Dorsal raphe dopaminergic neurons target CaMKII+ neurons in dorsal bed nucleus of the stria terminalis for mediating depression-related behaviors.

Dopamine (DA) neurons play a crucial role in the development and manifestation of depression, as well as in response to antidepressant treatments. While the function of the predominantly distributed DA neurons in the ventral tegmental area (VTA) is well established, the contribution of a small fraction of DA neurons in the dorsal raphe nucleus (DRN) during depression remains unclear. In this study, we found that chronic unpredictable stress (CUS) induces depression-related behaviors and decreases spontaneous firing rates, excitatory and inhibitory postsynaptic currents of DA neurons in the DRN associated with reduced excitatory synaptic transmission in male and female mice. The chemogenetic inhibition of DA neurons in the DRN produces depressive phenotypes. Conversely, their activation completely reversed the anhedonic and despair behaviors induced by CUS. Furthermore, we showed that a DRN dopaminergic projecting to the dorsal bed nucleus of the stria terminalis (dBNST) selectively controls depressive behaviors by influencing the neural activity and N-methyl-D-aspartate receptor (NMDAR) mediating EPSC of calcium/calmodulin-dependent protein kinase II+ (CaMKII+) target neurons by regulating dopamine neurotransmitter and dopamine receptor 2 (DR2) in the dBNST. Overall, these findings highlight the essential role of the DRNDA → dBNSTCaMKII+ neural circuit in bi-directionally mediating stress-induced depression-related behaviors. Our findings indicate that DRN DA neurons are a key component of the neural circuitry involved in regulating depression-related behaviors, making them a potential therapeutic target for depression.

A circuit from lateral hypothalamic to dorsal hippocampal dentate gyrus modulates behavioral despair in mice.

Behavioral despair is one of the clinical manifestations of major depressive disorder and an important cause of disability and death. However, the neural circuit mechanisms underlying behavioral despair are poorly understood. In a well-established chronic behavioral despair (CBD) mouse model, using a combination of viral tracing, in vivo fiber photometry, chemogenetic and optogenetic manipulations, in vitro electrophysiology, pharmacological profiling techniques, and behavioral tests, we investigated the neural circuit mechanisms in regulating behavioral despair. Here, we found that CBD enhanced CaMKIIα neuronal excitability in the dorsal dentate gyrus (dDG) and dDGCaMKIIα neurons involved in regulating behavioral despair in CBD mice. Besides, dDGCaMKIIα neurons received 5-HT inputs from median raphe nucleus (MRN) and were mediated by 5-HT1A receptors, whereas MRN5-HT neurons received CaMKIIα inputs from lateral hypothalamic (LH) and were mediated by AMPA receptors to regulate behavioral despair. Furthermore, fluvoxamine exerted its role in resisting behavioral despair through the LH-MRN-dDG circuit. These findings suggest that a previously unidentified circuit of LHCaMKIIα-MRN5-HT-dDGCaMKIIα mediates behavioral despair induced by CBD. Furthermore, these support the important role of AMPA receptors in MRN and 5-HT1A receptors in dDG that might be the potential targets for treatment of behavioral despair, and explain the neural circuit mechanism of fluvoxamine-resistant behavioral despair.

Loss of mitochondrial Ca2+ response and CaMKII/ERK activation by LRRK2R1441G mutation correlate with impaired depolarization-induced mitophagy.

BACKGROUND: Stress-induced activation of ERK/Drp1 serves as a checkpoint in the segregation of damaged mitochondria for autophagic clearance (mitophagy). Elevated cytosolic calcium (Ca2+) activates ERK, which is pivotal to mitophagy initiation. This process is altered in Parkinson's disease (PD) with mutations in leucine-rich repeat kinase 2 (LRRK2), potentially contributing to mitochondrial dysfunction. Pathogenic LRRK2 mutation is linked to dysregulated cellular Ca2+ signaling but the mechanism involved remains unclear. METHODS: Mitochondrial damages lead to membrane depolarization. To investigate how LRRK2 mutation impairs cellular response to mitochondrial damages, mitochondrial depolarization was induced by artificial uncoupler (FCCP) in wild-type (WT) and LRRK2R1441G mutant knockin (KI) mouse embryonic fibroblasts (MEFs). The resultant cytosolic Ca2+ flux was assessed using live-cell Ca2+ imaging. The role of mitochondria in FCCP-induced cytosolic Ca2+ surge was confirmed by co-treatment with the mitochondrial sodium-calcium exchanger (NCLX) inhibitor. Cellular mitochondrial quality and function were evaluated by Seahorse™ real-time cell metabolic analysis, flow cytometry, and confocal imaging. Mitochondrial morphology was visualized using transmission electron microscopy (TEM). Activation (phosphorylation) of stress response pathways were assessed by immunoblotting. RESULTS: Acute mitochondrial depolarization induced by FCCP resulted in an immediate cytosolic Ca2+ surge in WT MEFs, mediated predominantly via mitochondrial NCLX. However, such cytosolic Ca2+ response was abolished in LRRK2 KI MEFs. This loss of response in KI was associated with impaired activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and MEK, the two upstream kinases of ERK. Treatment of LRRK2 inhibitor did not rescue this phenotype indicating that it was not caused by mutant LRRK2 kinase hyperactivity. KI MEFs exhibited swollen mitochondria with distorted cristae, depolarized mitochondrial membrane potential, and reduced mitochondrial Ca2+ store and mitochondrial calcium uniporter (MCU) expression. These mutant cells also exhibited lower cellular ATP: ADP ratio albeit higher basal respiration than WT, indicating compensation for mitochondrial dysfunction. These defects may hinder cellular stress response and signals to Drp1-mediated mitophagy, as evident by impaired mitochondrial clearance in the mutant. CONCLUSIONS: Pathogenic LRRK2R1441G mutation abolished mitochondrial depolarization-induced Ca2+ response and impaired the basal mitochondrial clearance. Inherent defects from LRRK2 mutation have weakened the cellular ability to scavenge damaged mitochondria, which may further aggravate mitochondrial dysfunction and neurodegeneration in PD.

