Calmodulin-dependent Protein Kinase II Inhibitor Research Articles

Ca2+/Calmodulin-dependent Protein Kinase II Inhibitor KN-93 Enhances Chondrogenesis of Bone Marrow Mesenchymal Stem Cells and Delays Chondrogenic Hypertrophy.

Cartilage tissue engineering has been popularly applied in the treatment of articular cartilage defect because it is more effective in generating functional engineered cartilage than traditional methods. Although the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) is well established, it is often accompanied by undesired hypertrophy. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator in the ion channel pathway which is known to be involved in chondrogenic hypertrophy. Therefore, this study aimed to reduce the hypertrophy of BM-MSCs by inhibiting CaMKII activation. BM-MSCs were cultured in three-dimensional (3D) scaffold under chondrogenic induction with and without CaMKII inhibitor, KN-93. After cultivation, markers of chondrogenesis and hypertrophy were investigated. KN-93 at a concentration of 2.0 μM had no effect on the viability of BM-MSCs, while the activation of CaMKII was suppressed. A long period of KN-93 treatment significantly up-regulated the expression of SRY-box transcription factor 9 and aggrecan on day 28 compared to untreated BM-MSCs. Furthermore, KN-93 treatment significantly down-regulated the expression of RUNX family transcription factor 2 and collagen type X alpha 1 chain on days 21 and 28. Immunohistochemistry showed increased expression of aggrecan and type II collagen while the expression of type X collagen was reduced. A CaMKII inhibitor, KN-93 is able to enhance chondrogenesis of BM-MSCs and suppress chondrogenic hypertrophy, suggesting its potential applicability in cartilage tissue engineering.

Pathological implication of CaMKII in NF-κB pathway and SASP during cardiomyocytes senescence

Senescence-associated secretory phenotype (SASP) could be developed during heart ageing. But the role of SASP in cardiomyocytes senescence and its molecular mechanism remains undetermined. In this study, we observed elevated Ca2+/calmodulin -dependent protein kinase II (CaMKII) activation in both physiological aged heart and premature senescent cardiomyocytes. Notably, we confirmed the gradual SASP development induced by NF-κB activation in long-term cultured cardiomyocytes. Transgenic inhibition of CaMKII in mice (AC3-I mice) alleviated the NF-κB activation, chronic sterile inflammation and ageing-associated cardiomyopathy. Correspondingly, pharmacological inhibition of CaMKII with KN93 mitigated SASP and hindered cardiomyocytes senescence. Meanwhile, increased NF-κB activation and exacerbated cardiomyocytes senescence were observed with transgenic CaMKII activation. Collectively, our results indicated that the increased CaMKII activation accompanying ageing could aggravate NF-κB activation and SASP development and facilitate cardiomyocytes senescence and heart ageing.

Novel CaMKII biosensor enables identification of potent CaMKII inhibitors

The multifunctional Ca2+/Calmodulin-dependent protein kinase II (CaMKII) is a highly validated driver of cardiac illnesses including heart failure, ischemia/reperfusion injury, and arrhythmias. Excessive CaMKII activity causes intracellular Ca2+ dysregulation, inflammation, maladaptive reprogramming, and cell death. Multiple animal studies show that CaMKII inhibition is cardioprotective in various cardiac injury models. Despite this, there are currently no approved CaMKII inhibiting therapies. Thus, development of safe and effective CaMKII inhibitors is a translational priority.

CaMKIIα as a Promising Drug Target for Ischemic Grey Matter.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of Ca2+-dependent signaling pathways in various cell types throughout the body. Its neuronal isoform CaMKIIα (alpha) centrally integrates physiological but also pathological glutamate signals directly downstream of glutamate receptors and has thus emerged as a target for ischemic stroke. Previous studies provided evidence for the involvement of CaMKII activity in ischemic cell death by showing that CaMKII inhibition affords substantial neuroprotection. However, broad inhibition of this central kinase is challenging because various essential physiological processes like synaptic plasticity rely on intact CaMKII regulation. Thus, specific strategies for targeting CaMKII after ischemia are warranted which would ideally only interfere with pathological activity of CaMKII. This review highlights recent advances in the understanding of how ischemia affects CaMKII and how pathospecific pharmacological targeting of CaMKII signaling could be achieved. Specifically, we discuss direct targeting of CaMKII kinase activity with peptide inhibitors versus indirect targeting of the association (hub) domain of CaMKIIα with analogues of γ-hydroxybutyrate (GHB) as a potential way to achieve more specific pharmacological modulation of CaMKII activity after ischemia.

