Calmodulin-dependent Protein Kinase II Expression Research Articles

PFOS Elicits Cytotoxicity in Neuron Through Astrocyte-Derived CaMKII-DLG1 Signaling In Vitro Rat Hippocampal Model.

Both epidemiological investigation and animal experiments demonstrated that pre-/postnatal exposure to perfluorooctane sulfonic acid (PFOS) could induce neurodevelopmental disorders. Previous studies showed that astrocyte was involved in PFOS-induced neurotoxicity, while little information is available. In the present study, the role of astrocyte-derived calmodulin-dependent protein kinase II (CaMKII)-phosphorylated discs large homolog 1 (DLG1) signaling in PFOS eliciting cytotoxicity in neuron was explored with primary cultured hippocampal astrocyte and neuron. The application of PFOS showed a decreased cell viability, synapse length and glutamate transporter 1 (GLT-1) expression, but an increased CaMKII, DLG1 and cyclic AMP response element binding protein (CREB) expression in primary cultured astrocyte. With 2-(2-hydroxyethylamino)-6-aminohexylcarbamic acid tert-butyl ester-9-isopropylpurine (CK59), the CaMKII inhibitor, the disturbed cell viability and molecules induced by PFOS could be alleviated (CREB expression was excluded) in astrocytes. The cytotoxic effect of neuron exposed to astrocyte conditional medium collected from PFOS (PFOS-ACM) pretreated with CK59 was also decreased. These results indicated that PFOS mediated GLT-1 expression through astrocyte-derived CaMKII-DLG signaling, which might be associated with injuries on neurons. The present study gave an insight in further exploration of mechanism in PFOS-induced neurotoxicity.

Docosahexaenoic acid contributes to increased CaMKII protein expression and a tendency to increase nNOS protein expression in differentiated NG108-15 cells.

Docosahexaenoic acid (DHA; 22:6n-3), an n-3 polyunsaturated fatty acid, has various important roles in brain functions. Nitric oxide (NO) produced by neuronal NO synthase (nNOS) and Ca2+/calmodulindependent protein kinase II (CaMKII) is also involved in brain functions. We investigated the influence of DHA on nNOS and CaMKII protein expression in differentiated NG108-15 cells. NG108-15 cells were seeded in 12-well plates, and after 24 h, the medium was replaced with Dulbecco's modified Eagle's medium containing 1% fetal bovine serum, 0.2 mM dibutyryl cyclic adenosine monophosphate and 100 nM dexamethasone as differentiation-inducing medium. When cells were cultured in differentiation-inducing medium, neurite-like outgrowths were observed on days 5 and 6. However, no significant difference in morphology was observed in cells with or without DHA treatment. With or without DHA addition, nNOS protein expression was increased on days 5 and 6 compared with day 0. This increase tended to be enhanced by DHA. CaMKII protein expression did not change after differentiation without DHA, but was significantly increased on day 6 compared with day 0 with DHA addition. These data indicate that DHA is involved in brain functions by regulating CaMKII and nNOS protein expression.

The Effects of Ninjinyoeito on Impaired Spatial Memory and Prefrontal Cortical Synaptic Plasticity through α-Amino-3-hydroxy-5-4-isoxazole Propionic Acid Receptor Subunit in a Rat Model with Cerebral Ischemia and β-Amyloid Injection.

