KN-93 promotes HDAC4 nucleus translocation to promote fatty acid oxidation in myocardial infarction.

Myocardial infarction (MI) is a potentially fatal disease that causes a significant number of deaths worldwide. The strategy of increasing fatty acid oxidation in myocytes is considered a therapeutic avenue to accelerate metabolism to meet energy demands. We conducted the study aiming to investigate the effect of KN-93, which induces histone deacetylase (HDAC)4 shuttling to the nucleus, on fatty acid oxidation and the expression of related genes. A mouse model of myocardial infarction was induced by isoprenaline administration. Heart damage was assessed by the detection of cardiac injury markers. The level of fatty acid oxidation level was evaluated by testing the expression of related genes. Both immunofluorescence and immunoblotting in the cytosol or nucleus were utilized to observe the distribution of HDAC4. The interaction between HDAC4 and specificity protein (SP)1 was confirmed by co-immunoprecipitation. The acetylation level of SP1 was tested after KN-93 treatment and HDAC4 inhibitor. Oxygen consumption rate and immunoblotting experiments were used to determine whether the effect of KN-93 on increasing fatty acid oxidation is through HDAC4 and SP1. Administration of KN-93 significantly reduced cardiac injury in myocardial infarction and promoted fatty acid oxidation both in vitro and in vivo. KN-93 was shown to mediate nuclear translocation of HDAC4. HDAC4 was found to interact with SP1 and reduce SP1 acetylation. HDAC4 or SP1 inhibitors attenuated the effect of KN-93 on fatty acid oxidation. In conclusion, KN-93 promotes HDAC4 translocation to the nucleus, thereby potentially enhancing fatty acid oxidation by SP1.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

Activation of the N-methyl-D-aspartate receptor and calcium/calmodulin-dependent protein kinase IIα signal in the rostral anterior cingulate cortex is involved in pain-related aversion in rats with peripheral nerve injury.

The rostral anterior cingulate cortex (rACC) of rat brain is associated with pain-related emotions. However, the underlying molecular mechanism remains unclear. Here, we investigated the effects of the N-methyl-D-aspartate (NMDA) receptor and Ca2+/Calmodulin-dependent protein kinase type II (CaMKII)α signal on pain-related aversion in the rACC of a rat model of neuropathic pain (NP). Mechanical and thermal hyperalgesia were examined using von Frey and hot plate tests in a rat model of NP induced by spared nerve injury (SNI) of the unilateral sciatic nerve. Bilateral rACC pretreatment with the CaMKII inhibitor tat-CN21 (derived from the cell-penetrating tat sequence and CaM-KIIN amino acids 43-63) or tat-Ctrl (the tat sequence and the scrambled sequence of CN21) was performed on postoperative days 29-35 in Sham rats or rats with SNI. Spatial memory performance was tested using an eight-arm radial maze on postoperative days 34-35. Pain-related negative emotions (aversions) were evaluated using the place escape/avoidance paradigm on postoperative day 35 following the spatial memory performance test. The percentage of time spent in the light area was used to assess pain-related negative emotions (i.e., aversion). The expression levels of the NMDA receptor GluN2B subunit, CaMKIIα, and CaMKII-Threonine at position 286 (Thr286) phosphorylation in contralateral rACC specimens were detected by Western blot or real time PCR following the aversion test. Our data showed that pretreatment of the rACC with tat-CN21 increased determinate behavior but did not alter hyperalgesia or spatial memory performance in rats with SNI. In addition, tat-CN21 reversed the enhanced CaMKII-Thr286 phosphorylation and had no effect on the upregulated expression of GluN2B, CaMKIIα protein, and mRNA. Our data suggested that activation of the NMDA receptor-CaMKIIα signal in rACC is associated with pain-related aversion in rats with NP. These data may provide a new approach for the development of drugs that modulate cognitive and emotional pain aspects.

Inhibition of phosphorylated calcium/calmodulin-dependent protein kinase IIα relieves streptozotocin-induced diabetic neuropathic pain through regulation of P2X3 receptor in dorsal root ganglia.

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

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.