Involvement of basolateral amygdala-rostral anterior cingulate cortex in mechanical allodynia and anxiety-like behaviors and potential mechanisms of electroacupuncture.

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.

PKM2 regulates metabolic flux and oxidative stress in the murine heart.

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.

CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction.

Protein quality control serves as the primary defense mechanism for cells against proteotoxicity induced by proteasome dysfunction. While cells can limit the build-up of ubiquitinated misfolded proteins during proteasome inhibition, the precise mechanism is unclear. Here, we find that protein kinase Ca2+/Calmodulin (CaM)-dependent protein kinase II (CaMKII) maintains proteostasis during proteasome inhibition. We show that proteasome inhibition activates CaMKII, which phosphorylates B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) at residues S173, S377, and S386. Phosphorylated BAG3 activates the heme-regulated inhibitor (HRI)- eukaryotic initiation factor-2α (eIF2α) signaling pathway, suppressing protein synthesis and the production of aggregated ubiquitinated misfolded proteins, ultimately mitigating the proteotoxic crisis. Inhibition of CaMKII exacerbates the accumulation of aggregated misfolded proteins and paraptosis induced by proteasome inhibitors. Based on these findings, we validate that combined targeting of proteasome and CaMKII accelerates tumor cell death and enhances the efficacy of proteasome inhibitors in tumor treatment. Our data unveil a new proteasomal inhibition-induced misfolded protein quality control mechanism and propose a novel therapeutic intervention for proteasome inhibitor-mediated tumor treatment.

Signal flow in the NMDA receptor-dependent phosphoproteome regulates postsynaptic plasticity for aversive learning.

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.

A cholinergic signaling pathway underlying cortical circuit activation of quiescent neural stem cells in the lateral ventricle.

Neural stem cells (NSCs) in the subventricular zone (SVZ) located along the lateral ventricles (LVs) of the mammalian brain continue to self-renew to produce new neurons after birth and into adulthood. Quiescent LV cells, which are situated close to the ependymal cells lining the LVs, are activated by choline acetyltransferase-positive (ChAT+) neurons within the subependymal (subep) region of the SVZ when these neurons are stimulated by projections from the anterior cingulate cortex (ACC). Here, we uncovered a signaling pathway activated by the ACC-subep-ChAT+ circuit responsible for the activation and proliferation of quiescent LV NSCs specifically in the ventral area of the SVZ. This circuit activated muscarinic M3 receptors on quiescent LV NSCs, which subsequently induced signaling mediated by the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1). Downstream of IP3R1 activation, which would be expected to increase intracellular Ca2+, Ca2+-/calmodulin-dependent protein kinase II δ and the MAPK10 signaling pathway were stimulated and required for the proliferation of quiescent LV NSCs in the SVZ. These findings reveal the mechanisms that regulate quiescent LV NSCs and underscore the critical role of projections from the ACC in promoting their proliferative activity within the ventral SVZ.

Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity.

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.

Verapamil inhibits inflammation and promotes autophagy to alleviate ureteral scar by regulation of CaMK IIδ/STAT3 axis.

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.

The phosphorylation of Smad3 by CaMKIIγ leads to the hepatocyte pyroptosis under perfluorooctane sulfonate exposure.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant and accumulated in the liver of mammals. PFOS exposure is closely associated with the development of pyroptosis. Nevertheless, the underlying mechanism is unclear. We found here that PFOS induced pyroptosis in the mice liver and L-02 cells as demonstrated by activation of the NOD-like receptor protein 3 inflammasome, gasdermin D cleavage and increased release of interleukin-1ß and interleukin-18. The level of cytoplasmic calcium was accelerated in hepatocytes upon exposure to PFOS. The phosphorylated/activated form of calcium/calmodulin-dependent protein kinase II (CaMKII) was augmented by PFOS in vivo and in vitro. PFOS-induced pyroptosis was relieved by CaMKII inhibitor. Among various CaMKII subtypes, we identified that CaMKIIγ was activated specifically by PFOS. CaMKIIγ interacted with Smad family member 3 (Smad3) under PFOS exposure. PFOS increased the phosphorylation of Smad3, and CaMKII inhibitor or CaMKIIγ siRNA alleviated PFOS-caused phosphorylation of Smad3. Inhibiting Smad3 activity was found to alleviate PFOS-induced hepatocyte pyroptosis. This study puts forward that CaMKIIγ-Smad3 is the linkage between calcium homeostasis disturbance and pyroptosis, providing a mechanistic explanation for PFOS-induced pyroptosis.