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

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

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.

An in vivo and in vitro model on the protective effect of cilnidipine on contrast-induced nephropathy via regulation of apoptosis and CaMKⅡ/mPTP pathway.

Contrast-induced nephropathy (CIN) is an acute kidney injury (AKI) observed after the administration of contrast media. Calcium channel blockers (CCBs) have been reported to exert a renal protective effect. This study aims to investigate the role of cilnidipine, a novel CCBs, on CIN by regulating the calcium/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)/mitochondrial permeability transition pore(mPTP) pathway. Here, iohexol, a representative contrast media, was used to establish CIN model. KN-93 (CaMKⅡ inhibitor) and atractyloside (mPTP opener) were administered in rats, and CaMKⅡ overexpression was used in Human proximal tubular epithelial cells. Markers of renal injury (serum creatinine, blood urea nitrogen, and urinary NAGL), hematoxylin-eosin stain, oxidative stress (ROS, superoxide dismutase[SOD], and malondialdehyde [MDA] levels), cell death (MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling[TUNEL]), mitochondrial function (mPTP, mitochondrial membrane potential[MMP], and ATP) were assessed. Western blots were used to measure the expression levels of Bax/Bcl-2, caspase-3, CaMKⅡ/mPTP signaling pathways. Results showed that cilnidipine markedly improved kidney function, and alleviated tubular cell apoptosis, oxidative stress and mitochondrial damage induced by iohexol in vitro and in vivo. The underlying mechanism may be that cilnidipine relieved CaMKⅡ activation and mPTP opening induced by iohexol. All of these protective effects of cilnidipine were attenuated by CaMKⅡ overexpression and atractyloside (mPTP opener) pretreatment. Moreover, KN-93 (CaMKⅡ inhibitor) treatment showed a similar renal protective effect with cilnidipine, while the protective effect of cilnidipine on kidney in CIN rats was not further suppressed by KN-93 cotreatment. These in vitro and in vivo results point towardthe fact that cilnidipine might be a novel therapeutic drug against contrast-induced nephrotoxicity in a CaMKⅡ-dependent manner.

CaMKII orchestrates endoplasmic reticulum stress and apoptosis in doxorubicin‐induced cardiotoxicity by regulating the IRE1α/XBP1s pathway

Doxorubicin (Dox), an anthracycline antibiotic with potent antitumor effects, has limited clinical applications due to cumulative cardiotoxicity. Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is implicated in the pathological progression of Dox‐induced cardiotoxicity. This study examined the hypothesis that CaMKII exacerbates Dox‐induced cardiotoxicity by promoting endoplasmic reticulum stress and apoptosis through regulation of the inositol‐requiring enzyme 1α (IRE1α)/spliced X‐box binding protein 1 (XBP1s) pathway. Our results demonstrated that CaMKII activation and IRE1α/XBP1s pathway were involved in Dox‐treated hearts. CaMKII inhibition with KN‐93 ameliorated Dox‐induced cardiac dysfunction and pathological myocardial changes. In addition, CaMKII inhibition prevented Dox‐induced endoplasmic reticulum stress and apoptosis. Moreover, CaMKII inhibition increased the expression of IRE1α and XBP1s in Dox‐treated hearts. The IRE1α inhibitor 4μ8C blocked the protective effect of CaMKII inhibition against Dox‐induced cardiotoxicity. Mechanistically, 4μ8C prevented the effects of CaMKII inhibition on Dox‐induced endoplasmic reticulum stress and apoptosis by inhibiting the expression of IRE1α and XBP1s. Additionally, treatment with rhADAMTS13 decreased the protein level of thrombospondin 1 (TSP1) and the phosphorylation of CaMKII in Dox‐treated human AC16 cardiomyocytes. Taken together, these results demonstrate that the ADAMTS13‐TSP1 axis regulates CaMKII activation and exacerbates Dox‐induced cardiotoxicity by triggering endoplasmic reticulum stress and apoptosis by inhibiting the IRE1α/XBP1s pathway.

