Role Of Calmodulin-dependent Protein Kinase II Research Articles

Calmodulin-dependent protein kinase II activation promotes kidney mesangial expansion in streptozotocin-induced diabetic mice

Calcium-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 plays a role in the development of diabetic nephropathy (DN). Streptozotocin (STZ) injection into FVB wild-type mice led to mild mesangial matrix expansion, reproducing an essential feature of early human DN. Mesangial matrix measurements were performed on trichrome-stained paraffin sections using a trainable segmentation method based on WEKA (Waikato Environment for Knowledge Analysis) Image J-Fiji plugin (TWS plugin), and the electron micrographs of the whole glomeruli stitched from individual 4800x partial glomerular images. Both methods demonstrated that the statistically significant mesangial matrix expansion seen in the diabetic mice was prevented by chronic pretreatment with KN-93, a small molecule CaMKII inhibitor. This study indicates a role for CaMKII in the development of mesangial alterations in diabetes and suggests a possible new therapeutic target.

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca2+-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.

The Role of CaMKII and ERK Signaling in Addiction.

Nicotine is the predominant addictive compound of tobacco and causes the acquisition of dependence through its interactions with nicotinic acetylcholine receptors and various neurotransmitter releases in the central nervous system. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) play a pivotal role in synaptic plasticity in the hippocampus. CaMKII is involved in long-term potentiation induction, which underlies the consolidation of learning and memory; however, the roles of CaMKII in nicotine and other psychostimulant-induced addiction still require further investigation. This article reviews the molecular mechanisms and crucial roles of CaMKII and ERK in nicotine and other stimulant drug-induced addiction. We also discuss dopamine (DA) receptor signaling involved in nicotine-induced addiction in the brain reward circuitry. In the last section, we introduce the association of polyunsaturated fatty acids and cellular chaperones of fatty acid-binding protein 3 in the context of nicotine-induced addiction in the mouse nucleus accumbens and provide a novel target for the treatment of drug abuse affecting dopaminergic systems.

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.

CaMKII as a key regulator of contrast-induced nephropathy through mPTP opening in HK-2 cells

Contrast-induced nephropathy (CIN), refers to acute kidney injury observed after administration of contrast media during angiographic or other medical procedures such as urography, and accounting for 12% of all causes of acute renal failure, but no specific prevention or treatment strategy exists for its obscure pathophysiology. The aim of our study was to explore the influence of calcium/calmodulin-dependent protein kinase II (CaMKII) in CIN by using HK-2 cells. Knockdown of CypD was achieved by lentivirus, and CaMKII overexpression by transfection with the plasmid. In this study, we have demonstrated that CypD-mediated mPTP opening triggered mitochondrial dysfunction and tubule cells apoptosis in CIN. We also found that iohexol treatment was associated with mitochondrial ROS overloading, ATP depletion and LDH release. Inhibition of CypD with the pharmacologic inhibitor or knockdown of CypD abrogated mPTP opening, oxidative stress, mitochondria damage, and cell apoptosis induced by iohexol. In addition, we found that inhibition of the CaMKII activity alleviated iohexol-induced CypD expression, whereas also decreased mPTP opening, oxidative stress, mitochondria damage, and cell apoptosis, similarly to the inhibition of CypD did. Moreover, CaMKII overexpression enhanced iohexol-induced mPTP opening, mitochondrial damage and renal tubular epithelial cells apoptosis. These findings first identified the novel role of CaMKII in iohexol-induced tubular cells apoptosis and delineated the CaMKII-CypD/mPTP pathway during contrast-induced tubular cell damage. Hence, these results could provide a new strategy for CIN protection.

TRPV4 disrupts mitochondrial transport and causes axonal degeneration via a CaMKII-dependent elevation of intracellular Ca2+

The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular mechanisms are unknown. Here, we show that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4R269C) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity. TRPV4R269C triggers increased intracellular Ca2+ through a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism, and CaMKII inhibition prevents both increased intracellular Ca2+ and neurotoxicity in Drosophila and cultured primary mouse neurons. Importantly, TRPV4 activity impairs axonal mitochondrial transport, and TRPV4-mediated neurotoxicity is modulated by the Ca2+-binding mitochondrial GTPase Miro. Our data highlight an integral role for CaMKII in neuronal TRPV4-associated Ca2+ responses, the importance of tightly regulated Ca2+ dynamics for mitochondrial axonal transport, and the therapeutic promise of TRPV4 antagonists for patients with TRPV4-related neurodegenerative diseases.