Enhanced Late INa Induces Intracellular Ion Disturbances and Automatic Activity in the Guinea Pig Pulmonary Vein Cardiomyocytes.

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.

Electroacupuncture Alleviates Streptozotocin-Induced Diabetic Neuropathic Pain via the TRPV1-Mediated CaMKII/CREB Pathway in Rats.

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.

Involvement of posterior hypothalamic CaMKII-positive neurons in ADHD-like behaviors in mice.

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.

Salidroside derivative SHPL-49 attenuates glutamate excitotoxicity in acute ischemic stroke via promoting NR2A-CAMKâ ¡α-Akt /CREB pathway.

BACKGROUND: Ischemic stroke is a significant cause of death and disability with a limited treatment time window. The reduction of early glutamate excitotoxicity using neuroprotective agents targeting N-methyl-d-aspartic acid (NMDA) receptors have attracted recent research attention. SHPL-49, a structurally modified derivative of salidroside, was synthesized by our team. Previous studies have confirmed the neuroprotective efficacy of SHPL-49 in rats with ischemic stroke. However, the underlying mechanisms need to be clarified. METHODS: We conducted in vivo experiments using the permanent middle cerebral artery occlusion rat model to investigate the role of SHPL-49 in glutamate release at different time points and treatment durations. Glutamate transporters and receptor proteins and neural survival proteins in the brain were also examined at the same time points. In vitro, primary neurons and the coculture system of primary neurons-astrocytes were subjected to oxygen-glucose deprivation and glutamate injury. Proteomics and parallel reaction monitoring analyses were performed to identify potential therapeutic targets of SHPL-49, which were further confirmed through in vitro experiments on the inhibition and mutation of the target. RESULTS: SHPL-49 significantly reduced glutamate release caused by hypoxia-ischemia. One therapeutic pathway of SHPL-49 was promoting the expression of glutamate transporter-1 to increase glutamate reuptake and further reduce the occurrence of subsequent neurotoxicity. In addition, we explored the therapeutic targets of SHPL-49 and its regulatory effects on glutamate receptors for the first time. SHPL-49 enhanced neuroprotection by activating the NMDA subunit NR2A, which upregulated the cyclic-AMP response binding protein (CREB) neural survival pathway and Akt phosphorylation. Since calcium/calmodulin-dependent kinase IIα (CaMKIIα) is necessary for synaptic transmission of NMDA receptors, we explored the interaction between CaMKIIα and SHPL-49, which protected CaMKIIα from hypoxia-ischemia-induced autophosphorylation damage. CONCLUSION: Overall, SHPL-49 enhanced neuronal survival and attenuated acute ischemic stroke by promoting the NR2A-CAMKⅡα-Akt/CREB pathway. Our study provides the first evidence demonstrating that the neuroprotective effect of SHPL-49 is achieved by promoting the NR2A subunit to extend the treatment time window, making it a promising drug for ischemic stroke.

Cytosolic calcium regulates hepatic mitochondrial oxidation, intrahepatic lipolysis, and gluconeogenesis via CAMKII activation.

To examine the roles of mitochondrial calcium Ca2+ ([Ca2+]mt) and cytosolic Ca2+ ([Ca2+]cyt) in the regulation of hepatic mitochondrial fat oxidation, we studied a liver-specific mitochondrial calcium uniporter knockout (MCU KO) mouse model with reduced [Ca2+]mt and increased [Ca2+]cyt content. Despite decreased [Ca2+]mt, deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux, α-ketoglutarate dehydrogenase flux, and succinate dehydrogenase flux in vivo. Rates of [14C16]palmitate oxidation and intrahepatic lipolysis were increased in MCU KO liver slices, which led to decreased hepatic triacylglycerol content. These effects were recapitulated with activation of CAMKII and abrogated with CAMKII knockdown, demonstrating that [Ca2+]cyt activation of CAMKII may be the primary mechanism by which MCU deletion promotes increased hepatic mitochondrial oxidation. Together, these data demonstrate that hepatic mitochondrial oxidation can be dissociated from [Ca2+]mt and reveal a key role for [Ca2+]cyt in the regulation of hepatic fat mitochondrial oxidation, intrahepatic lipolysis, gluconeogenesis, and lipid accumulation.

Lamin A/C deficiency-mediated ROS elevation contributes to pathogenic phenotypes of dilated cardiomyopathy in iPSC model.

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.

CACNA1H restrains chemotherapy resistance in ovarian clear cell carcinoma cells by repressing autophagy.

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.

TRPC6 regulates necroptosis in myocardial ischemia/reperfusion injury via Ca2+/CaMKII signaling pathway.

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.

CaMK II in Cardiovascular Diseases, Especially CaMK II-δ: Friends or Enemies.

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.