TRPM2 mediates CaMKⅡ-Beclin-1 signaling in early cortical injury after induced subarachnoid hemorrhage in mice

BackgroundThough early brain injury (EBI) is the primary cause of poor outcomes among patients with subarachnoid hemorrhage (SAH), its exact molecular mechanisms remain unclear. Improved the understanding of how transient receptor potential melastatin-related 2 (TRPM2) is involved in SAH-induced EBI will help develop novel interventions. MethodsWild type (WT) male C57BL/6J mice were subjected to SAH for 12 h, 24 h or 48 h, after which neurological scores and pathological changes in the hippocampus (CA3, DG, and CA1) and temporal base cortex were observed. Expressions of TRPM2, Ca2+/calmodulin (CaM)-dependent protein kinase Ⅱ (CaMKⅡ), and Beclin-1 in hippocampus (CA3, DG, and CA1) and temporal base cortex were compared across post-SAH timepoints. TRPM2-deficient (TRPM2−/−) male C57BL/6 J mice and a CaMKⅡ inhibitor (KN-93) were used to analyze the effects oTRPM2 on the CaMKⅡ-Beclin-1 signaling post SAH. ResultsNeurological and temporal base cortex deterioration were more severe with increased time post-SAH induction, whereas hippocampal damage was not observed. Post-SAH, TRPM2-CaMKⅡ-Beclin-1 cascade was activated in the temporal base cortex, but not the hippocampus. Using TRPM2−/− mice and KN-93 administration, SAH-induced EBI was improved, and CaMKⅡ and Beclin-1 expressions in the temporal base cortex were significantly decreased compared with WT mice. TRPM2−/− mice also showed better neurological improvement compared with KN-93 treated mice. ConclusionTRPM2 mediates CaMKⅡ-Beclin-1 signaling that aggravates SAH-induced EBI in the temporal base cortex. TRPM2 may be an alternative therapy target in EBI after SAH. Data availabilityThe datasets generated and/or analysed during the current study are available from the corresponding author

CaMKII as a Therapeutic Target in Cardiovascular Disease.

CaMKII (the multifunctional Ca2+ and calmodulin-dependent protein kinase II) is a highly validated signal for promoting a variety of common diseases, particularly in the cardiovascular system. Despite substantial amounts of convincing preclinical data, CaMKII inhibitors have yet to emerge in clinical practice. Therapeutic inhibition is challenged by the diversity of CaMKII isoforms and splice variants and by physiological CaMKII activity that contributes to learning and memory. Thus, uncoupling the harmful and beneficial aspects of CaMKII will be paramount to developing effective therapies. In the last decade, several targeting strategies have emerged, including small molecules, peptides, and nucleotides, which hold promise in discriminating pathological from physiological CaMKII activity. Here we review the cellular and molecular biology of CaMKII, discuss its role in physiological and pathological signaling, and consider new findings and approaches for developing CaMKII therapeutics.

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.

CaMKII Inhibition Attenuates Distinct Gain-of-Function Effects Produced by Mutant Nav1.6 Channels and Reduces Neuronal Excitability.

Aberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the SCN8A gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.

Kalirin mediates Rac1 activation downstream of calcium/calmodulin-dependent protein kinase II to stimulate glucose uptake during muscle contraction.