Ninjinyoeito (NYT), a traditional Japanese medicine, is effective for improving physical strength and treating fatigue and anorexia. Recently, a clinical report revealed that NYT ameliorates cognitive dysfunction in Alzheimer's disease (AD) patients, although the mechanisms remain unclear. AD is a neurodegenerative disorder accompanied by a progressive deficit in memory. Current therapeutic agents are largely ineffective in treating cognitive dysfunction in AD patients. In this study, we investigated the effects of NYT on spatial memory impairment in a rat model of dementia. Rats were prepared with transient cerebral ischemia and intraventricular injection of β-amyloid1-42 for 7 days (CI + Aβ). NYT was orally administered for 7 days after cerebral ischemia. We evaluated spatial memory using the Morris water maze and investigated the expression of α-amino-3-hydroxy-5-4-isoxazole propionic acid receptor subunits, the phosphorylation level of glutamate receptor A (GluA)1 at serine sites S831 and S845, and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the hippocampus and prefrontal cortex of CI + Aβ rats. In the CI + Aβ rats, NYT treatment shortened the extended time to reach the platform. However, NYT did not restore the decrease in the hippocampal GluA1, GluA2, or CaMKII expression but increased prefrontal cortical phosphorylation levels of S845-GluA1 and CaMKII. Therefore, NYT may alleviate spatial memory impairment by promoting glutamatergic transmission involved in the phosphorylation of S845-GluA1 and CaMKII in the prefrontal cortex of CI + Aβ rats. Our results suggest that NYT is a valuable treatment for AD patients.

Effects and potential mechanism of Ca2+/calmodulin‑dependent 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+/calmodulin-dependent 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 post-coitus (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 DEAD-box 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 NADP-dependent steroid dehydrogenase-like (Nsdhl), lanosterol synthase (Lss), farnesyl-diphosphate farnesyltransferase 1 (Fdft1), cytochrome P450 family 51 family A member 1 (Cyp51a1), hydroxymethylglutaryl-CoA 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.

Effects of Sacubitril/Valsartan on the Expression of CaMKII/Cav1.2 in Atrial Fibrillation Stimulation Rabbit Model.

Overall, 18 rabbits were randomly divided into control, pacing (600 beats/min), and pacing+sac/val groups. The rabbits in the pacing+sac/val cohort received oral sac/val (10 mg/kg twice daily) across the 21-day investigation period. After three weeks, the atrial effective refractory period (AERP) and AF induction rate were compared. HL-1 cultures were exposed to fast pacing (24 h) with and without LBQ657 (active sacubitril form)/valsartan. Western blots were used for detecting Cav1.2 and CaMKII expression within atrial muscles of the rabbits and HL-1 cultures of AF model. In comparison to the sham cohort, the AF induction rate was markedly increased together with AERP reduction within pacing cohort. Such changes were markedly rescued through sac/val treatment in pacing+sac/val cohort. The proteomic expression profiles of CaMKII and Cav1.2 showed that the CaMKII expression was markedly upregulated, while Cav1.2 expression was downregulated in the pacing cohort. Importantly, these effects were absent in pacing+sac/val cohort. Results of this study show that sac/val treatment regulates the expression of CaMKII/Cav1.2 and could alter this pathway in atrial rapid electrical stimulation models. Therefore, this investigation could contribute to a novel strategy in AF therapeutics in clinical settings.

Xiaoyu Jiangzhi capsule protects against heart failure via Ca2+/CaMKII signaling pathways in mice

Heart failure (HF), a worldwide health condition, is the result of many cardiovascular diseases. The traditional Chinese medicine (TCM) Xiaoyu Jiangzhi capsule (XYC) has long been in use in China to treat hyperlipidemia and inhibit platelet aggregation. This study explores the effects of XYC on heart failure (HF) and its detailed mechanisms. Isoproterenol (ISO, 30 mg/kg) was injected intraperitoneally for 7 days to copy a HF model of 10–12 weeks old, 20–30 g male mice. We then compared the CON (control) group, ISO (HF model) group, MET (metoprolol) group, and XYC group. Cardiac systolic function and left wall thickness were evaluated by echocardiograph. Using western blot analysis, we detected the proteins of calmodulin dependent protein kinase II (CaMKII) and sarco/endoplasmic reticulum Ca 2+ -ATPase (Serca). Furthermore, tsA201 cells were cultured and the human CaV1.2 calcium channel current (hCaV1.2) were detected by patch clamp experiments. XYC reduced HF, inhibiting the protein expression of CaMKII, but Serca did not change significantly. Moreover, XYC inhibited the peak amplitude of the hCaV1.2 current, depolarizing shifted the activation curve 27.6 mV, and shifted the inactivation curve toward a positive potential 17.6 mV. The fraction recovered from inaction was reduced in XYC group compared with that in CON group. XYC could inhibit ISO-induced HF by reducing the Ca 2+ /CaMKII signaling pathway in mice.

