod photoreceptor terminals, the ratio of pSTX3 to STX3 was significantly higher in dark-adapted mice, when rods are active, than in light-exposed mice. By contrast, in RBC terminals, the ratio of pSTX3 to STX3 was higher in light-exposed mice, when these terminals are active, than in dark-adapted mice. These results were recapitulated in the isolated eyecup preparation, but only when Ca2+ was included in the external medium. In the absence of external Ca2+, pSTX3 levels remained low regardless of light/dark exposure. Using the isolated RBC preparation, we next showed that elevation of intraterminal Ca2+ alone was sufficient to increase STX3 phosphorylation at T14. Furthermore, both the non-specific kinase inhibitor staurosporine and the selective CaMKII inhibitor AIP inhibited the Ca2+-dependent increase in the pSTX3/STX3 ratio in isolated RBC terminals, while in parallel experiments, AIP suppressed RBC depolarization-evoked exocytosis, measured using membrane capacitance measurements. Our data support a novel, illumination-regulated modulation of retinal ribbon-style synapse function in which activity-dependent Ca2+ entry drives the phosphorylation of STX3B at T14 by CaMKII, which in turn, modulates the ability to form SNARE complexes required for exocytosis.

Tilianin Protects against Ischemia/Reperfusion-Induced Myocardial Injury through the Inhibition of the Ca2+/Calmodulin-Dependent Protein Kinase II-Dependent Apoptotic and Inflammatory Signaling Pathways.

Tilianin is a naturally occurring phenolic compound with a cardioprotective effect against myocardial ischemia/reperfusion injury (MIRI). The aim of our study was to determine the potential targets and mechanism of action of tilianin against cardiac injury induced by MIRI. An in silico docking model was used in this study for binding mode analysis between tilianin and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Oxygen-glucose deprivation/reperfusion- (OGD/R-) injured H9c2 cardiomyocytes and ischemia/reperfusion- (I/R-) injured isolated rat hearts were developed as in vitro and ex vivo models, respectively, which were both treated with tilianin in the absence or presence of a specific CaMKII inhibitor KN93 for target verification and mechanistic exploration. Results demonstrated the ability of tilianin to facilitater the recovery of OGD/R-induced cardiomyocyte injury and the maintenance of cardiac function in I/R-injured hearts. Tilianin interacted with CaMKIIδ with an efficient binding performance, a favorable binding score, and restraining p-CaMKII and ox-CaMKII expression in cardiomyocytes injured by MIRI. Importantly, inhibition of CaMKII abolished tilianin-mediated recovery of OGD/R-induced cardiomyocyte injury and maintenance of cardiac function in I/R-injured hearts, accompanied by the disability to protect mitochondrial function. Furthermore, the protective effects of tilianin towards mitochondrion-associated proapoptotic and antiapoptotic protein counterbalance and c-Jun N-terminal kinase (JNK)/nuclear factor- (NF-) κB-related inflammation suppression were both abolished after pharmacological inhibition of CaMKII. Our investigation indicated that the inhibition of CaMKII-mediated mitochondrial apoptosis and JNK/NF-κB inflammation might be considered as a pivotal mechanism used by tilianin to exert its protective effects on MIRI cardiac damage.

Wnt5b/Ryk-mediated membrane trafficking of P2X3 receptors contributes to bone cancer pain

Wnt5b, a member of Wnt family, plays multiple roles in tumor progression and metastasis. However, whether Wnt5b contributes to the sensitization of dorsal root ganglia (DRG) neurons and pathogenesis of bone cancer pain still remains unclear. Here, we found that the protein expression of Wnt5b and its atypical tyrosine protein kinase receptor Ryk was upregulated in ipsilateral DRGs in tumor-bearing mice. Application of Wnt5b evoked an increased discharge frequency in isolated DRG neurons and pain hypersensitivity in naïve mice which were almost completely prevented by anti-Ryk antibody. Moreover, intrathecal injection of anti-Ryk antibody to tumor-bearing mice significantly inhibited bone cancer-induced mechanic allodynia and thermal hyperalgesia. Subsequently, we also demonstrated that application of Wnt5b to cultured DRG neurons could enhance membrane P2X3 receptors and α,β-meATP-induced currents. Intrathecal injection of calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 or P2X3 receptors antagonist A317491 almost completely abolished Wnt5b-induced mechanical allodynia and thermal hyperalgesia in mice. Meanwhile, pretreatment with anti-Ryk antibody or CaMKII inhibitor KN93 can attenuate bone-cancer induced the upregulation of P2X3 membrane protein as well as pain hypersensitivity. These findings suggested that Wnt5b/Ryk promoted the trafficking of P2X3 receptors to the membrane via the activation of CaMKII in primary sensory neurons, resulting in peripheral sensitization and bone cancer-induced pain. Our results may offer a potential therapeutic strategy for bone cancer pain.