The CaMKIIα hub ligand Ph-HTBA promotes neuroprotection after focal ischemic stroke by a distinct molecular interaction.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a potential target for acute neuroprotection due to its key role in physiological and pathological glutamate signaling. The hub domain organizes the CaMKII holoenzyme into large oligomers, and additional functional effects on holoenzyme activation have lately emerged. We recently reported that compounds related to the proposed neuromodulator γ-hydroxybutyrate (GHB) selectively bind to the CaMKIIα hub domain and increase hub thermal stabilization, which is believed to have functional consequences and to mediate neuroprotection. However, the detailed molecular mechanism is unknown. In this study, we functionally characterize the novel and brain permeable GHB analog (E)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (Ph-HTBA). Administration of a single dose of Ph-HTBA at a clinically relevant time point (3-6 h after photothrombotic stroke) promotes neuroprotection with a superior effect at low doses compared to the smaller GHB analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA). In contrast to HOCPCA, Ph-HTBA reduces Ca2+-stimulated CaMKIIα Thr286 autophosphorylation in primary cortical neurons and substrate phosphorylation of recombinant CaMKIIα, potentially contributing to its neuroprotective effect. Supported by previous in silico docking studies, we suggest that Ph-HTBA makes distinct molecular interactions with the hub cavity, which may contribute to its differential functional profile and superior neuroprotective effect compared to HOCPCA. Together, this highlights Ph-HTBA as a promising tool to study hub functionality, but also as a good candidate for clinical development.

Effects and potential mechanism of Ca2+/calmodulin­dependent protein kinase II pathway inhibitor KN93 on the development of ovarian follicle.

Adequate regulation of the speed of follicular development has been reported to prolong the reproductive life of the ovary. The aim of the present study was to assess the potential effects and mechanism of the Ca2+/calmodulin­dependent protein kinase II (CaMKII) pathway on the development of ovarian follicle. In the present study, the expression of CaMKII was measured in the ovary of mice at different developmental stages by immunofluorescence, confirming that CaMKII has a role in follicular development. Subsequently, the 17.5 days post­coitus (dpc) embryonic ovaries were collected and cultured with KN93 for 4 days in vitro. It was revealed that KN93 inhibited the development of follicles, where it reduced the expression levels of oocyte and granulosa cell markers DEAD­box helicase 4 (DDX4) and forkhead box L2 (FOXL2). These results suggested that KN93 could delay follicular development. Proteomics technology was then used to find that 262 proteins of KN93 treated 17.5 dpc embryonic ovaries were significantly altered after in vitro culture. Bioinformatics analysis was used to analyze these altered proteins. In total, four important Kyoto Encyclopedia of Genes and Genome pathways, namely steroid biosynthesis, p53 signaling pathway and retinol metabolism and metabolic pathways, were particularly enriched. Further analysis revealed that the upregulated proteins NADP­dependent steroid dehydrogenase­like (Nsdhl), lanosterol synthase (Lss), farnesyl­diphosphate farnesyltransferase 1 (Fdft1), cytochrome P450 family 51 family A member 1 (Cyp51a1), hydroxymethylglutaryl­CoA synthase 1 (Hmgcs1), fatty acid synthase (Fasn) and dimethylallyltranstransferase (Fdps) were directly interacting with each other in the four enriched pathways. In summary, the potential mechanism of KN93 in slowing down follicular development most likely lies in its inhibitory effects on CaMKII, which upregulated the expression of Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps. This downregulated the expression of oocyte and granulosa cell markers DDX4 and FOXL2 in the follicles, thereby delaying follicular development. Overall, these results provide novel insight into the potential mechanism by which KN93 and CaMKII can delay follicular development.

[Role of CaMK II in pancreatic injury in mice with severe acute pancreatitis].