CaMKII is Necessary for Proliferation and Migration of Pulmonary Arterial Smooth Muscle Cells

ObjectiveCa2+/calmodulin‐dependent protein kinase II (CaMKII) is a calcium‐ and oxidation‐activated serine/threonine kinase, with four known isoforms. CaMKII has been shown to be necessary for remodeling involving smooth muscle cell proliferation and migration in response to injury in the systemic vasculature. However, the role of CaMKII in pulmonary arterial smooth muscle cell (PASMC) function remains unknown.MethodsPASMCs were isolated from male Wistar rats and expanded in culture. In cell lysates, mRNA expression of CaMKII isoforms was measured by real‐time quantitative reverse transcription PCR; protein expression was assessed via immunoblot. PASMC proliferation was measured via BrdU incorporation. PASMC migration was assessed by the Transwell assay. PASMCs were treated with 10 μM KN‐93 in vitro to inhibit CaMKII or with either vehicle or 10 μM KN‐92 (an inactive analog of KN‐93) for control experiments. To deplete CaMKIIδ protein, PASMCs were treated with 100nM siRNA targeted to CaMKIIδ while PASMCs treated with scrambled siRNA were used for control experiments.ResultsThe CaMKIIδ and CaMKIIγ isoforms showed mRNA and protein expression in rat PASMCs. Both proliferation and migration of rat PASMCs were inhibited by pharmacologic inhibition of CaMKII using KN‐93, but were not inhibited by the inactive analog KN‐92. siRNA‐mediated knockdown of CaMKIIδ resulted in reduction of CaMKIIδ protein expression but did not have an effect on either PASMC proliferation or migration.ConclusionsCaMKII activity is necessary for PASMC proliferation and migration. As the CaMKIIδ isoform is not required for these functions, future work will examine the role of the CaMKIIγ isoform.Support or Funding InformationFunding SourcesNIH K08HL133475 & Pulmonary Hypertension Association K08 Supplement

Involvement of CaMKII in regulating the release of diplotene-arrested mouse oocytes by pAkt1 (Ser473)

ABSTRACT Calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) had been reported to play a role in the process of fertilization. However, the role of CaMKII in the release of diplotene-arrested oocytes is poorly understood. In this study, we explored the potential effect of CaMKII on Akt1 and the relationship among CaMKII, Akt1 and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) during the meiotic resumption of mouse oocytes. We found that inhibition of CaMKII aggravated diplotene arrest. We detected the expression and distribution of pCaMKII (Thr286), pAkt1 (Ser473), Cdc25B and pCdc2 (Tyr15) when oocytes were treated with KN-93, SH-6, LY294002 or PIP3, respectively. Our data showed that down-regulated CaMKII by KN-93 decreased the levels of pAkt1 (Ser473) and rearranged the distribution of pAkt1 (Ser473). Meanwhile, down-regulated pAkt1 (Ser473) by SH-6 also decreased the levels of pCaMKII (Thr286), Cdc25B and pCdc2 (Tyr15) significantly and rearranged the distributions of pCaMKII (Thr286). Furthermore, our data showed that exogenous PIP3 up-regulated GVBD rates significantly and increased the levels of pCaMKII (Thr286) and pAkt1 (Ser473). On the contrary, down-regulation of PIP3 by LY294002 decreased GVBD rates and the levels of pCaMKII (Thr286) and pAkt1 (Ser473), respectively. Our results showed that Akt1 and CaMKII regulated each other, and PIP3 may be involved in these regulations during the release of mouse oocytes from diplotene arrest.

Molecular mechanism of TMEM16A regulation: role of CaMKII and PP1/PP2A.