In this study, we investigated the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in contraction-stimulated glucose uptake in skeletal muscle. C2C12 myotubes were contracted by electrical pulse stimulation (EPS), and treadmill running was used to exercise mice. The activities of CaMKII, the small G protein Rac1, and the Rac1 effector kinase PAK1 were elevated in muscle by running exercise or EPS, while they were lowered by the CaMKII inhibitor KN-93 and/or small interfering RNA (siRNA)-mediated knockdown. EPS induced the mRNA and protein expression of the Rac1-GEF Kalirin in a CaMKII-dependent manner. EPS-induced Rac1 activation was lowered by the Kalirin inhibitor ITX3 or siRNA-mediated Kalirin knockdown. KN-93, ITX3, and siRNA-mediated Kalirin knockdown reduced EPS-induced glucose uptake. These findings define a CaMKII-Kalirin-Rac1 signaling pathway that contributes to contraction-stimulated glucose uptake in skeletal muscle myotubes and tissue.

Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is expressed in hearts and implicated in cardiac remodeling and dysfunction. However, the effects of TRPV4 on pressure overload-induced cardiac hypertrophy remain unclear. In this study, we found that TRPV4 expression was significantly increased in mouse hypertrophic hearts, human failing hearts, and neurohormone-induced hypertrophic cardiomyocytes. Deletion of TRPV4 attenuated transverse aortic constriction (TAC)-induced cardiac hypertrophy, cardiac dysfunction, fibrosis, inflammation, and the activation of NFκB - NOD - like receptor pyrin domain-containing protein 3 (NLRP3) in mice. Furthermore, the TRPV4 antagonist GSK2193874 (GSK3874) inhibited cardiac remodeling and dysfunction induced by TAC. In vitro, pretreatment with GSK3874 reduced the neurohormone-induced cardiomyocyte hypertrophy and intracellular Ca2+ concentration elevation. The specific TRPV4 agonist GSK1016790A (GSK790A) triggered Ca2+ influx and evoked the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). But these effects were abolished by removing extracellular Ca2+ or GSK3874. More importantly, TAC or neurohormone stimulation-induced CaMKII phosphorylation was significantly blocked by TRPV4 inhibition. Finally, we show that CaMKII inhibition significantly prevented the phosphorylation of NFκB induced by GSK790A. Our results suggest that TRPV4 activation contributes to pressure overload-induced cardiac hypertrophy and dysfunction. This effect is associated with upregulated Ca2+/CaMKII mediated activation of NFκB-NLRP3. Thus, TRPV4 may represent a potential therapeutic drug target for cardiac hypertrophy and dysfunction after pressure overload.

Activation of the N-methyl-D-aspartate receptor contributes to orofacial neuropathic and inflammatory allodynia by facilitating calcium-calmodulin-dependent protein kinase II phosphorylation in mice

Neuropathic and inflammatory pain are major clinical challenges due to their ambiguous mechanisms and limited treatment approaches. N-methyl-D-aspartate receptor (NMDAR) and calcium-calmodulin-dependent protein kinase II (CaMKII) are responsible for nerve system sensation and are required for the induction and maintenance of pain. However, the roles of NMDAR and CaMKII in regulating orofacial pain are still less well known. Here, we established a neuropathic pain model by transecting a mouse inferior alveolar nerve (IAN) and an inflammatory pain model by injecting complete Freund’s adjuvant (CFA) into its whisker pad. The Cre/loxp site-specific recombination system was used to conditionally knock out (KO) NR2B in the trigeminal ganglion (TG). Von Frey filament behavioral tests showed that IANX and CFA-induced mechanical allodynia were altered in NR2B-deficient mice. CFA upregulated CaMKIIα and CaMKIIβ in the mouse TG and spinal trigeminal caudate nucleus (SpVc). CaMKIIα first decreased and then increased in the TG after IANX, and CaMKIIβ decreased in the TG and SpVc. CFA and IANX both greatly enhanced the expression of phospho (p)-NR2B, p-CaMKII, cyclic adenosine monophosphate (cAMP), p-ERK, and p-cAMP response element binding protein (CREB) in the TG and SpVc. These neurochemical signal pathway alterations were reversed by the conditional KO of NR2B and inhibition of CaMKII. Similarly, IANX- and CFA-related behavioral alterations were reversed by intra-ganglionic (i.g.) -application of inhibitors of CaMKII, cAMP, and ERK. These findings revealed novel molecular signaling pathways (NR2B-CaMKII-cAMP-ERK-CREB) in the TG- and SpVc-derived latent subsequent peripheral and spinal central sensitization under nerve injury and inflammation, which might be beneficial for the treatment of orofacial allodynia.

