Calmodulin-dependent Protein Kinase II Inhibitor KN-93 Research Articles

Abstract 9396: Calmodulin-Dependent Protein Kinase II Activation Promotes Kidney Mesangial Expansion in Streptozotocin-Induced Diabetic Mice

Background: Calmodulin-dependent protein kinase II (CaMKII) is upregulated in diabetes mellitus (DM), leading to the overproduction of collagen in the myocardium. We hypothesized that CaMKII also stimulates the extracellular matrix production in the kidneys leading to mesangial matrix expansion, a hallmark of diabetic nephropathy and a major cause of renal failure. To test this hypothesis, we developed an automated process of mesangial matrix measurement. Methods: We induced Type 1 diabetes (DM1) in wild type female FVB mice via an intraperitoneal injection of Streptozotocin (STZ, 200 mg/kg ip) and studied the dynamics of mesangial matrix fractional area (MMFA) over 10 weeks (W) in mice with: DM , DM/KN93 , 6 W after STZ, followed by 4 W treatment with a CaMKII inhibitor KN93 (70 μg/kg/day, via mini pump), and Control (C) (vehicle only) mice. Both kidneys were retrieved. MMFA measurements were performed on 50 glomeruli from each animal on trichrome-stained paraffin sections using a trainable segmentation WEKA (Waikato Environment of Knowledge Analysis) plugin for Fiji, as well as on the electron micrographs of 4-5 whole glomeruli/animal. Results: Both DM and DM/KN93 mice were severely hyperglycemic (517 and 538 mg/dL compared to 161 mg/dL in C). In DM mice, the kidney weight increased by 76% and the MMFA significantly increased by 81% compared to C (9.4 ± 1% vs 5.2 ± 2.8%). In DM/KN93 mice, MMFA was similar to that in controls (6.2 ± 2.3%). The electron microscopy study has confirmed these MMFA changes. Other features of human diabetic nephropathy were not seen and the fractional mesangial cell area and the fractional podocyte area were relatively unchanged in DM and DM/KN93 mice. Conclusions: Our DM1 mouse model reproduces the key features of early human diabetic nephropathy. KN93 prevents DM-induced extracellular matrix production and kidney mesangial matrix expansion, suggesting that chronic inhibition of CaMKII could be a potential treatment for the early diabetic nephropathy. Reliable automated measurement of the MM area can be achieved after training by the WEKA plugin.

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.

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.

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.

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.

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.

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.

Tau hyperphosphorylation induced by the anesthetic agent ketamine/xylazine involved the calmodulin-dependent protein kinase II.

Tau hyperphosphorylation is a major neuropathological hallmark of many neurodegenerative disorders such as Alzheimer's disease. Several anesthetics have been shown previously to induced marked tau hyperphosphorylation. Although the ketamine/xylazine mixture is one of the most commonly used anesthetic agents in animal research and veterinary practice, the effect of this anesthetic agent on tau phosphorylation still remains to be determined. Here, we found that ketamine-/xylazine-induced a rapid and robust hyperphosphorylation of tau in a dose-dependent manner under normothermic and hypothermic conditions in mice. When used together, ketamine and xylazine exerted a synergistic action on tau phosphorylation most strongly not only on epitopes S396 and S262, but also on other residues (T181, and S202/T205). We observed that activation of the calmodulin-dependent protein kinase II (CaMKII) is the major upstream molecular event leading to tau hyperphosphorylation following ketamine/xylazine anesthesia in mice. Moreover, we observed that intracerebroventricular injection of the selective CaMKII inhibitor KN93 attenuated tau hyperphosphorylation. Since ketamine/xylazine also had a marked impact on other key molecular signaling pathways involving the MAP/microtubule affinity-regulating kinase (MARK), extracellular signal-regulated kinase (ERK), and glycogen synthase kinase-3 (GSK3), our study calls for high caution and careful monitoring when using this anesthetic agent in laboratory animal settings across all fields of biological sciences in order to avoid artifactual results.