OBJECTIVE: To investigate the expression of Ca2+/calmodulin-dependent protein kinase II (CaMK Ⅱ) in pancreatic tissues of mice with severe acute pancreatitis (SAP) and explore the protective effect of KN93, a CaMK Ⅱ inhibitor, against pancreatic injury in SAP and the possible mechanism. METHODS: Thirty-six healthy male C57 mice were randomly divided into sham operation group, SAP group, KN93 group and SAP + KN93 group (n=9). Serum and pancreatic tissue samples were collected 24 h after modeling. The pathological changes in the pancreatic tissues were observed using HE staining. Serum lipase and amylase activities and the levels of inflammatory factors were detected using ELISA. Western blotting was used to detect the expressions of CaMK Ⅱ, p-CaMK Ⅱ, p-NF-κB, MAPK and p-MAPK in mouse pancreas. RESULTS: Compared with those in sham operation group, the expressions of p-CaMK Ⅱ, p-NF-κB and p-MAPK were significantly increased in SAP group (P < 0.05). KN93 treatment obviously alleviated pathological injuries of the pancreas in SAP mice, and significantly lowered serum levels of lipase, amylase and inflammatory factors (TNF-α and IL-6) and phosphorylation levels of NF-κB, ERK and MAPK proteins (P < 0.05). CONCLUSION: The activity of CaMK Ⅱ is significantly increased in the pancreatic tissue of SAP mice. KN93 can alleviate pancreatic injury and inflammation in SAP mice possibly through the ERK/MAPK signaling pathway.

CaMKII inhibitor KN-93 impaired angiogenesis and aggravated cardiac remodelling and heart failure via inhibiting NOX2/mtROS/p-VEGFR2 and STAT3 pathways.

Persistent cardiac Ca2+ /calmodulin-dependent Kinase II (CaMKII) activation was considered to promote heart failure (HF) development, some studies believed that CaMKII was a target for therapy of HF. However, CaMKII was an important mediator for the ischaemia-induced coronary angiogenesis, and new evidence confirmed that angiogenesis inhibited cardiac remodelling and improved heart function, and some conditions which impaired angiogenesis aggravated ventricular remodelling. This study aimed to investigate the roles and the underlying mechanisms of CaMKII inhibitor in cardiac remodelling. First, we induced cardiac remodelling rat model by ISO, pre-treated by CaMKII inhibitor KN-93, evaluated heart function by echocardiography measurements, and performed HE staining, Masson staining, Tunel staining, Western blot and RT-PCR to test cardiac remodelling and myocardial microvessel density; we also observed ultrastructure of cardiac tissue with transmission electron microscope. Second, we cultured HUVECs, pre-treated by ISO and KN-93, detected cell proliferation, migration, tubule formation and apoptosis, and carried out Western blot to determine the expression of NOX2, NOX4, VEGF, VEGFR2, p-VEGFR2 and STAT3; mtROS level was also measured. In vivo, we found KN-93 severely reduced myocardial microvessel density, caused apoptosis of vascular endothelial cells, enhanced cardiac hypertrophy, myocardial apoptosis, collagen deposition, aggravated the deterioration of myocardial ultrastructure and heart function. In vitro, KN-93 inhibited HUVECs proliferation, migration and tubule formation, and promoted apoptosis of HUVECs. The expression of NOX2, NOX4, p-VEGFR2 and STAT3 were down-regulated by KN-93; mtROS level was severely reduced by KN-93. We concluded that KN-93 impaired angiogenesis and aggravated cardiac remodelling and heart failure via inhibiting NOX2/mtROS/p-VEGFR2 and STAT3 pathways.

Effects of CaMKII phosphorylation on myocardial ischemia-reperfusion injury in rats / 中华急诊医学杂志

