RESUMEN
AIMS: Chronic pain is highly associated with anxiety. Electroacupuncture (EA) is effective in relieving pain and anxiety. Currently, little is known about the neural mechanisms underlying the comorbidity of chronic pain and anxiety and the EA mechanism. This study investigated a potential neural circuit underlying the comorbid and EA mechanisms. METHODS: Spared nerve injury (SNI) surgery established the chronic neuropathic pain mouse model. The neural circuit was activated or inhibited using the chemogenetic method to explore the relationship between the neural circuit and mechanical allodynia and anxiety-like behaviors. EA combined with the chemogenetic method was used to explore whether the effects of EA were related to this neural circuit. RESULTS: EA attenuated mechanical allodynia and anxiety-like behaviors in SNI mice, which may be associated with the activity of CaMKII neurons in the basolateral amygdala (BLA). Inhibition of BLACaMKII-rACC induced mechanical allodynia and anxiety-like behaviors in sham mice. Activation of the BLACaMKII-rACC alleviated neuropathic pain and anxiety-like behaviors in SNI mice. The analgesic and anxiolytic effects of 2 Hz EA were antagonized by the inhibition of the BLACaMKII-rACC. CONCLUSION: BLACaMKII-rACC mediates mechanical allodynia and anxiety-like behaviors. The analgesic and anxiolytic effects of 2 Hz EA may be associated with the BLACaMKII-rACC.
Asunto(s)
Ansiedad , Complejo Nuclear Basolateral , Electroacupuntura , Giro del Cíngulo , Hiperalgesia , Animales , Electroacupuntura/métodos , Hiperalgesia/terapia , Ansiedad/terapia , Ansiedad/psicología , Masculino , Ratones , Complejo Nuclear Basolateral/metabolismo , Ratones Endogámicos C57BL , Neuralgia/terapia , Neuralgia/psicología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Vías NerviosasRESUMEN
γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Dominios Proteicos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Dispersión del Ángulo Pequeño , Triptófano/química , Triptófano/metabolismo , Piridazinas/química , Piridazinas/metabolismo , FosforilaciónRESUMEN
Cardiac metabolism ensures a continuous ATP supply, primarily using fatty acids in a healthy state and favoring glucose in pathological conditions. Pyruvate kinase muscle (PKM) controls the final step of glycolysis, with PKM1 being the main isoform in the heart. PKM2, elevated in various heart diseases, has been suggested to play a protective role in cardiac stress, but its function in basal cardiac metabolism remains unclear. We examined hearts from global PKM2 knockout (PKM2-/-) mice and found reduced intracellular glucose. Isotopic tracing of U-13C glucose revealed a shift to biosynthetic pathways in PKM2-/- cardiomyocytes. Total ATP content was two-thirds lower in PKM2-/- hearts, and functional analysis indicated reduced mitochondrial oxygen consumption. Total reactive oxygen species (ROS) and mitochondrial superoxide were also increased in PKM2-/- cardiomyocytes. Intriguingly, PKM2-/- hearts had preserved ejection fraction compared to controls. Mechanistically, increased calcium/calmodulin-dependent kinase II activity and phospholamban phosphorylation may contribute to higher sarcoendoplasmic reticulum calcium ATPase 2 pump activity in PKM2-/- hearts. Loss of PKM2 led to altered glucose metabolism, diminished mitochondrial function, and increased ROS in cardiomyocytes. These data suggest that cardiac PKM2 acts as an important rheostat to maintain ATP levels while limiting oxidative stress. Although loss of PKM2 did not impair baseline contractility, its absence may make hearts more sensitive to environmental stress or injury.
Asunto(s)
Miocitos Cardíacos , Estrés Oxidativo , Animales , Miocitos Cardíacos/metabolismo , Ratones , Ratones Noqueados , Glucosa/metabolismo , Masculino , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Mitocondrias Cardíacas/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Ratones Endogámicos C57BL , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Adenosina Trifosfato/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Miocardio/metabolismoRESUMEN
Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.