Hyperglycemia Induces Osteoclastogenesis and Bone Destruction Through the Activation of Ca2+/Calmodulin-Dependent Protein Kinase II.

Hyperglycemia induces osteoclastogenesis and bone resorption through complicated, undefined mechanisms. Ca2+/calmodulin-dependent protein kinase II (CaMKII) promotes osteoclastogenesis, and could be activated by hyperglycemia. Here, we investigated whether CaMKII is involved in hyperglycemia-induced osteoclastogenesis and subsequent bone resorption. Osteoclast formation, bone resorption, CaMKII expression and phosphorylation were measured under high glucose in vitro and in streptozotocin-induced hyperglycemia rats with or without CaMKII inhibitor KN93. The results showed that 25mmol/L high glucose in vitro promoted cathepsin K and tartrate-resistant acid phosphatase expression (p < 0.05) and osteoclast formation (p < 0.01) associated with enhancing β isoform expression (p < 0.05) and CaMKII phosphorylation (p < 0.001). Hyperglycemia promoted the formation of osteoclasts and resorption of trabecular and alveolar bone, and inhibited sizes of femur and mandible associated with enhanced CaMKII phosphorylation (p < 0.001) in rats. All these changes could be alleviated by KN93. These findings imply that CaMKII participates not only in hyperglycemia-induced osteoclastogenesis and subsequent bone resorption, but also in the hyperglycemia-induced developmental inhibition of bone.

Linagliptin improved myocardial function recovery in rat hearts after a prolonged hypothermic preservation

AimsTo determine whether linagliptin, a dipeptidyl peptidase 4 inhibitor, can promote the recovery of cardiac function after hypothermic preservation. Main methodsRat hearts were preserved in cold Celsior solution with or without linagliptin for 9 h. Cardiac function was evaluated at 60 min of reperfusion after hypothermic preservation. Cardiac mitochondrial morphology was observed using transmission electron microscope. The expression of dynamin-related protein 1 (Drp1), NADPH oxidase 2 (NOX2), calmodulin-dependent protein kinase II (CaMKII) were detected using Western blot. Key findingsCompared with Celsior group, supplement of Celsior solution with linagliptin (0.25–0.75 nM) could significantly prevent hypothermic preservation-induced cardiac dysfunction. The expression of NOX2 protein, ROS level and MDA content in cardium were increased after hypothermic preservation, which was inhibited by linagliptin. Although the mitofusin1, 2, optic atrophy type 1, and total Drp1 expression in myocardium did not change, the level of p-Drp1 S616 and mitochondrial Drp1 were enhanced after hypothermic preservation. Linagliptin supplement could inhibit the hypothermic preservation-induced increase in p-Drp1 S616 and mitochondrial Drp1 protein, and mitigate the mitochondrial fragmentation. Level of p-CaMKII protein enhanced after hypothermic preservation, which could be prevented by linagliptin or a NOX2 inhibitor Phox-I2. Both Phox-I2 and a CaMKII inhibitor KN-93 could reduce the hypothermic preservation-induced increase in p-Drp1 S616 and mitochondrial Drp1 protein. SignificanceSupplement Celsior solution with linagliptin could improve cardiac function recovery in 9-h hypothermic preserved rat hearts. The cardioprotective effect of linagliptin might be due to the inhibition of Drp1 phosphorylation and mitochondrial translocation by preventing NOX2-mediated CaMKII activation.

Roles of Ca(2+)/calmodulin-dependent protein kinase II in subcellular expression of striatal N-methyl-D-aspartate receptors in l-3, 4-dihydroxyphenylalanine-induced dyskinetic rats.