SIRT1‐mediated Lysine Crotonylation is Involved in the Regulation of CaMKII Activity in Myocardium

ObjectivesPost‐translational modifications (PTMs) can modify proteins and regulate their functions. Lysine crotonylation is a novel protein PTM that can be regulated by silent mating type information regulation 2 homolog 1 (SIRT1). Ca2+/calmodulin‐dependent protein kinase II (CaMKII) plays a significant role in mediating cellular responses to external signaling molecules. Little is known regarding the effect of SIRT1 on CaMKII crotonylation and the impact of crotonylation on the activity of this enzyme.MethodsCre‐Loxp technology was used to generate conditional cardiac‐specific SIRT1 knockout mice (SIRT1 KO). Protein crotonylation modification was studied by PTMomics. CaMKII activity was determined by ELISA. Western blot and co‐immunoprecipitation (CO‐IP) were performed to examine the expression and crotonylation of CaMKIIResultsPTMomics showed changes in protein crotonylation profiles in the myocardium of cardiac‐specific SIRT1 knockout mice. Crotonylation of CaMKII was significantly increased at lysine 301 and the enhanced CaMKII crotonylation was further confirmed by CO‐IP (2.37±0.25 in SIRT1 KO vs. 0.96±0.10 in wild‐type and 1.04±0.10 in SIRT1fl/fl mice, p<0.01). Cardiac‐specific knockout of SIRT1 did not affect the protein expression of CaMKII whereas significantly increased CaMKII activity (2.75±0.17 m units/μL in SIRT1 KO vs. 2.10±0.13 m units/μL in wild‐type and 1.98±0.17 m units/μL in SIRT1fl/fl mice, p<0.05). CO‐IP showed no physical interaction between SIRT1 and CaMKII.ConclusionsSIRT1 deficiency increases CaMKII activity in myocardium at least partially through enhancing lysine crotonylation of CaMKII, which advances our understanding of the role played by PTM in the regulation of CaMKII.

Retinal ganglion cells expressing CaM kinase II in human and nonhuman primates.

Immunoreactivity for calcium-/calmodulin-dependent protein kinase II (CaMKII) in the primate dorsal lateral geniculate nucleus (dLGN) has been attributed to geniculocortical relay neurons and has also been suggested to arise from terminals of retinal ganglion cells. Here, we combined immunostaining with single-cell injections to investigate the expression of CaMKII in retinal ganglion cells of three primate species: macaque (Macaca fascicularis, M. nemestrina), human, and marmoset (Callithrix jacchus). We found that in all species, about 2%-10% of the total ganglion cell population expressed CaMKII. In all species, CaMKII was expressed by multiple types of wide-field ganglion cell including large sparse, giant sparse (melanopsin-expressing), broad thorny, and narrow thorny cells. Three other ganglion cells types, namely, inner and outer stratifying maze cells in macaque and tufted cells in marmoset were also found. Double labeling experiments showed that CaMKII-expressing cells included inner and outer stratifying melanopsin cells. Nearly all CaMKII-expressing ganglion cell types identified here are known to project to the koniocellular layers of the dLGN as well as to the superior colliculus. The best characterized koniocellular projecting cell type-the small bistratified (blue ON/yellow OFF) cell-was, however, not CaMKII-positive in any species. Our results indicate that the pattern of CaMKII expression in retinal ganglion cells is largely conserved across different species of primate suggesting a common functional role. But the results also show that CaMKII is not a marker for all koniocellular projecting retinal ganglion cells.