This study explored the mechanism by which Ca2+-activated Cl- channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl- currents (IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

Molecular Characterization of Ca2+/Calmodulin-Dependent Protein Kinase II Isoforms in Three Rice Planthoppers-Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

This study reports the identification of splice variants for the calcium/calmodulin-dependent protein kinase II (CaMKII) gene from Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera. CaMKII is a multifunctional serine/threonine protein kinase that transduces Ca2+ signals in cells to control a range of cellular processes in the nervous system and muscular tissue. Sequence analysis showed that CaMKII was 99.0% identical at the amino acid level among three rice planthoppers, with the exception of a variable region located in the association domain. Four kinds of 20–81 amino acid “inserts” were found in the variable region. The phylogenetic tree of the deduced amino acid sequences showed that the NlCaMKII isoforms were more closely related to the LsCaMKII isoforms and were slightly distinct from SfCaMKII. CaMKII-E was the dominant type among the five main isoforms. CaMKII genes were constitutively expressed in various nymphal and adult stages and in tested tissues with the predominant transcription occurring in the head. There was no major tissue specificity of isoform expression, but the expression pattern and relative abundance of isoforms varied when compared with the RT-PCR between tissues. In addition, RNAi in N. lugens with dsRNA at a concentration of 200 ng nymph−1 induced a mortality of 77.7% on the 10th day and a reduction in the mRNA expression level of 67.2%. Unlike the holometabolous insect Helicoverpa armigera, the knockdown of NlCaMKII did not suppress the expression of 20E response genes, such as ECR, USP1, and HR3, in N. lugens. These results indicate that the role of CaMKII in hemimetabolous insects may be different from that in holometabolous insects.

Calcium/calmodulin-dependent protein kinase II causes atrial structural remodeling associated with atrial fibrillation and heart failure

Atrial fibrillation (AF) is sustained by reentrant mechanisms that depend, in part, on atrial structural remodeling. Increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity occurs in persistent AF. A general consensus has been that electrophysiological actions of CaMKII must be the contributing factor, but electrical remodeling in AF differs considerably with electrophysiological effects of CaMKII. CaMKII has been associated with structural remodeling in several tissues, but not the cardiac atria. The role of CaMKII in sustaining AF remains undefined. The purpose of this study was to assess the effects of CaMKII on AF-related structural remodeling. We evaluated the objective in a porcine AF-heart failure model using atrial gene transfer of the CaMKII inhibitory peptide CaMKIIn. We used conventional methods including invivo electrophysiological study, telemetry, western blot, echocardiography, and histology to quantify rhythm, function, microstructure, and signaling pathways relevant to CaMKII and structural remodeling. CaMKII levels and activity increased progressively in the early stages of AF-heart failure. Inhibiting CaMKII preserved atrial contractile function and attenuated atrial hypertrophy, fibrosis, and apoptosis but did not affect inflammation or myolysis. These effects were accompanied by significantly decreased phosphorylation of HDAC4, decreased expression of p38MAP-kinase, and alterations in the phosphorylation pattern and relative ratios of JNK isoforms. Our findings suggest that CaMKII mediates signaling pathways related to atrial contractile function and structural remodeling in AF. CaMKII inhibition is potentially a novel therapy for AF. These findings are of importance because no clinically relevant mediators of either atrial contractile function or structural remodeling have yet been identified.

The role of CaMKII in neuropathic pain and fear memory in chronic constriction injury in rats