Objective:To explore the effects of calcium/calmodulin dependent protein kinase II (CaMKII) on myocardial ischemia-reperfusion injury in vitro, and apoptosis and autophagy of myocardial cells in isolated rats.Methods:Seventy female SD rats (250-280 g) with normal electrocardiogram were selected to establish the myocardial IR injury model by Langendorff perfusion system. These SD rats were randomly divided into five groups (n=14): cardiac ischemia reperfusion group (IR group), CaMKII phosphorylation activator group (IR+ isoproterenol group), CaMKII phosphorylation inhibitor analogue group (IR+KN92 group), CaMKII phosphorylation inhibitor group (IR+KN93 group), and control group. After reperfusion, the left ventricular function and myocardial morphology were measured to assess the myocardial injury, and TUNEL was performed to assess the apoptosis index. Western blot was used to determine the phosphorylation levels of CaMKII and PLN (p-CaMKII/CaMKII and p-PLN/PLN), and the expression levels of apoptosis-related proteins Bax, Bcl-2, cleaved caspase-3, and autophagy marker proteins LC3II/LC3I, Beclin-1 and P62.Results:Compared with the control group, the left ventricular function of the IR group was decreased, morphological arrangement of myocardial fibers was disordered, and the apoptosis index was increased. The levels of p-CaMKII/CaMKII, p-PLN/PLN, cleaved caspase-3, Bax/Bcl-2, LC3II/LC3I, and Beclin-1 were increased significantly, while the level of P62 was decreased significantly, and apoptosis and autophagy were increased significantly (all P<0.05). Compared with the IR group, the myocardial damage of rats in the IR+KN93 group was significantly improved, the apoptosis index was decreased, and the expression of p-CaMKII/CaMKII, p-PLN/PLN, Cleaved Caspase-3, Bax/Bcl-2, LC3II/LC3I and Beclin-1 were significantly decreased and the level of p62 was remarkable increased, and apoptosis and autophagy decreased significantly (all P< 0.05). Compared with the IR group, the left ventricular function was further decreased in the IR+ isoproterenol group, while the levels of apoptosis and autophagy were further increased ( P < 0.05), while there was no significant difference in myocardial indexes between the IR+ KN92 group and the IR group ( P > 0.05). Conclusions:Inhibition of CaMKII phosphorylation attenuates isolated myocardial ischemia-reperfusion injury by reducing apoptosis and autophagy.

Role of CaMK II in pancreatic injury in mice with severe acute pancreatitis / 南方医科大学学报

OBJECTIVE@#To investigate the expression of Ca2+/calmodulin-dependent protein kinase II (CaMK Ⅱ) in pancreatic tissues of mice with severe acute pancreatitis (SAP) and explore the protective effect of KN93, a CaMK Ⅱ inhibitor, against pancreatic injury in SAP and the possible mechanism.@*METHODS@#Thirty-six healthy male C57 mice were randomly divided into sham operation group, SAP group, KN93 group and SAP + KN93 group (n=9). Serum and pancreatic tissue samples were collected 24 h after modeling. The pathological changes in the pancreatic tissues were observed using HE staining. Serum lipase and amylase activities and the levels of inflammatory factors were detected using ELISA. Western blotting was used to detect the expressions of CaMK Ⅱ, p-CaMK Ⅱ, p-NF-κB, MAPK and p-MAPK in mouse pancreas.@*RESULTS@#Compared with those in sham operation group, the expressions of p-CaMK Ⅱ, p-NF-κB and p-MAPK were significantly increased in SAP group (P < 0.05). KN93 treatment obviously alleviated pathological injuries of the pancreas in SAP mice, and significantly lowered serum levels of lipase, amylase and inflammatory factors (TNF-α and IL-6) and phosphorylation levels of NF-κB, ERK and MAPK proteins (P < 0.05).@*CONCLUSION@#The activity of CaMK Ⅱ is significantly increased in the pancreatic tissue of SAP mice. KN93 can alleviate pancreatic injury and inflammation in SAP mice possibly through the ERK/MAPK signaling pathway.

Emerging roles of protein O-GlcNAcylation in cardiovascular diseases: Insights and novel therapeutic targets.

Cardiovascular diseases (CVDs) are the leading cause of death globally. While the major focus of pharmacological and non-pharmacological interventions has been on targeting disease pathophysiology and limiting predisposing factors, our understanding of the cellular and molecular mechanisms underlying the pathogenesis of CVDs remains incomplete. One mechanism that has recently emerged is protein O-GlcNAcylation. This is a dynamic, site-specific reversible post-translational modification of serine and threonine residues on target proteins and is controlled by two enzymes: O-linked ß-N-acetylglucosamine transferase (OGT) and O-linked ß-N-acetylglucosaminidase (OGA). Protein O-GlcNAcylation alters the cellular functions of these target proteins which play vital roles in pathways that modulate vascular homeostasis and cardiac function. Through this review, we aim to give insights on the role of protein O-GlcNAcylation in cardiovascular diseases and identify potential therapeutic targets in this pathway for development of more effective medicines to improve patient outcomes.