BackgroundThe role of N-Methyl-D-aspartate (NMDA) receptors is critical to the development of l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) in Parkinson’s disease (PD). Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to regulate the expression and activation of NMDA receptors in LID, but the interaction between LID and CaMKII-modulated NMDA receptor activity is not clear so far.MethodsWe used 6-hydroxydopamine-lesioned rats to create PD rat model, and at least 21 days of l-DOPA was administrated followed with or without microinjection of CaMKII inhibitor KN-93 into the lesioned striatum of all the PD rats and sham rats. A surface receptor cross-linking assay was used to distinguish expression of striatal NMDA receptors in surface and intracellular compartments.Resultsl-DOPA treatment enhanced surface levels of GluN1 expression and reduced its intracellular expression, but did not change total levels of GluN1 protein in the lesioned striatum. In contrast, l-DOPA decreased GluN2A surface expression but increased its intracellular expression. l-DOPA increased GluN2B expression preferentially in the surface compartment. We also found that l-DOPA increased CaMKII autophosphorylation at T286 in striatal neurons. The inhibition of CaMKII by microinjecting CaMKII inhibitor KN-93 into the lesioned striatum largely reversed the l-DOPA-induced changes in three subunits. In addition, dyskinetic behaviors of animals were observed alleviated after treatment of KN-93.ConclusionOur research indicates that long-term l-DOPA administration activates CaMKII in striatal neurons. Activated CaMKII is involved at least in part in mediating l-DOPA-induced changes of NMDA receptors surface/intracellular expression.

Protein kinaseC and Ca(2+) -calmodulin-dependent protein kinaseII mediate the enlarged reverse INCX induced by ouabain-increased late sodium current in rabbit ventricular myocytes.

What is the central question of this study? What are the effects of protein kinaseC (PKC) and Ca(2+) -calmodulin-dependent protein kinaseII (CaMKII) on late sodium current (INaL ), reverse Na(+) -Ca(2+) exchange current (reverse INCX ) or intracellular Ca(2+) levels changed by ouabain? What is the main finding and its importance? Ouabain, even at low concentrations (0.5-8.0μm), can increase INaL and reverse INCX , and these effects may contribute to the effect of the glycoside to increase Ca(2+) transients and contractility. Both PKC and CaMKII activities may mediate or modulate these processes. It has been reported that the cardiac glycoside ouabain can increase the late sodium current (INaL ), as well as the diastolic intracellular calcium concentration and contractile shortening. Whether an increase of INaL participates in a pathway that can mediate the positive inotropic response to ouabain is unknown. We therefore determined the effects of ouabain on INaL , reverse Na(+) -Ca(2+) exchange current (reverse INCX ), intracellular Ca(2+) ([Ca(2+) ]i ) levels and contractile shortening in rabbit isolated ventricular myocytes. Ouabain (0.1-8μm) markedly increased INaL and reverse INCX in a concentration-dependent manner, with significant effects at concentrations as low as 0.5 and 1μm. These effects of ouabain were suppressed by the INaL inhibitors TTX and ranolazine, the protein kinaseC inhibitor bisindolylmaleimide and the Ca(2+) -calmodulin-dependent protein kinaseII inhibitor KN-93. The enhancement by 0.5μm ouabain of ventricular myocyte contractility and intracellular Ca(2+) transients was suppressed by 2.0μm TTX. We conclude that ouabain, even at low concentrations (0.5-8.0μm), can increase INaL and reverse INCX , and these effects may contribute to the effect of the glycoside to increase Ca(2+) transients and contractility. Both protein kinaseC and Ca(2+) -calmodulin-dependent protein kinaseII activities may mediate or modulate these processes.

RETRACTED: Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca(2+) signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca(2+) through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca(2+) signaling insitu, by analyzing Ca(2+) sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca(2+) spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca(2+) release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca(2+) depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site onRyR2. The appearance of long-lasting Ca(2+) release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca(2+) release events.