Purpose/Aim of the study In this study, we sought to observe the effects of Ca2+/calmodulin-dependent protein kinase II (CaMKII) on neuropathic pain and fear memory in a rat model of chronic constriction injury (CCI).Materials and methods Rats were randomly divided into the Sham, Control, CCI and m-AIP groups. In the m-AIP group, an intrathecal injection of m-AIP, the specific antagonist of CaMKII, was given either pretreatment or posttreatment in rats. Mechanical allodynia and thermal hyperalgesia tests were used to test pain behavior, and the passive avoidance test was used to measure fear memory in rats.Results The right side of hippocampus tissues were taken at varying time points. The expression levels of CaMKII-α, pCaMKII-α, CaMKII-β, pCaMKII-β, NR2A, pNR2A, NR2B and pNR2B were detected by Western blot analysis. Significant pain behaviors and impaired cognitive function were shown after CCI surgery, accompanied by the upregulation of proteins in the hippocampus. Pretreatment with m-AIP appeared to provide a temporary improvement in pain and fear memory and decreased the expression of the above proteins in the hippocampus seven days after surgery. Furthermore, postoperative treatment with m-AIP provided relief for pain behavior and protein expression but did not affect fear memory.Conclusions These data suggested that CaMKII played an important role in the crosstalk between neuropathic pain and fear memory, indicating that CaMKII may be a potential therapeutic target for neuropathic pain treatment.

Calcium/calmodulin-dependent kinase II and memory destabilization: a new role in memory maintenance.

In this review, we discuss the poorly explored role of calcium/calmodulin‐dependent protein kinase II (CaMKII) in memory maintenance, and its influence on memory destabilization. After a brief review on CaMKII and memory destabilization, we present critical pieces of evidence suggesting that CaMKII activity increases retrieval‐induced memory destabilization. We then proceed to propose two potential molecular pathways to explain the association between CaMKII activation and increased memory destabilization. This review will pinpoint gaps in our knowledge and discuss some ‘controversial’ observations, establishing the basis for new experiments on the role of CaMKII in memory reconsolidation. The role of CaMKII in memory destabilization is of great clinical relevance. Still, because of the lack of scientific literature on the subject, more basic science research is necessary to pursue this pathway as a clinical tool.

Endothelial CaMKII as a regulator of eNOS activity and NO-mediated vasoreactivity.

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase important in transducing intracellular Ca2+ signals. While in vitro data regarding the role of CaMKII in the regulation of endothelial nitric oxide synthase (eNOS) are contradictory, its role in endothelial function in vivo remains unknown. Using two novel transgenic models to express CaMKII inhibitor peptides selectively in endothelium, we examined the effect of CaMKII on eNOS activation, NO production, vasomotor tone and blood pressure. Under baseline conditions, CaMKII activation was low in the aortic wall. Consistently, systolic and diastolic blood pressure, heart rate and plasma NO levels were unaltered by endothelial CaMKII inhibition. Moreover, endothelial CaMKII inhibition had no significant effect on NO-dependent vasodilation. These results were confirmed in studies of aortic rings transduced with adenovirus expressing a CaMKII inhibitor peptide. In cultured endothelial cells, bradykinin treatment produced the anticipated rapid influx of Ca2+ and transient CaMKII and eNOS activation, whereas CaMKII inhibition blocked eNOS phosphorylation on Ser-1179 and dephosphorylation at Thr-497. Ca2+/CaM binding to eNOS and resultant NO production in vitro were decreased under CaMKII inhibition. Our results demonstrate that CaMKII plays an important role in transient bradykinin-driven eNOS activation in vitro, but does not regulate NO production, vasorelaxation or blood pressure in vivo under baseline conditions.

A New Insight into the Role of CART in Cocaine Reward: Involvement of CaMKII and Inhibitory G-Protein Coupled Receptor Signaling.

Cocaine- and amphetamine-regulated transcript (CART) peptides are neuropeptides that are expressed in brain regions associated with reward, such as the nucleus accumbens (NAc), and play a role in cocaine reward. Injection of CART into the NAc can inhibit the behavioral effects of cocaine, and injecting CART into the ventral tegmental area (VTA) reduces cocaine-seeking behavior. However, the exact mechanism of these effects is not clear. Recent research has demonstrated that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and inhibitory G-protein coupled receptor (GPCR) signaling are involved in the mechanism of the effect of CART on cocaine reward. Hence, we review the role of CaMKII and inhibitory GPCR signaling in the effect of CART on cocaine reward and provide a new insight into the mechanism of that effect. In this article, we will first review the biological function of CART and discuss the role of CART in cocaine reward. Then, we will focus on the role of CaMKII and inhibitory GPCR signaling in cocaine reward. Furthermore, we will discuss how CaMKII and inhibitory GPCR signaling are involved in the mechanistic action of CART in cocaine reward. Finally, we will provide our opinions regarding the future directions of research on the role of CaMKII and inhibitory GPCR signaling in the effect of CART on cocaine reward.