PKC, AKT and ERK1/2-Mediated Modulations of PARP1, NF-κB and PEA15 Activities Distinctly Regulate Regional Specific Astroglial Responses Following Status Epilepticus.

Status epilepticus (SE, a prolonged seizure activity) leads to reactive astrogliosis and astroglial apoptosis in the regional specific manners, independent of hemodynamics. Poly(ADP-ribose) polymerase-1 (PARP1) activity is relevant to these distinct astroglial responses. Since various regulatory signaling molecules beyond PARP1 activity may be involved in the distinct astroglial response to SE, it is noteworthy to explore the roles of protein kinases in PARP1-mediated reactive astrogliosis and astroglial apoptosis following SE, albeit at a lesser extent. In the present study, inhibitions of protein kinase C (PKC), AKT and extracellular signal-related kinases 1/2 (ERK1/2), but not calcium/calmodulin-dependent protein kinase II (CaMKII), attenuated CA1 reactive astrogliosis accompanied by reducing PARP1 activity following SE, respectively. However, inhibition of AKT and ERK1/2 deteriorated SE-induced dentate astroglial loss concomitant with the diminished PARP1 activity. Following SE, PKC- and AKT inhibitors diminished phosphoprotein enriched in astrocytes of 15 kDa (PEA15)-S104 and -S116 phosphorylations in CA1 astrocytes, but not in dentate astrocytes, respectively. Inhibitors of PKC, AKT and ERK1/2 also abrogated SE-induced nuclear factor-κB (NF-κB)-S311 and -S468 phosphorylations in CA1 astrocytes. In contrast, both AKT and ERK1/2 inhibitors enhanced NF-κB-S468 phosphorylation in dentate astrocytes. Furthermore, PARP1 inhibitor aggravated dentate astroglial loss following SE. AKT inhibition deteriorated dentate astroglial loss and led to CA1 astroglial apoptosis following SE, which were ameliorated by AKT activation. These findings suggest that activities of PARP1, PEA15 and NF-κB may be distinctly regulated by PKC, AKT and ERK1/2, which may be involved in regional specific astroglial responses following SE.

The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of NaV1.5 and RyR2 function.

Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca2+-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM-protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na+ channel (NaV1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-NaV1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca2+ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.

Expression and role of CaMKII and Cx43 in a rat model of post-stroke depression.

Expression of Ca2+/CaM-dependent protein kinase II (CaMKII) and connexin 43 (Cx43) in a rat model of post-stroke depression (PSD) was investigated. Rats were separated into control group (10 rats underwent a sham operation and were not ligated after incision), PSD group (13 PSD rats) and KN93 group (12 rats were treated with KN93, an inhibitor of CaMKII, on the basis of the PSD group). After PSD modeling, Longa scoring was performed, and an open field test as well as a step-through test were carried out to observe rat behavior. RT-qPCR and western blot analysis were used to detect the expression of CaMKII and CX43 in the hippocampus tissue. On the 14th day, the Longa scores in the PSD and KN93 groups were higher than that in the control group (P<0.05), while on the 18th day, Longa score was higher in the PSD group than that in the control and KN93 groups, and higher in the KN93 group than that in the control group (both P<0.05). In the PSD group, the Longa score on the 18th day was significantly higher than that on the 14th day, whereas in the KN93 group, the Longa score on the 18th day was significantly lower than that on the 14th day (both P<0.05). Compared with the PSD group on the 18th day, the passive avoidance defects in the KN93 group were improved, and the frequency of activity in the open field test was significantly increased. On the 18th day, the expression levels of the mRNA and protein of CaMKII were higher in the PSD group than in the control group, whereas those of Cx43 were lower in the PSD group than those in the control group (P<0.05). The mRNA and protein expression levels of CaMKII in the KN93 group were lower than those in the PSD group, but higher than those in the control group. In PSD rats, CaMKII expression is upregulated, but Cx43 expression is downregulated, and both CaMKII and Cx43 may participate in PSD. The inhibitor of CaMKII, KN93, can improve the depression in PSD rats.