Biodentine Induces Human Dental Pulp Stem Cell Differentiation through Mitogen-activated Protein Kinase and Calcium-/Calmodulin-dependent Protein Kinase II Pathways

IntroductionBiodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. MethodshDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. ResultsBiodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. ConclusionsBiodentine is a bioactive and biocompatible material capable of inducing odontoblast differentiation of hDPSCs. Our results indicate that this induction is regulated via MAPK and CaMKII pathways.

Reducing Oxidative Stress and CaMKII Activity as an Antiarrhythmic Strategy in Woodchucks in Winter

Multiple lines of evidence have suggested that increased oxidative stress and activation of Ca2+-calmodulin-dependent protein kinase II (CaMKII) are proarrhythmic in various diseased heart models. In the present study we have evaluated the involvement of oxidative stress and CaMKII pathway in antiarrhythmic mechanisms in a unique coronary artery occlusion (CAO)-induced arrhythmia model in woodchucks. Woodchucks, are true hibernators, and in winter they survive without food or water or activity and their hearts are more resistant to cardiac arrhythmias than in summer. We conducted Western blotting analyses and compared the expression levels of key antioxidant enzymes. In control hearts (without CAO) the expression level of catalase was higher in winter than in summer, while the MnSOD levels were the same. While CAO (24 hrs) upregulated both catalase and MnSOD, the increase in catalase was greater in winter than in summer. Meanwhile, oxidized CaMKII (activated form) levels were lower in winter than in summer. The effects of the CaMKII inhibitor KN-93 on CAO-induced arrhythmias in summer were also evaluated. We carried out CAO surgeries and monitored ECG by telemetry for 24 hrs. KN-93 was administrated with an initial bolus injection followed by a continuous delivery via osmotic minipumps. Arrhythmia scores were evaluated and compared to the control group. KN-93 significantly decreased the arrhythmia score in woodchucks in summer (3.4 ± 0.4) to a lower level (1.3 ± 0.6, p<0.05), which is similar to that observed in winter (1.6 ± 0.3), suggesting the reduction of CaMKII activity may account for the adaptation for the heart's resistance to CAO-induced arrhythmias during winter in woodchucks. These results suggest that manipulation of CaMKII signaling may hold therapeutic potential for treating cardiac arrhythmias.

Mitigation of postischemic cardiac contractile dysfunction by CaMKII inhibition: effects on programmed necrotic and apoptotic cell death

While Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been suggested to be an important protein regulating heart function upon ischemia/reperfusion (I/R), the mechanisms responsible are not fully known. Furthermore, it is not known whether CaMKII activation can modulate necroptosis, a recently described form of programmed cell death. In order to investigate these issues, Langendroff-perfused rat hearts were subjected to global ischemia and reperfusion, and CaMKII inhibition was achieved by adding the CaMKII inhibitor KN-93 (0.5μmol/dm(3)) to the perfusion solution before the induction of ischemia. Immunoblotting was used to detect changes in expression of proteins modulating both necroptotic and apoptotic cell death. CaMKII inhibition normalized I/R induced increases in expression of necroptotic RIP1 and caspase-8 along with proteins of the intrinsic apoptotic pathway, namely cytochrome c and caspase-9. In addition, it increased the Bcl-2/Bax ratio and reduced caspase-3 and cleaved PARP1 content suggesting reduction of cell death. These changes coexisted with improvement of postischemic contractile function. On the other hand, there was no correlation between levels of pT287-CaMKIIδ and LVDP recovery after I/R. These results demonstrate for the first time that CaMKII inhibition may mitigate cardiac contractile dysfunction, at least partially, by limiting the contents of not only apoptotic, but also necroptotic proteins. Phosphorylation of CaMKII seems unlikely to determine the degree of postischemic recovery of contractile function.

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ-aminobutyric acid type A receptor (GABAA R) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABAA R antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N-methyl-D-aspartate (NMDA)-subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA-subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN-93, NMDA receptor antagonist D-APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization.