IMPACTS OF STATIC ELECTRIC FIELD PRODUCED BY ULTRA-HIGH-VOLTAGE DIRECT-CURRENT TRANSMISSION LINES ON HIPPOCAMPAL PROTEIN EXPRESSION AND MORPHOLOGICAL STRUCTURE IN MICE

Static electric field (SEF) from ultra-high-voltage direct-current (UHVDC) transmission lines has the potential to produce neurobiological effects. To explore these effects and elucidate their potential mechanisms, protein expression levels and morphological structure in the hippocampi of mice were investigated after SEF exposure. Mice from the Institute of Cancer Research were exposed to an environmental SEF induced by UHVDC transmission lines with the strength of 9.20–21.85[Formula: see text]kV/m for 35 days. Mouse body weight was measured weekly during the exposure. After the exposure, hippocampal Ca[Formula: see text]/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) expression levels were assayed by Western blot. Hippocampal pathologic morphology and ultrastructure were observed using light microscopy and transmission electron microscopy, respectively. No significant differences in body weight, CaMKII and CaN expression levels, and hippocampal pathologic morphology were observed between mice in the exposed and the control groups. However, cytoplasmic vacuolization of the hippocampal neurons was observed in the exposed group. Thus, hippocampal neuron ultrastructure damage may be a mechanism of SEF-exposure-induced memory decline in mice.

Characterization of six CaMKIIα variants found in patients with schizophrenia

SummaryThe Ca2+/Calmodulin-dependent protein kinase II (CaMKII) is a central regulator of synaptic plasticity and has been implicated in various neurological conditions, including schizophrenia. Here, we characterize six different CaMKIIα variants found in patients with schizophrenia. Only R396stop disrupted the 12-meric holoenzyme structure, GluN2B binding, and synaptic localization. Additionally, R396stop impaired T286 autophosphorylation that generates Ca2+-independent “autonomous” kinase activity. This impairment in T286 autophosphorylation was shared by the R8H mutation, the only mutation that additionally reduced stimulated kinase activity. None of the mutations affected the levels of CaMKII expression in HEK293 cells. Thus, impaired CaMKII function was detected only for R396stop and R8H. However, two of the other mutations have been later identified also in the general population, and not all mutations found in patients with schizophrenia would be expected to cause disease. Nonetheless, for the R396stop mutation, the severity of the biochemical effects found here would predict a neurological phenotype.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Effect of acupoint thread-embedding on tight junction of intestinal mucosa epithelium in rats with ulcerative colitis

To observe the effect of acupoint thread-embedding on tight junction of intestinal mucosal epithelial barrier in rats with ulcerative colitis (UC) under the state of "deficiency and stasis", and to explore its mechanism. Sixty male SD rats were randomly divided into a control group (n=12) and a UC model group (n=48). The rats were gavaged with adenine and folium sennae by stages to prepare the "deficiency and stasis" state, and then 5% 2,4,6-trinitrobenzene sulfonic acid (TNBS, 100 mg/kg) and 0.25 mL 50% ethanol were used to induce UC model. Thirty rats with successful model establishment were selected and randomly divided into a model group, a medication group and a thread-embedding group, 10 rats in each group. The rats in the thread-embedding group were intervened with thread-embedding at "Zusanli" (ST 36), "Tianshu" (ST 25), "Geshu" (BL 17), "Pishu" (BL 20), "Shenshu" (BL 23) and "Dachangshu" (BL 25), once every 14 days, totaling 3 times. The rats in the medication group were gavaged with sulfasalazine, once a day. The rats in the control group, model group and thread-embedding group were gavaged with the same amount of 0.9% sodium chloride solution at the same time for 42 days. The general condition and body weight of rats in each group were observed. The appearance and morphological changes of colonic tissue were observed by naked eye and HE staining. The serum levels of calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and myosin light chain kinase (MLCK) were detected by ELISA. The mRNA levels of colonic tight junction (TJ) proteins including occludin, claudin1, claudin2 and ZO-1were detected by quantitative real-time PCR. The protein expressions of occludin, claudin1, claudin2, ZO-1 and CaMKⅡ, MLCK were detected by Western blot. Compared with the control group, in the model group the body weight was decreased (P<0.01), colon tissue was significantly swelled with bleeding and a large number of inflammatory cells infiltration, and the colon tissues damage index score was increased (P<0.01), serum CaMKⅡ and MLCK levels were increased (P<0.01), and the mRNA and protein expression levels of occludin, claudin1 and ZO-1 in colon tissue were decreased (P<0.01), the mRNA and protein expression levels of claudin2 were increased (P<0.01), and the protein expression levels of CaMKⅡ and MLCK were increased (P<0.01). Compared with the model group, in the medication group and thread-embedding group the weight was increased (P<0.01), the colonic lesions were significantly alleviated, the tissues damage index scores were decreased (P<0.01), the serum CaMKⅡ and MLCK contents were decreased (P<0.01), the mRNA and protein expression levels of occludin, claudin1 and ZO-1 in colonic tissue were increased (P<0.01, P<0.05), the mRNA and protein expression levels of claudin2 were decreased (P<0.01), and the protein expression levels of CaMKⅡ and MLCK were decreased (P<0.01, P<0.05). There was no significant difference of each index between the medication group and thread-embedding group (P>0.05). The thread-embedding could repair the tight junction of intestinal mucosa epithelium and reduce the permeability of intestinal mucosa epithelium, which may be related to the decrease of the expression of CaMKⅡ, MLCK and other protein kinases.