Assessment of the protective effect of KN-93 drug in systemic epilepsy disorders induced by pilocarpine in male rat.

BACKGROUND AND AIMS: Epileptic seizures occur as a consequence of a sudden imbalance between the stimuli and inhibitors within the network of cortical neurons in favor of the stimulus. One of the drugs that induce epilepsy is pilocarpine. Systemic injection of pilocarpine affects on muscarinic receptors. Increasing evidence has addressed the implication of KN-93 by blocking Ca2+ /calmodulin-dependent protein kinase II (CaMKII), suppressing oxidative stress and inflammation, and also reducing neuron decay. So, we aimed to evaluate the potential preventive effects of KN-93 in systemic epilepsy disorders induced by pilocarpine. MATERIALS AND METHODS: In this animal study, male rats were divided into five groups including treatment group (KN-93 with the dose of 5 mM/10 µL dimethyl sulfoxide (DMSO) before inducing epilepsy by 380 mg/kg pilocarpine) KN-93 group (received 5 mM KN-93), control group, epilepsy group (received 380 mg/kg pilocarpine Intraperitoneal), and sham group (received 10 µL DMSO). Oxidative stress was assessed by measuring its indicators including the concentration of malondialdehyde (MDA), nitrite, glutathione (GSH), as well as the antioxidant activity of catalase. In addition, serum levels of proinflammatory mediators including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were determined. RESULTS: Pretreatment with KN-93 significantly reduced oxidative stress index by reducing the concentration of MDA, nitrite, and increasing the level of GSH. In addition, low concentrations of TNF-α and IL-1ß were observed in hippocampus supernatant of KN-93 pretreated rats in comparison with the pilocarpine groups. Moreover, administration of KN-93 improved neuronal density and attenuated the seizure activity and behavior. CONCLUSIONS: Overall, our findings suggest that KN-93 can effectively suppress oxidative stress and inflammation. Furthermore, KN-93 is able to attenuate seizure behaviors by preventing its effects on neuron loss, so, it is valuable for the treatment of epileptic seizures.

Perampanel Affects Up-Stream Regulatory Signaling Pathways of GluA1 Phosphorylation in Normal and Epileptic Rats.

To elucidate the pharmacological properties of perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile, a novel non-competitive antagonist of AMPA receptor], we investigated its effects on the up-stream regulatory pathways of GluA1 phosphorylation including protein kinase C (PKC), Ca2+-calmodulin-dependent protein kinase II (CAMKII), protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), protein phosphatase (PP) 1, PP2A, and PP2B in normal and pilocarpine-induced epileptic rat model using Western blot analysis. In normal animals, perampanel affected GluA1 expression/phosphorylation, PKC, CAMKII, PKA, ERK1/2, JNK, and PPs activities. In epileptic rats, perampanel effectively inhibited spontaneous seizure activities. Perampanel enhanced phospho (p)-GluA1-S831 and -S845 ratios (phosphoprotein/total protein), while it reduced GluA1 expression. Perampanel also increased pCAMKII and pPKA ratios, which phosphorylate GluA1-S831 and -S845 site, respectively. Perampanel elevated pJNK and pPP2B ratios, which phosphorylates and dephosphorylates both GluA1-S831 and -S845 sits. Perampanel also increased pERK1/2 ratio in epileptic animals, while U0126 (an ERK1/2 inhibitor) did not affect pGluA1 ratios. Perampanel did not influence PKC, PP1, and PP2A expression levels and their phosphorylation ratios. In addition, perampanel did not have a detrimental impact on cognitive abilities of epileptic and normal rats in Morris water maze test. These findings suggest that perampanel may regulate AMPA receptor functionality via not only blockade of AMPA receptor but also the regulations of multiple molecules (CAMKII, PKA, JNK, and pPP2B)-mediated GluA1 phosphorylations without negative effects on cognition, although the effects of perampanel on PKC, PP1, and PP2A activities were different between normal and epileptic rats.