Asunto(s)
Proteínas del Tejido Nervioso , Plasticidad Neuronal , Proteoma , Receptores de N-Metil-D-Aspartato , Animales , Receptores de N-Metil-D-Aspartato/metabolismo , Plasticidad Neuronal/fisiología , Ratones , Fosforilación , Proteoma/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Masculino , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Ratones Endogámicos C57BL , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Aprendizaje/fisiología , Reacción de Prevención/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Sinapsis/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Espinas Dendríticas/metabolismoRESUMEN
BACKGROUND: Ureteral stricture (US) is a pathological stenosis in the urinary tract characterized by increased collagen synthesis and inflammation. Autophagy activation has been shown to ameliorate tissue fibrosis and protect against fibrotic diseases. Verapamil has beneficial therapeutic benefits on fibrotic disorders. The pharmacological effects of verapamil on fibroblast autophagy in US and the underlying mechanism need to be investigated further. METHODS: US patients were recruited to isolate scar tissues, hematoxylin-eosin (HE) and Masson trichrome staining were performed to analyze histopathological changes. The US animal model was established and administered with verapamil (0.05 mg/kg) in the drinking water. Transforming growth factor (TGF)-ß1 was adopted to facilitate collagen synthesis in fibroblasts. The mRNA and protein expressions were examined by qRT-PCR, western blot, immunofluorescence and immunohistochemistry. ELISA was adopted to measure interleukin (IL)-1ß and IL-6 levels. Molecular interaction experiments like dual luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to analyze the interaction between signal transducers and activators of transcription 3 (STAT3) and RNA polymerase II associated factor 1 (PAF1). RESULTS: Herein, our results revealed that verapamil activated TGF-ß1-treated fibroblast autophagy and inhibited inflammation and fibrosis by repressing Ca2+/calmodulin-dependent protein kinase II (CaMK II) δ-mediated STAT3 activation. Our following tests revealed that STAT3 activated PAF1 transcription. PAF1 upregulation abrogated the regulatory effect of verapamil on fibroblast autophagy and fibrosis during US progression. Finally, verapamil mitigated US in vivo by activating fibroblast autophagy. CONCLUSION: Taken together, verapamil activated TGF-ß1-treated fibroblast autophagy and inhibited fibrosis by repressing the CaMK IIδ/STAT3/PAF1 axis.
Asunto(s)
Autofagia , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Fibroblastos , Fibrosis , Factor de Transcripción STAT3 , Factor de Crecimiento Transformador beta1 , Obstrucción Ureteral , Verapamilo , Verapamilo/farmacología , Verapamilo/uso terapéutico , Autofagia/efectos de los fármacos , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/metabolismo , Factor de Transcripción STAT3/metabolismo , Humanos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Masculino , Factor de Crecimiento Transformador beta1/metabolismo , Cicatriz/patología , Cicatriz/metabolismo , Cicatriz/tratamiento farmacológico , Cicatriz/etiología , Cicatriz/prevención & control , Modelos Animales de Enfermedad , Inflamación/metabolismo , Transducción de Señal/efectos de los fármacos , Femenino , Persona de Mediana EdadRESUMEN
Ovarian clear cell carcinoma (OCCC) is a subtype of ovarian cancer and is highly malignant with high chemoresistance. CACNA1H is pivotal in tumor development. However, the role of CACNA1H in the acquisition process of chemotherapeutic resistance in OCCC cells is rarely reported. Therefore, this study aimed to explore the role of CACNA1H in chemotherapy resistance of OCCC cells and its related mechanism. Based on bioinformatics analysis, we found that CACNA1H was downregulated in chemoresistant OCCC patients compared to chemosensitive OCCC patients. Comparing DDP-resistant and sensitive OCCC cell lines, the resistant strain showed lower CACNA1H mRNA expression. CACNA1H expression was associated with calcium signaling pathways in chemoresistant OCCC patients. CACNA1H mRNA expression was significantly downregulated in OCCC cells compared to normal ovarian epithelial cells. When CACNA1H was overexpressed, intracellular Ca2+ concentration and protein levels of p-CaMKII and p-Akt were significantly upregulated, while protein levels of LC3-II/LC3-I and Beclin1 were downregulated, indicating a repression of autophagy. The rescue experiment revealed that CACNA1H overexpression in drug-resistant OCCC cells reduced autophagy-induced DDP resistance via CaMKII/Akt signaling. Overall, CACNA1H increased intracellular Ca2+ concentration and activated CaMKII/Akt signaling pathway in OCCC, thereby repressing autophagy to maintain the sensitivity of OCCC cells to DDP.