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) with Lidocaine Provokes Pyroptosis of Glioblastoma Cells.

The study examines the problem whether pyroptosis of U87-MG glioblastoma cells can result from activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by a local anesthetic. Glioblastoma cells exposed to various concentrations of typical local anesthetic lidocaine demonstrated augmented cytosolic flux of Ca2+, while suppression of CaMKII expression with the corresponding siRNA significantly inhibited this effect in cells treated with 2 mM lidocaine. Lidocaine up-regulated the expression of mRNA caspase-3 and gasdermin GSDME proteins, whereas silencing of CaMKII gene with siRNA significantly moderated this effect. In addition, lidocaine inhibited proliferation of U87-MG cells, and this effect was prevented by silencing CaMKII gene. Thus, lidocaine activated protein kinase CaMKII, which phosphorylated TRPV1 ion channels and induced calcium overload of U87-MG glioblastoma cells, thereby provoking their pyroptosis.

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

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

The pathophysiological mechanism of ischemic stroke after hypobaric hypoxia simulation at high altitude.

Tibet is an area in China with a high incidence of stroke, typically attributed to hypobaric hypoxia. The present study aimed to observe the neuronal injury of ischemic stroke after hypobaric hypoxia and explore the mechanism by which N-methyl-D-aspartate receptor (NMDAR) and its downstream pathways are involved. This study employed a hypobaric chamber to imitate high altitude at 4000m. After hypoxia, the middle cerebral artery occlusion (MCAO) model was used to mimic ischemic stroke. Behavioral tests and measurements of infarct area were used to observe neuronal injuries. The expression of NMDAR, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphorylated CaMKII (Threonine 286) (P-CaMKII) was tested by western blot, and hematological tests were used to count the number of red blood cells (RBCs) and hemoglobin. Compared with the plain+MCAO group, the neurological deficit scores and infarct area of rats in the 4000m + MCAO group were all decreased, and the protein expression of NMDAR, CaMKII and P-CaMKII was reduced. Compared with the plain group, the numbers of RBCs, hemoglobin and hematocrit were increased in the 4000m group; compared with the 4000m groups, the three indexes were increased in the 4000m + MCAO groups. The neuronal injuries after hypoxia were not more serious than those in rats enduring ischemia and reperfusion in plain. The underlying mechanisms were related to the decreased expression of NMDAR and CaMKII; furthermore, the increased numbers of RBCs and hemoglobin may be crucial mechanisms for the incidence and development of ischemic stroke at high altitude.