Autonomous CaMKII Activity as a Drug Target for Histological and Functional Neuroprotection after Resuscitation from Cardiac Arrest

SUMMARYThe Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling, but its role in pathological glutamate signaling (excitotoxicity) remains less clear, with indications for both neurotoxic and neuro-protective functions. Here, the role of CaMKII in ischemic injury is assessed utilizing our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). CaMKII inhibition (with tatCN21 or tatCN19o) at clinically relevant time points (30 min after resuscitation) greatly reduces neuronal injury. Importantly, CaMKII inhibition also works in combination with mild hypothermia, the current standard of care. The relevant drug target is specifically Ca2+-independent “autonomous” CaMKII activity generated by T286 autophosphorylation, as indicated by substantial reduction in injury in autonomy-incompetent T286A mutant mice. In addition to reducing cell death, tatCN19o also protects the surviving neurons from functional plasticity impairments and prevents behavioral learning deficits, even at extremely low doses (0.01 mg/kg), further highlighting the clinical potential of our findings.

Roles of calcium/calmodulin-dependent kinase II in long-term memory formation in crickets.

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca2+/CaM and cAMP signaling participates in long-term memory (LTM) formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM). Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca2+ influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC) activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca2+ signaling and cAMP signaling for LTM formation, a new role of CaMKII in learning and memory.

Calcium/calmodulin‑dependent protein kinaseII enhances metastasis of human gastric cancer by upregulating nuclear factor‑κB and Akt‑mediated matrix metalloproteinase‑9 production.

Calcium/calmodulin‑dependent protein kinaseII (CaMKII) is a multi-functional serine/threonine protein kinase, involved in processes that cause tumor progression, including cell cycle regulation, apoptosis and differentiation. However, the role of CaMKII in cancer cell metastasis has not been fully elucidated. In the present study, the function of CaMKII in gastric cancer cell metastasis is reported. Firstly, it was demonstrated that the overexpression of H282R (constitutively active CaMKII) enhanced gastric cancer cell migration and invasion, and the inhibition of CaMKII activity by KN‑62 decreased gastric cancer cell metastasis. Furthermore, H282R upregulated matrix metalloproteinase‑9 (MMP‑9) expression and production, which were dependent on CaMKII‑mediated increase in nuclear factor (NF)‑κB and Akt activation. Finally, CaMKII activation, through phosphorylation of the Thr286 site, was significantly increased in the metastatic gastric cancer tissues compared with non‑metastatic tissues, suggesting that CaMKII has an important function in the regulation of gastric cancer cell metastasis. Collectively, the present study demonstrated that CaMKII promotes gastric cancer cell metastasis by NF‑κB and Akt‑mediated‑MMP‑9 production. These findings suggest a novel function of CaMKII in the control of gastric cancer metastasis, offering a promising target for future therapeutics to treat and prevent gastric cancer metastases via the inhibition of CaMKII activity.

247 AT1 RECEPTORS AND CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE II INHIBITION UNLIKE THEIR COMBINATION DURING ISCHEMIA-REPERFUSION IS ASSOCIATED WITH CHANGES IN CONTENT OF OXIDATIVE STRESS AND APOPTOTIC MARKERS AND IMPROVED CONTRACTILE FUNCTION IN HEART