Abstract WP259: CaMKII Inhibitor tatCN21 Decreases Neuronal Damage Following Cardiac Arrest and Cardiopulmonary Resuscitation in Mice

INTRODUCTION: Glutamate excitotoxicity is considered an important mechanism of ischemic neuronal damage. However, directly inhibiting glutamate receptors can cause severe side effects due to their key physiological functions in the central nervous system (CNS). Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major downstream target of glutamate-induced Ca2+ signaling. The aim of this study is to examine the neuroprotective potential of the CaMKII inhibitor tatCN21 on experimental cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Methods: Global cerebral ischemia was induced with 8 min of cardiac arrest followed by CPR in C57BL/6 male mice. The mice were randomly administered either tatCN21 (1 mg/kg, n=14) or vehicle (saline, n=13) 2 minutes after resuscitation (intravenous). Hippocampal neuronal damage was analyzed three days after resuscitation by H&amp;E staining. Primary hippocampal neurons were insulted with 400 μm glutamate for 5 min and treated with tatCN21 (5 μm), the scrambled CN peptide (tatRev, 5 μm) or KN93 (10 μm) 1 hr after removal of glutamate, cell viability was measure by LDH release 24 hrs later. Statistical analyses were performed using ANOVA and t tests, with P&lt;0.05 considered statistically significant. Results: Analysis of histological damage 72 hrs after resuscitation revealed that tatCN21 significantly reduces hippocampal CA1 neuronal injury compared to vehicle-treated mice, reducing damage from 72.9±4.5% (n=13) for vehicle to 54.1±7.6% (n=14, P&lt;0.05) for tatCN21 treated mice. tatCN21 significantly reduced glutamate-induced neuronal cell death, but not the classical CaMKII inhibitor KN93 (P&lt;0.05). No difference in body weight, cardiac arrest parameters and survival rates were observed between each groups. Conclusions: Our data showed that the novel CaMKII inhibitor tatCN21 decreases neuronal damage both in vivo and in vitro. Therefore, this study indicated that CaMKII may have an important role in glutamate excititoxicity and that tatCN21 may serve as a potential therapeutic strategy for minimizing brain injury following cardiac arrest.

Oxidative activation of Ca2+/calmodulin-activated kinase II mediates ER stress-induced cardiac dysfunction and apoptosis

Endoplasmic reticulum (ER) stress elicits oxidative stress and intracellular Ca(2+) derangement via activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). This study was designed to examine the role of CaMKII in ER stress-induced cardiac dysfunction and apoptosis as well as the effect of antioxidant catalase. Wild-type FVB and transgenic mice with cardiac-specific overexpression of catalase were challenged with the ER stress inducer tunicamycin (3 mg/kg ip for 48 h). Presence of ER stress was verified using the ER stress protein markers immunoglobulin binding protein (BiP) and C/EBP homologous protein (CHOP), the effect of which was unaffected by catalase overexpression. Echocardiographic assessment revealed that tunicamycin elicited cardiac remodeling (enlarged end-systolic diameter without affecting diastolic and ventricular wall thickness), depressed fractional shortening, ejection fraction, and cardiomyocyte contractile capacity, intracellular Ca(2+) mishandling, accumulation of reactive oxygen species (superoxide production and NADPH oxidase p47phox level), CaMKII oxidation, and apoptosis (evidenced by Bax, Bcl-2/Bax ratio, and TUNEL staining), the effects of which were obliterated by catalase. Interestingly, tunicamycin-induced cardiomyocyte mechanical anomalies and cell death were ablated by the CaMKII inhibitor KN93, in a manner reminiscent of catalase. These data favored a permissive role of oxidative stress and CaMKII activation in ER stress-induced cardiac dysfunction and cell death. Our data further revealed the therapeutic potential of antioxidant or CaMKII inhibition in cardiac pathological conditions associated with ER stress. This research shows for the first time that contractile dysfunction caused by ER stress is a result of the oxidative activation of the CaMKII pathway.