Asunto(s)
Adenocarcinoma de Células Claras , Autofagia , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas , Femenino , Humanos , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/patología , Adenocarcinoma de Células Claras/metabolismo , Autofagia/genética , Autofagia/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio/genética , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
This study explores the behavioral effects of modulating CaMKII-positive (CaMKII+) neurons in the posterior hypothalamus (PH). Utilizing a chemogenetic approach in mice, we discovered that the activation of CaMKII + neurons within the PH is associated with heightened locomotor activity, reduced social interaction, and impulsive behavior unrelated to anxiety or avoidance. These observed behaviors share a significant resemblance with characteristics commonly found in attention deficit and hyperactivity disorder (ADHD). Notably, treatment with clonidine, which is frequently prescribed for ADHD, effectively reduced impulsive behaviors in our mouse model. Our findings uncover the role of the PH that has not been previously explored and suggest a possible involvement of the PH in the manifestation of ADHD-like behaviors.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad , Conducta Animal , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Hipotálamo Posterior , Neuronas , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Neuronas/metabolismo , Trastorno por Déficit de Atención con Hiperactividad/patología , Masculino , Ratones Endogámicos C57BL , Conducta Impulsiva , Conducta Social , Clonidina/farmacología , Ratones , AnsiedadRESUMEN
Cardiovascular diseases (CVDs) tend to affect the young population and are associated with a significant economic burden and psychological distress to the society and families. The physiological and pathological processes underlying CVDs are complex. Ca2+/calmodulin-dependent kinase II (CaMK II), a protein kinase, has multiple biological functions. It participates in multiple pathological processes and plays a central role in the development of CVDs. Based on this, this paper analyzes the structural characteristics and distribution of CaMK II, the mechanism of action of CaMK II, and the relationship between CaMK II and CVDs, including ion channels, ischemia-reperfusion injury, arrhythmias, myocardial hypertrophy, cardiotoxicity, hypertension, and dilated cardiomyopathy. Given the different regulatory mechanisms of different isoforms of CaMK II, the clinical use of specific targeted inhibitors or novel compounds should be evaluated in future research to provide new directions.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Enfermedades Cardiovasculares , Humanos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Myocardial ischemia-reperfusion injury (MIRI) frequently complicates postoperative cardiovascular disease treatment. Necroptosis, a cell death mechanism similar to apoptosis, is regulated by specific signaling pathways and plays an important role in MIRI. Receptor-interacting protein 3 (RIP3), a key protein regulating necroptosis during MIRI, directly phosphorylates calmodulin-dependent protein kinase II (CaMKII). Leading to mitochondrial permeablity transition pore (mPTP) opening and inducing necroptosis. Transient receptor potential canonical channel 6 (TRPC6) regulats Ca2+ entry, is linked to CaMKII as an important upstream effector. However, the connection between TRPC6 and MIRI necroptosis remains unclear. The study aimed to investigate the relationship between TRPC6 and MIRI necroptosis, with a specific focus on elucidating the role of TRPC6 in regulating CaMKII phosphorylation during cardiac necroptosis via Ca2+ modulation. METHODS AND RESULTS: The experiment used wild-type (WT) and TRPC6 knockout (TRPC6-/-) mice for I/R model construction, and H9c2 myocardial cell line for H/R model. After ischemia-reperfusion (I/R), TRPC6 protein levels in mice significantly increased, exacerbating myocardial injury, infarct size (IS), and cardiac function in WT mice. In contrast, TRPC6 knockout attenuated myocardial injury, IS, and improved cardiac function. The results showed a significant correlation between changes in CaMKII and TRPC6. TRPC6 knockout led to decreased intracellular calcium levels, CaMKII phosphorylation, reactive oxygen species levels, mPTP opening, and improve mitochondrial structure. CONCLUSION: I/R upregulates TRPC6, which mediates Ca2+ entry and CaMKII phosphorylation, exacerbates oxidative stress, and induces necroptosis. These findings suggest a potential therapeutic avenue for mitigating MIRI by targeting TRPC6.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calcio , Daño por Reperfusión Miocárdica , Necroptosis , Canal Catiónico TRPC6 , Animales , Masculino , Ratones , Calcio/metabolismo , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fosforilación , Transducción de Señal , Canal Catiónico TRPC6/metabolismoRESUMEN