The KN-93 Molecule Inhibits Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity by Binding to Ca2+/CaM.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.

The differential roles of PEA15 phosphorylations in reactive astrogliosis and astroglial apoptosis following status epilepticus.

Up to this day, the roles of PEA15 expression and its phosphorylation in seizure-related events have not been still unclear. In the present study, we found that PEA15 was distinctly phosphorylated in reactive astrocytes and apoptotic astrocytes in the rat hippocampus following LiCl-pilocarpine-induced status epilepticus (SE, a prolonged seizure activity). PEA15-serine (S) 104 phosphorylation was up-regulated in reactive astrocytes following SE, although PEA15 expression and its S116 phosphorylation were unaltered. Bisindolylmaleimide (BIM), a protein kinase C (PKC) inhibitor, attenuated SE-induced reactive astrogliosis, but phorbol 12-myristate 13-acetate (PMA, a PKC activator) aggravated it. Unlike reactive astrocytes, PEA15-S116 phosphorylation was reduced in apoptotic astrocytes. However, PEA15 expression and its S104 phosphorylation were unchanged in apoptotic astrocyte. Neither BIM nor PMA affected SE-induced astroglial apoptosis. PEA15 expression and its phosphorylations were not relevant to SE-induced CA1 neuronal death. These findings indicate that PEA15-S104 and S116 phosphorylations may play a role in reactive astrogliosis and prevention of astroglial apoptosis, respectively. Therefore, we suggest that the selective manipulation of PEA15 phosphorylations may regulate apoptotic and/or proliferative signals in astrocytes.

Effect of KN93 on delayed afterdepolarization and calcium ion in ventricular myocytes of rabbits with heart failure / 中华急诊医学杂志

Objective To observe the effect of KN93, a CaMK Ⅱ inhibitor, on delayed afterdepolarization (DAD) and calcium ion in ventricular myocytes of rabbits with heart failure, and to investigate the effect of CaMK Ⅱ signaling pathway on trigged arrhythmia after heart failure. Methods Thirty male New Zealand White rabbits were randomized(random number) into the sham operated group (sham group), heart failure group (HF group) and heart failure with KN93 group (HF+KN93 group) (n=10 each group). The rabbit heart failure model was established by abdominal aortic constriction combined with aortic valve regurgitation. The ventricular myocytes were isolated by double enzyme digestion. The action potential and the transient inward current (Iti) were recorded by the whole-cell patch-clamp. The intracellular calcium transient was measured by the ion concentration measurement system. The main calcium transporter protein was detected by Western blotting. Data were analyzed by pCLAMP10.2. Statistical analysis was performed using SPSS 17.0. Comparisons among groups were conducted using ANOVA, and SNK-q multiple comparison procedure was utilized for post-hoc analysis.Results (1) After induction of heart failure, DAD and increment of trigger activity (TA) were observedin rabbit ventricular myocytes. Treatment of KN93 with 1.0 μmol/L reduced the events of DAD and TA.(2) After induction of heart failure, Iti densities were increased from -0.12±0.02 pA/pF to -0.95±0.06pA/pF at the polarization potential of -50 mV (n=10, P<0.01). The current densities were reduced to -0.44±0.04 pA/pF after application of 1.0 μmol/L of KN93 (n=10, P<0.01). (3) KN93 led to decrementof intracellular calcium ion concentration and calcium transient amplitude, and acceleration of the decayprocess of calcium transient. (4) KN93 upregulated the expression of pPLN and SERCA2a, increased the uptake of intracellular calcium ion, downregulated the expression of NCX, decreased the Iti, and reduced the occurrence of DAD and TA. Conclusions KN93 can reduce the intracellular calcium ion concentration of the heart failure animal model, and the occurrence of the DAD and TA. CaMK Ⅱ may be a new therapeutic target for arrhythmias in the heart failure.