SCN10A-knock-out improves survival and proarrhythmia in a transgenic heart failure mouse model

Abstract Background In heart failure (HF) both Ca2+/Calmodulin-dependent protein-kinase II (CaMKII) and late sodium current (INaL) are known to contribute to arrhythmogenesis as they contribute to action-potential (AP) prolongation and the occurrence of early- (EADs) and delayed afterdepolarizations (DADs). Further, augmented CaMKII and INaL maintain a vicious cycle as they both can activate each other. We recently found that the sodium channel isoform NaV1.8 is upregulated in HF and hypertrophy and that it is involved in INaL-generation. In the current study we investigated the effects of NaV1.8-knock-out (KO) on HF-progression and arrhythmogenesis in a CaMKII-overexpressing HF mouse model. Methods/Results CaMKII overexpressing mice (CaMKII+/T) were crossbred with NaV1.8-KO mice (SCN10A−/−). To our surprise knock-out of NaV1.8 in CaMKII+/T mice (SCN10A−/−/CaMKII+/T) significantly improved survival (median survival 103 days vs 74.5 CaMKII+/T, p&amp;lt;0.01). CaMKII+/T mice exhibited a strong HF phenotype compared to WT with increased heart-weight to tibia length ratio as well as reduced ejection fraction and left-ventricular end-diastolic diameter obtained by echocardiography. However, these structural parameters did not differ between SCN10A−/−/CaMKII+/T and CaMKII+/T. Therefore, cellular electrophysiology experiments were performed in isolated cardiomyocytes for a better understanding of the observed improvement in survival. INaL, measured by patch-clamp technique, was significantly augmented in CaMKII+/T vs WT and SCN10A−/−, while SCN10A−/−/CaMKII+/T showed significantly less INaL than CaMKII+/T alone. Further, AP-duration (APD) was significantly reduced in SCN10A−/−/CaMKII+/T vs CaMKII+/T while AP-amplitude, resting membrane-potential and upstroke velocity (dv/dtmax) remained unchanged. In addition, the occurrence of afterdepolarizations was significantly lower in SCN10A−/−/ CaMKII+/T vs CaMKII+/T. Confocal microscopy using the dye Fluo-4AM was performed and significantly less diastolic Ca2+-waves occurred in SCN10A−/−/CaMKII+/T compared to CaMKII+/T. In order to analyze an organ-specific SCN10A-KO, we generated homozygous SCN10A-KO lines of induced pluripotent stem cells by using CRISPR/Cas9 technology. 2-month old iPSC-cardiomyocytes lacking NaV1.8 were treated with low dose isoprenaline and showed significantly less INaL, thereby serving as a final proof of the relevant role of this Na+-channel on INaL-generation in the cardiomyocyte. Conclusion We found a survival benefit by selective knock-out of the neuronal sodium channel isoform NaV1.8 in a proarrhythmic HF mouse model with augmented CaMKII expression. However, in our model NaV1.8-knock-out showed no effects on HF progression, while cellular proarrhythmic triggers were attenuated. Taken together with our findings in IPS-cardiomyocytes treated with the CRSIPR/Cas9 technology NaV1.8 plays a significant role for the generation of INaL and cellular arrhythmogenic triggers in the cardiomyocyte. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Stiftung für Herzforschung

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

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

Cardioprotective effect of pioglitazone and curcumin against diabetic cardiomyopathy in type 1 diabetes mellitus: impact on CaMKII/NF-κB/TGF-β1 and PPAR-γ signaling pathway.