Diabetic neuropathic pain (DNP) is a diabetic complication that causes severe pain and deeply impacts the quality of the sufferer's daily life. Currently, contemporary clinical treatments for DNP generally exhibit a deficiency in effectiveness. Electroacupuncture (EA) is recognized as a highly effective and safe treatment for DNP with few side effects. Regrettably, the processes via which EA alleviates DNP are still poorly characterized. Transient receptor potential vanilloid 1 (TRPV1) and phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) are overexpressed on spinal cord dorsal horn (SCDH) in DNP rats, and co-localization is observed between them. Capsazepine, a TRPV1 antagonist, effectively reduced nociceptive hypersensitivity and downregulated the overexpression of phosphorylated CaMKIIα in rats with DNP. Conversely, the CaMKII inhibitor KN-93 did not have any impact on TRPV1. EA alleviated heightened sensitivity to pain caused by nociceptive stimuli and downregulated the level of TRPV1, p-CaMKIIα, and phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) in DNP rats. Intrathecal injection of capsaicin, on the other hand, reversed the above effects of EA. These findings indicated that the CaMKII/CREB pathway on SCDH is located downstream of TRPV1 and is affected by TRPV1. EA alleviates DNP through the TRPV1-mediated CaMKII/CREB pathway.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Neuropatías Diabéticas , Electroacupuntura , Ratas Sprague-Dawley , Canales Catiónicos TRPV , Animales , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Electroacupuntura/métodos , Ratas , Masculino , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Neuropatías Diabéticas/terapia , Neuropatías Diabéticas/metabolismo , Capsaicina/farmacología , Capsaicina/análogos & derivados , Transducción de Señal , Asta Dorsal de la Médula Espinal/metabolismo , Bencenosulfonamidas , BencilaminasRESUMEN
The effects of enhanced late INa, a persistent component of the Na+ channel current, on the intracellular ion dynamics and the automaticity of the pulmonary vein cardiomyocytes were studied with fluorescent microscopy. Anemonia viridis toxin II (ATX- II), an enhancer of late INa, caused increases in the basal Na+ and Ca2+ concentrations, increases in the number of Ca2+ sparks and Ca2+ waves, and the generation of repetitive Ca2+ transients. These phenomena were inhibited by eleclazine, a blocker of the late INa; SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX); H89, a protein kinase A (PKA) inhibitor; and KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results suggest that enhancement of late INa in the pulmonary vein cardiomyocytes causes disturbance of the intracellular ion environment through activation of the NCX and Ca2+-dependent enzymes. Such mechanisms are probably involved in the ectopic electrical activity of the pulmonary vein myocardium.
Asunto(s)
Calcio , Venenos de Cnidarios , Miocitos Cardíacos , Venas Pulmonares , Intercambiador de Sodio-Calcio , Animales , Venas Pulmonares/metabolismo , Venas Pulmonares/citología , Venas Pulmonares/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Cobayas , Calcio/metabolismo , Venenos de Cnidarios/farmacología , Intercambiador de Sodio-Calcio/metabolismo , Sodio/metabolismo , Masculino , Potenciales de Acción/efectos de los fármacos , Canales de Sodio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Compuestos de Anilina/farmacología , Sulfonamidas/farmacología , Señalización del Calcio/efectos de los fármacos , Isoquinolinas , Éteres FenílicosRESUMEN
Mutations in the nuclear envelope (NE) protein lamin A/C (encoded by LMNA), cause a severe form of dilated cardiomyopathy (DCM) with early-onset life-threatening arrhythmias. However, molecular mechanisms underlying increased arrhythmogenesis in LMNA-related DCM (LMNA-DCM) remain largely unknown. Here we show that a frameshift mutation in LMNA causes abnormal Ca2+ handling, arrhythmias and disformed NE in LMNA-DCM patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). Mechanistically, lamin A interacts with sirtuin 1 (SIRT1) where mutant lamin A/C accelerates degradation of SIRT1, leading to mitochondrial dysfunction and oxidative stress. Elevated reactive oxygen species (ROS) then activates the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-ryanodine receptor 2 (RYR2) pathway and aggravates the accumulation of SUN1 in mutant iPSC-CMs, contributing to arrhythmias and NE deformation, respectively. Taken together, the lamin A/C deficiency-mediated ROS disorder is revealed as central to LMNA-DCM development. Manipulation of impaired SIRT1 activity and excessive oxidative stress is a potential future therapeutic strategy for LMNA-DCM.