Discovery of a New CaMKII-Targeted Synthetic Lethal Therapy against Glioblastoma Stem-like Cells

Simple SummaryThe poor prognoses of patients with glioblastoma multiforme (GBM) are attributed to glioblastoma stem-like cells (GSCs), which induce drug resistance and rapid tumor recurrence. Accordingly, the discovery of promising therapies targeting GSCs is important for the treatment of GBM. The purpose of this study is to explore a novel synthetic lethal therapy targeting calcium/calmodulin-dependent protein kinase II (CaMKII), an emerging target for combating GSCs. Through high-throughput drug combination screening using CaMKII inhibitors and a library of bioactive compounds in GSCs, we find that the combined treatment with CaMKII and neurokinin 1 receptor (NK1R) inhibitors exhibit chemical synthetic lethal effects both in vitro and in vivo. Further analyses at the molecular level demonstrate that NK1R is a potential synthetic lethal partner of CaMKIIγ in its role in eradicating GSCs. Therefore, we propose a novel CaMKII-targeted combination therapy for the effective treatment of GBM.Glioblastoma stem-like cells (GSCs) drive tumor initiation, cancer invasion, immune evasion, and therapeutic resistance and are thus a key therapeutic target for improving treatment for glioblastoma multiforme (GBM). We previously identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an emerging molecular target for eliminating GSCs. In this study, we aim to explore a new CaMKII-targeted synthetic lethal therapy for GSCs. Through high-throughput drug combination screening using CaMKII inhibitors and a bioactive compound library in GSCs, neurokinin 1 receptor (NK1R) inhibitors such as SR 140333 and aprepitant are found to be potential anticancer agents that exhibit chemical synthetic lethal interactions with CaMKII inhibitors, including hydrazinobenzoylcurcumin (HBC), berbamine, and KN93. Combined treatment with NK1R and CaMKII inhibitors markedly suppresses the viability and neurosphere formation of U87MG- and U373MG-derived GSCs. In addition, the combination of HBC and NK1R inhibitors significantly inhibits U87MG GSC tumor growth in a chick embryo chorioallantoic membrane (CAM) model. Furthermore, the synthetic lethal interaction is validated using RNA interference of CaMKIIγ and NK1R. Notably, the synthetic lethal effects in GSCs are associated with the activation of caspase-mediated apoptosis by inducing p53 expression and reactive oxygen species generation, as well as the suppression of stemness marker expression by reducing nuclear factor-kappa B (NF-κB) activity. This follows the downregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling and a decrease in intracellular calcium concentration. Moreover, NK1R affects CaMKIIγ activation. These findings demonstrate that NK1R is a potential synthetic lethal partner of CaMKII that is involved in eradicating GSCs, and they suggest a new CaMKII-targeted combination therapy for treating GBM.

Ca2+/calmodulin-dependent protein kinase II inhibition reduces myocardial fatty acid uptake and oxidation after myocardial infarction