CaMKII antagonism in the ventral tegmental area impairs acquisition of conditioned approach learning in rats

This study investigated the role of calcium2+/calmodulin-dependent protein kinase II (CaMKII), a protein in the second messenger pathway of NMDA receptors, in the ventral tegmental area (VTA) in the acquisition and performance of conditioned approach learning. Male Long-Evans rats (N = 79) were exposed to 3 (to test acquisition) or 7 (to test performance) conditioning sessions in which they received 30 paired presentations of a light stimulus (CS) and a food pellet (US) on a random time schedule. These conditioning sessions were then followed by one 30-min session without the CS or US and lastly by a CS-only test session, where only the light stimulus was presented (without food) according to the same schedule as the conditioning sessions. Bilateral intra-VTA injections of KN93 (vehicle, 3.0, 4.5 or 6.0 μg/0.5 μL), a CaMKII inhibitor, were administered prior to each conditioning session to test effects on the acquisition of conditioned approach or prior to the CS-only test session to test effects on the performance of conditioned approach. KN93, when given prior to conditioning sessions, significantly reduced the number of conditioned approach responses emitted during CS presentations in the CS-only test. When KN93 was given prior to the CS-only test it had no effect. These results suggest that CaMKII activation in the VTA is necessary for the acquisition, but not the performance, of reward-related learning.

Atherogenic L5 LDL induces cardiomyocyte apoptosis and inhibits KATP channels through CaMKII activation

BackgroundCardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation plays a critical role in cardiomyocyte (CM) apoptosis and arrhythmia. Functional ATP-sensitive potassium (KATP) channels are essential for cardiac protection during ischemia. In cultured CMs, L5 low-density lipoprotein (LDL) induces apoptosis and QTc prolongation. L5 is a highly electronegative and atherogenic aberrant form of LDL, and its levels are significantly higher in patients with cardiovascular-related diseases. Here, the role of L5 in cardiac injury was studied by evaluating the effects of L5 on CaMKII activity and KATP channel physiology in CMs.MethodsCultured neonatal rat CMs (NRCMs) were treated with a moderate concentration (ie, 7.5 μg/mL) of L5 or L1 (the least electronegative LDL subfraction). NRCMs were examined for apoptosis and viability, CaMKII activity, and the expression of phosphorylated CaMKIIδ and NOX2/gp91phox. The function of KATP and action potentials (APs) was analyzed by using the patch-clamp technique.ResultsIn NRCMs, L5 but not L1 significantly induced cell apoptosis and reduced cell viability. Furthermore, L5 decreased Kir6.2 expression by more than 50%. Patch-clamp analysis showed that L5 reduced the KATP current (IKATP) density induced by pinacidil, a KATP opener. The partial recovery of the inward potassium current during pinacidil washout was susceptible to subsequent inhibition by the IKATP blocker glibenclamide. Suppression of IKATP by L5 significantly prolonged the AP duration. L5 also significantly increased the activity of CaMKII, the phosphorylation of CaMKIIδ, and the expression of NOX2/gp91phox. L5-induced apoptosis was prevented by the addition of the CaMKII inhibitor KN93 and the reactive oxygen species scavenger Mn (III)TBAP.ConclusionsL5 but not L1 induces CM damage through the activation of the CaMKII pathway and increases arrhythmogenicity in CMs by modulating the AP duration. These results help to explain the harmful effects of L5 in cardiovascular-related disease.