Asunto(s)
Cardiomiopatía Dilatada , Células Madre Pluripotentes Inducidas , Lamina Tipo A , Miocitos Cardíacos , Estrés Oxidativo , Especies Reactivas de Oxígeno , Sirtuina 1 , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Células Madre Pluripotentes Inducidas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Humanos , Sirtuina 1/metabolismo , Sirtuina 1/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fenotipo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Mutación del Sistema de Lectura , Calcio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Membrana Nuclear/metabolismo , Mitocondrias/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) signaling in the brain plays a critical role in regulating neuronal Ca2+ homeostasis. Its dysfunctional activity is associated with various neurological and neurodegenerative disorders, including Parkinson's disease (PD). Using computational modeling analysis, we predicted that, two essential cysteine residues contained in CaMKIIα, Cys30 and Cys289, may undergo redox modifications impacting the proper functioning of the CaMKIIα docking site for Ca2+/CaM, thus impeding the formation of the CaMKIIα:Ca2+/CaM complex, essential for a proper modulation of CaMKIIα kinase activity. Our subsequent in vitro investigations confirmed the computational predictions, specifically implicating Cys30 and Cys289 residues in impairing CaMKIIα:Ca2+/CaM interaction. We observed CaMKIIα:Ca2+/CaM complex disruption in dopamine (DA) nigrostriatal neurons of post-mortem Parkinson's disease (PD) patients' specimens, addressing the high relevance of this event in the disease. CaMKIIα:Ca2+/CaM complex disruption was also observed in both in vitro and in vivo rotenone models of PD, where this phenomenon was associated with CaMKIIα kinase hyperactivity. Moreover, we observed that, NADPH oxidase 2 (NOX2), a major enzymatic generator of superoxide anion (O2â-) and hydrogen peroxide (H2O2) in the brain with implications in PD pathogenesis, is responsible for CaMKIIα:Ca2+/CaM complex disruption associated to a stable Ca2+CAM-independent CaMKIIα kinase activity and intracellular Ca2+ accumulation. The present study highlights the importance of oxidative stress, in disturbing the delicate balance of CaMKIIα signaling in calcium dysregulation, offering novel insights into PD pathogenesis.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calmodulina , NADPH Oxidasa 2 , Oxidación-Reducción , Enfermedad de Parkinson , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Enfermedad de Parkinson/metabolismo , Humanos , Calmodulina/metabolismo , Animales , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Calcio/metabolismo , Cisteína/metabolismo , RatonesRESUMEN
OBJECTIVE: To investigate the effect of sevoflurane on neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve in mice, and to elucidate its mechanism by animal experiments. METHODS AND RESULTS: Thirty-two C57BL/6 mice were randomly divided into four groups: Sham group, Model group, Control group and Sevoflurane group. First, a mouse model of neuropathic pain was established. Then, the mice in each group were killed on Day 14 after operation to harvest the enlarged lumbosacral spinal cord. In contrast with the Model group, the Sevoflurane group displayed a significantly increased paw withdrawal mechanical threshold (PWMT) and significantly prolonged paw withdrawal thermal latency (PWTL) from Day 5 after operation. The morphological changes of lumbosacral spinal cord were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy. Pathological results showed that sevoflurane reduced nuclear pyknosis in lumbosacral spinal cord tissue, with a large number of mitochondrial crista disappearance and mitochondrial swelling. The results of Western blotting showed that sevoflurane significantly decreased the protein expressions of phosphorylated phospholipase Cγ (p-PLCγ), phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) and phosphorylated inositol 1,4,5-triphosphate receptor (p-IP3R), and reduced the protein expressions of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 (GRP78) and GRP94, oxidative stress-related proteins P22 and P47 and inflammatory factors nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), interleukin-1 ß (IL-1ß), and tumor necrosis factor-α (TNF-α). CONCLUSIONS: Sevoflurane inhibits neuropathic pain by maintaining ER stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Receptores de Inositol 1,4,5-Trifosfato , Ratones Endogámicos C57BL , Neuralgia , Estrés Oxidativo , Fosfolipasa C gamma , Sevoflurano , Transducción de Señal , Animales , Sevoflurano/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Neuralgia/metabolismo , Neuralgia/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Ratones , Fosfolipasa C gamma/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Homeostasis/efectos de los fármacos , Modelos Animales de Enfermedad , Nervio Ciático/efectos de los fármacos , Nervio Ciático/metabolismo , Nervio Ciático/lesionesRESUMEN
Comorbid chronic neuropathic pain and anxiety is a common disease that represents a major clinical challenge. The underlying mechanisms of chronic neuropathic pain and anxiety are not entirely understood, which limits the exploration of effective treatment methods. Glutamatergic neurons in the ventrolateral periaqueductal gray (vlPAG) have been implicated in regulating pain, but the potential roles of the vlPAG in neuropathic pain-induced anxiety have not been investigated. Herein, whole-cell recording and immunofluorescence showed that the excitability of CamkIIα neurons in the vlPAG (vlPAGCamkIIα+ neurons) was decreased in mice with spared nerve injury (SNI), while electroacupuncture (EA) activated these neurons. We also showed that chemogenetic inhibition of vlPAGCamkIIα+ neurons resulted in allodynia and anxiety-like behaviors in naive mice. Furthermore, chemogenetic activation of vlPAGCamkIIα+ neurons reduced anxiety-like behaviors and allodynia in mice with SNI, and EA had a similar effect in alleviating these symptoms. Nevertheless, EA combined with chemogenetic activation failed to further relieve allodynia and anxiety-like behaviors. Artificial inhibition of vlPAGCamkIIα+ neurons abolished the analgesic and anxiolytic effects of EA. Overall, our study reveals a novel mechanism of neuropathic pain-induced anxiety and shows that EA may relieve comorbid chronic neuropathic pain and anxiety by activating vlPAGCamkIIα+ neurons.