<h3>Introduction</h3> Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a crucial regulator of Ca2+-handling and excitation-contraction coupling in cardiomyocytes. Lately, an oxidative activation of CaMKII has been described. Oxidative stress is an integral component of ischemia/reperfusion (IR) injury which results in a complex of subcellular and molecular changes leading to impaired cardiac function and cell death. As AT1 receptor activation promotes reactive oxygen species production through NADPH oxidase system it may be hypothesized that this signaling cascade might play an important role in oxidative activation of CaMKII under IR conditions. <h3>Methods</h3> To test this, the isolated Langendorff-perfused hearts of Wistar rats treated with losartan (20 mg/kg, p.o., 14 days) as well as control ones were subjected to ischemia (30 min) followed by reperfusion (40 min). A role of CaMKII was studied by using a specific CaMKII inhibitor (KN-93, 0,5 µmol/dm3) which was present in the perfusion solution 10 min before onset of ischemia, during ischemia and first 10 min of reperfusion. During the whole IR protocol, myocardial parameters were being continuously recorded. Protein content of total, oxidized CaMKII, oxidative stress markers, such as pro-oxidant NADPH oxidase (NOX2) and antioxidant superoxide dismutase (MnSOD) and catalase (CAT) as well as apoptotic markers (Bax, Bcl-2, cyt c) was evaluated in left ventricles by Western blotting. <h3>Results</h3> In the IR hearts, inhibition of AT1 receptors or CaMKII alone normalized the protein content of antioxidant proteins MnSOD and CAT to the values of the non-ischemic hearts. Likewise, the protein content of oxidized CaMKII was modified. Interestingly, the protein content of NOX2 was significantly increased in IR-subjected group treated with losartan, suggesting a negative feedback due to a permanent AT1 inhibition. Moreover, the protein content of anti-apoptotic Bcl-2 was not changed upon losartan treatment nor CaMKII inhibition in the ischemic hearts. On the other hand, pro-apoptotic Bax and cytochrome C were upregulated in the non-ischemic losartan group. Simultaneous inhibition of AT1 receptors and CaMKII had no additive effect on content of any protein studied. On the other hand, although AT1 receptor inhibition or CaMKII inhibition improved postischemic recovery of left-ventricular developed pressure (LVDP), the simultaneous inhibition of AT1 receptors and CaMKII abolished this beneficial effect. <h3>Conclusion</h3> In conclusion, it is likely that AT1 receptor signaling cascade plays a role in oxidative activation of CaMKII documented by normalized anti-oxidant protein content and by improved contractile function of heart after AT1 inhibition. However, a simultaneous inhibition of CaMKII and AT1 receptors resulted in worsened LVDP recovery suggesting that excessive inhibition of CaMKII activation and therefore diminished activation of CaMKII target proteins of Ca2+-handling may be deleterious.

Role of CaMKII in post acidosis arrhythmias: A simulation study using a human myocyte model

Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca2+ load and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation. However, the molecular mechanisms underlying these arrhythmias are still unclear. To better understand this process, acidosis produced by CO2 increase from 5% to 30%, resulting in intracellular pH (pHi) change from 7.15 to 6.7, was incorporated into a myocyte model of excitation-contraction coupling and contractility, including acidotic inhibition of L-type Ca2+ channel (ICaL), Na+–Ca2+ exchanger, Ca2+ release through the SR ryanodine receptor (RyR2) (Irel), Ca2+ reuptake by the SR Ca2+ ATPase2a (Iup), Na+–K+ pump, K+ efflux through the inward rectifier K+ channel and the transient outward K+ flow (Ito) together with increased activity of the Na+–H+ exchanger (INHE). Simulated CaMKII regulation affecting Irel, Iup, ICaL, INHE and Ito was introduced in the model to partially compensate the acidosis outcome. Late Na+ current increase by CaMKII was also incorporated. Using this scheme and assuming that diastolic Ca2+ leak through the RyR2 was modulated by the resting state of this channel and the difference between SR and dyadic cleft [Ca2+], postacidotic delayed after depolarizations (DADs) were triggered upon returning to normal pHi after 6min acidosis. The model showed that DADs depend on SR Ca2+ load and on increased Ca2+ leak through RyR2. This postacidotic arrhythmogenic pattern relies mainly on CaMKII effect on ICaL and Iup, since its individual elimination produced the highest DAD reduction. The model further revealed that during the return to normal pHi, DADs are fully determined by SR Ca2+ load at the end of acidosis. Thereafter, DADs are maintained by SR Ca2+ reloading by Ca2+ influx through the reverse NCX mode during the time period in which [Na+]i is elevated.