An AMP-activated kinase (AMPK) signaling pathway is activated during myocardial ischemia and promotes cardiac fatty acid (FA) uptake and oxidation. Similarly, the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is also triggered by myocardial ischemia, but its function in FA metabolism remains unclear. Here, we explored the role of CaMKII in FA metabolism during myocardial ischemia by investigating the effects of cardiac CaMKII on AMPK-acetyl-CoA carboxylase (ACC), malonyl CoA decarboxylase (MCD), and FA translocase cluster of differentiation 36 (FAT/CD36), as well as cardiac FA uptake and oxidation. Moreover, we tested whether CaMKII and AMPK are binding partners. We demonstrated that diseased hearts from patients with terminal ischemic heart disease displayed increased phosphorylation of CaMKII, AMPK, and ACC and increased expression of MCD and FAT/CD36. AC3-I mice, which have a genetic myocardial inhibition of CaMKII, had reduced gene expression of cardiac AMPK. In post-MI (myocardial infarction) AC3-I hearts, AMPK-ACC phosphorylation, MCD and FAT/CD36 levels, cardiac FA uptake, and FA oxidation were significantly decreased. Notably, we demonstrated that CaMKII interacted with AMPK α1 and α2 subunits in the heart. Additionally, AC3-I mice displayed significantly less cardiac hypertrophy and apoptosis 2 weeks post-MI. Overall, these findings reveal a unique role for CaMKII inhibition in repressing FA metabolism by interacting with AMPK signaling pathways, which may represent a novel mechanism in ischemic heart disease.

P21-Activated Kinase 1 Promotes Breast Tumorigenesis via Phosphorylation and Activation of the Calcium/Calmodulin-Dependent Protein Kinase II.

p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.

The Pharmacological Inhibition of CaMKII Regulates Sodium Chloride Cotransporter Activity in mDCT15 Cells.

Simple SummaryThe renal sodium chloride cotransporter (NCC) plays an important role in the total body electrolyte balance and blood pressure control. The regulation of this protein by the actin cytoskeleton has not been thoroughly studied. Here, we investigate a novel association between the actin cytoskeleton protein filamin A and the NCC using a mouse cellular model and in the native kidney. Our results show for the first time that the disruption of the actin cytoskeleton reduces NCC activity and filamin A plays an essential role in NCC protein expression in cells of the distal convoluted tubule. We further show that the pharmacological inhibition of the Ca2+/calmodulin dependent protein kinase II (CAMKII) augments NCC protein expression. These results introduce a new mechanism for the regulation of the NCC.The thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule is responsible for reabsorbing up to one-tenth of the total filtered load of sodium in the kidney. The actin cytoskeleton is thought to regulate various transport proteins in the kidney but the regulation of the NCC by the actin cytoskeleton is largely unknown. Here, we identify a direct interaction between the NCC and the cytoskeletal protein filamin A in mouse distal convoluted tubule (mDCT15) cells and in the native kidney. We show that the disruption of the actin cytoskeleton by two different mechanisms downregulates NCC activity. As filamin A is a substrate of the Ca2+/calmodulin-dependent protein kinase II (CaMKII), we investigate the physiological significance of CaMKII inhibition on NCC luminal membrane protein expression and NCC activity in mDCT15 cells. The pharmacological inhibition of CaMKII with the compound KN93 increases the active form of the NCC (phospho-NCC) at the luminal membrane and also increases NCC activity in mDCT15 cells. These data suggest that the interaction between the NCC and filamin A is dependent on CaMKII activity, which may serve as a feedback mechanism to maintain basal levels of NCC activity in the distal nephron.

Rewarding Social Interaction in Rats Increases CaMKII in the Nucleus Accumbens.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is known to be involved in the sensitized locomotor responses and drug-seeking behavior to psychostimulants. However, little is known about the contribution of CaMKII signaling in the nucleus accumbens (NAc) in natural rewards such as social interaction. The present experiments explored the implication of CaMKII signaling in drug versus natural reward. In the NAc of rats expressing cocaine or social interaction conditioned place preference (CPP), αCaMKII activation was induced in those expressing social interaction but not cocaine CPP. In order to investigate the role of NAc CaMKII in the expression of reward-related learning of drug versus non-drug stimuli, we inhibited CaMKII through an infusion of KN-93, a CaMKII inhibitor, directly into the NAc shell or core, before the CPP test in a concurrent paradigm in which social interaction was made available in the compartment alternative to the one associated with cocaine during conditioning. Whereas vehicle infusions led to equal preference to both stimuli, inhibition of CaMKII by a KN-93 infusion before the CPP test in the shell but not the core of the NAc shifted the rats’ preference toward the cocaine-associated compartment. Altogether, these results suggest that social interaction reward engages CaMKII in the NAc.