Asunto(s)
Ansiedad , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Electroacupuntura , Neuralgia , Neuronas , Sustancia Gris Periacueductal , Animales , Neuralgia/terapia , Electroacupuntura/métodos , Neuronas/fisiología , Neuronas/metabolismo , Masculino , Ansiedad/terapia , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Hiperalgesia/terapia , Dolor Crónico/terapia , Ácido Glutámico/metabolismo , Modelos Animales de Enfermedad , Conducta Animal/fisiologíaRESUMEN
Zika virus (ZIKV) infection may lead to severe neurological consequences, including seizures, and early infancy death. However, the involved mechanisms are still largely unknown. TRPC channels play an important role in regulating nervous system excitability and are implicated in seizure development. We investigated whether TRPCs might be involved in the pathogenesis of ZIKV infection. We found that ZIKV infection increases TRPC4 expression in host cells via the interaction between the ZIKV-NS3 protein and CaMKII, enhancing TRPC4-mediated calcium influx. Pharmacological inhibition of CaMKII decreased both pCREB and TRPC4 protein levels, whereas the suppression of either TRPC4 or CaMKII improved the survival rate of ZIKV-infected cells and reduced viral protein production, likely by impeding the replication phase of the viral life cycle. TRPC4 or CaMKII inhibitors also reduced seizures and increased the survival of ZIKV-infected neonatal mice and blocked the spread of ZIKV in brain organoids derived from human-induced pluripotent stem cells. These findings suggest that targeting CaMKII or TRPC4 may offer a promising approach for developing novel anti-ZIKV therapies, capable of preventing ZIKV-associated seizures and death.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Canales Catiónicos TRPC , Infección por el Virus Zika , Virus Zika , Infección por el Virus Zika/virología , Infección por el Virus Zika/metabolismo , Animales , Humanos , Virus Zika/fisiología , Virus Zika/efectos de los fármacos , Ratones , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Células HEK293 , Proteínas Virales/metabolismo , Convulsiones/virología , Convulsiones/metabolismo , Convulsiones/tratamiento farmacológico , Proteasas Virales , Serina Endopeptidasas , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-boxRESUMEN
The objective of this investigation was to examine the impact of multiple exposures to general anesthesia (GA) with sevoflurane on the offspring of pregnant mice, as well as to elucidate the underlying mechanism. Neurodevelopmental assessments, including various reflexes and behavioral tests, were conducted on the offspring in the GA group to evaluate neuronal cell development. Furthermore, neonatal mouse neuronal cells were isolated and transfected with a high-expression CREB vector (pcDNA3.1-CREB), followed by treatment with sevoflurane (0.72 mol/L), ZD7288 (50 µmol/L), and KN-62 (10 µmol/L), or a combination of these compounds. The expression of relevant genes was then analyzed using qRT-PCR and western blot techniques. In comparison to the sham group, neonatal mice in the GA group exhibited significantly prolonged latencies in surface righting reflex, geotaxis test, and air righting reflex. Furthermore, there was a notable deceleration in the development of body weight and tail in the GA group. These mice also displayed impairments in social ability, reduced reciprocal social interaction behaviors, diminished learning capacity, and heightened levels of anxious behaviors. Additionally, synaptic trigger malfunction was observed, along with decreased production of c-Fos and neurotrophic factors. Sevoflurane was found to notably decrease cellular c-Fos and neurotrophic factor production, as well as the expression of HCN2 and CaMKII/CREB-related proteins. The inhibitory effects of sevoflurane on HCN2 or CaMKII channels were similar to those observed with ZD7288 or KN-62 inhibition. However, overexpression of CREB mitigated the impact of sevoflurane on neuronal cells. Repetitive exposure to sevoflurane general anesthesia while pregnant suppresses the CaMKII/CREB pathway, leading to the development of autism-like characteristics in offspring mice through the reduction of HCN2 expression.