Diabetic cardiomyopathy (DCM) is a leading cause of death in diabetic patients, which is currently without available specific treatment. This study aimed to investigate the potential protective effects of pioglitazone (Pio) and curcumin (Cur) against DCM in type 1 diabetes mellitus (T1DM), with pointing to their role on Ca+2/calmodulin-dependent protein kinase II (CaMKII) and peroxisome proliferator-activated receptor gamma (PPAR-γ) expression. Diabetes was induced in adult male Sprague Dawley rats by administration of single intraperitoneal injection of streptozotocin (STZ) (52.5mg/kg). Diabetic rats were administered either Pio (20mg/kg/day) or Cur (100mg/kg/day) orally for 6weeks. Treatment with Pio and/or Cur markedly reduced serum cardiac injury markers and lipid profile markers in diabetic animals. Additionally, Pio and/or Cur treatment mitigated oxidative stress and fibrosis in diabetic rats as evident from the significant suppression in myocardial lipid peroxidation and tumor growth factor beta 1 (TGF-β1) level, with concomitant significant elevation in total antioxidant capacity (TAC) and improvement in histopathological architecture of heart tissue. Pio/Cur treatment protocol accomplished its cardioprotective effect by depressing cardiac CaMKII/NF-κB signaling accompanied by enhancement in PPAR-γ expression. Conclusively, these findings demonstrated the therapeutic potential of Pio/Cur regimen in alleviating DCM in T1DM through modulation of CaMKII and PPAR-γ expression. Graphical Abstract.

Activation of CaMKII and GluR1 by the PSD-95-GluN2B Coupling-Dependent Phosphorylation of GluN2B in the Spinal Cord in a Rat Model of Type-2 Diabetic Neuropathic Pain

The mechanisms underlying type-2 diabetic neuropathic pain (DNP) are unclear. This study investigates the coupling of postsynaptic density-95 (PSD-95) to N-methyl-D-aspartate receptor subunit 2B (GluN2B), and the subsequent phosphorylation of GluN2B (Tyr1472-GluN2B) in the spinal cord in a rat model of type-2 DNP. Expression levels of PSD-95, Tyr1472-GluN2B, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its phosphorylated counterpart (Thr286-CaMKII), and α-amino-3-hydroxy-5-methyl-4-soxazole propionic acid receptor subtype 1 (GluR1) and its phosphorylated counterpart (Ser831-GluR1) were significantly increased versus controls in the spinal cord of type-2 DNP rats whereas the expression of total spinal GluN2B did not change. The intrathecal injection of Ro25-6981 (a specific antagonist of GluN2B) or Tat-NR2B9c (a mimetic peptide disrupting the interaction between PSD-95 and GluN2B) induced an antihyperalgesic effect and blocked the increased expression of Tyr1472-GluN2B, CaMKII, GluR1, Thr286-CaMKII, and Ser831-GluR1 in the spinal cords; the increase in spinal cord PSD-95 was not affected. These findings indicate that the PSD-95-GluN2B interaction may increase phosphorylation of GluN2B, and subsequently induce the expression of phosphorylation of CaMKII and GluR1 in the spinal cord of type-2 DNP rats. Targeting the interaction of PSD-95 with GluN2B may provide a new therapeutic strategy for type-2 DNP.

Distinct roles of calmodulin and Ca2+/calmodulin-dependent protein kinase II in isopreterenol-induced cardiac hypertrophy

Intracellular calcium is related to cardiac hypertrophy. The CaV1.2 channel and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaM regulate the intracellular calcium content. However, the differences in CaMKII and CaM in cardiac hypertrophy are still conflicting and are worthy of studying as drug targets. Therefore, in this study, we aim to investigate the roles and mechanism of CaM and CaMKII on CaV1.2 in pathological myocardial hypertrophy. The results showed that ISO stimulation caused SD rat heart and cardiomyocyte hypertrophy. In vivo, the HW/BW, LVW/BW, cross-sectional area, fibrosis ratio and ANP expression were all increased. There were no differences in CaV1.2 channel expression in the in vivo model or the in vitro model, but the ISO stimulation induced channel activity, and the [Ca2+]i increased. The protein expression levels of CaMKII and p-CaMKII were all increased in the ISO group, but the CaM expression level decreased. AIP inhibited ANP, CaMKII and p-CaMKII expression, and ISO-induced [Ca2+]i increased. AIP also reduced HDAC4, p-HDAC and MEF2C expression. However, CMZ did not play a cardiac hypertrophy reversal role in vitro. In conclusion, we considered that compared with CaM, CaMKII may be a much more important drug target in cardiac hypertrophy reversal.