Asunto(s)
Anestésicos por Inhalación , Trastorno Autístico , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Regulación hacia Abajo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Efectos Tardíos de la Exposición Prenatal , Sevoflurano , Animales , Sevoflurano/farmacología , Sevoflurano/toxicidad , Ratones , Embarazo , Femenino , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Anestésicos por Inhalación/farmacología , Anestésicos por Inhalación/toxicidad , Anestésicos por Inhalación/efectos adversos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Canales de Potasio/metabolismo , Canales de Potasio/genética , Células Cultivadas , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Masculino , Ratones Endogámicos C57BLRESUMEN
Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human CaV1.3 VGCC, however, is largely unexplored. We characterized modulation of CaV1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in CaV1.3 but absent from all other high-voltage activating calcium channel types co-expressed with CaV1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating CaV1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human CaV1.3. We obtained three main findings: (1) CaV1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype CaV1.3 and the phosphorylation-resistant CaV1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of CaV1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating CaV1.3 through S1475. (3) CaMKII activation led to CaV1.3_WT-current properties similar to those of CaV1.3_S1475D, but did not affect CaV1.3_S1475A, confirming that CaMKII modulates human CaV1.3 via S1475. Given the physiological and pathophysiological importance of CaV1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific CaV1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human CaV1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human CaV1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype CaV1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human CaV1.3 channels, and present a possible target for CaV1.3-specific gating modulation in physiological and pathophysiological conditions.
Asunto(s)
Canales de Calcio Tipo L , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calmodulina , Humanos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Fosforilación , Células HEK293 , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/fisiología , Calmodulina/metabolismo , Animales , Activación del Canal Iónico/fisiología , RatasRESUMEN
The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. In this study, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in homeostatic regulation of sleep in male mice. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. Here, we propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Corteza Cerebral , Homeostasis , Neuronas , Parvalbúminas , Sueño , Vigilia , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Parvalbúminas/metabolismo , Masculino , Sueño/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Ratones , Vigilia/fisiología , Corteza Cerebral/metabolismo , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine secreted by the pineal gland during the dark phase of the photoperiod. Its main function is the synchronization of different body rhythms with the dark-light cycle. Research on melatonin has significantly advanced since its discovery and we now know that it has considerable significance in various physiological processes, including immunity, aging, and reproduction. Moreover, in recent years evidence of the pharmacological possibilities of melatonin has increased. Indoleamine, on the other hand, has antidepressant-like effects in rodents, which may be mediated by the activation of calcium-calmodulin-dependent kinase II (CaMKII) and are also related to the regulation of neuroplasticity processes, including neurogenesis, synaptic maintenance, and long-term potentiation. Remarkably, patients with major depression show decreased levels of circulating melatonin in plasma. This review presents evidence of the antidepressant-like effects of melatonin in preclinical models and the participation of CaMKII in these actions. CaMKII's role in cognition and memory processes, which are altered in depressive states, are part of the review, and the effects of melatonin in these processes are also reviewed. Furthermore, participation of CaMKII on structural and synaptic plasticity and the effects of melatonin are also described. Finally, the advantages of using melatonin in combination with other antidepressants such as ketamine for neuroplasticity are described. Evidence supports that CaMKII is activated by melatonin and downstream melatonin receptors and may be the common effector in the synergistic effects of melatonin with other antidepressants. SIGNIFICANCE STATEMENT: This review compiled evidence supporting that melatonin causes antidepressant-like effects in mice through calmodulin kinase II stimulation of downstream melatonin receptors as well as the participation of this enzyme in neuroplasticity, memory, and cognition. Finally, we describe evidence about the effectiveness of antidepressant-like effects of melatonin in combination with ketamine.