Search

The outgrowth and stabilization of nascent dendritic spines are crucial processes underlying learning and memory. Most new spines retract shortly after growth; only a small subset is stabilized and integrated into the new circuit connections that support learning. New spine stabilization has been shown to rely upon activity-dependent molecular mechanisms that also contribute to long-term potentiation (LTP) of synaptic strength. Indeed, disruption of the activity-dependent targeting of the kinase CaMKIIα to the GluN2B subunit of the NMDA-type glutamate receptor disrupts both LTP and activity-dependent stabilization of new spines. Yet it is not known which of CaMKIIα's many enzymatic and structural functions are important for new spine stabilization. Here, we used two-photon imaging and photolysis of caged glutamate to monitor the activity-dependent stabilization of new dendritic spines on hippocampal CA1 neurons from mice of both sexes in conditions where CaMKIIα functional and structural interactions were altered. Surprisingly, we found that inhibiting CaMKIIα kinase activity either genetically or pharmacologically did not impair activity-dependent new spine stabilization. In contrast, shRNA knockdown of CaMKIIα abolished activity-dependent new spine stabilization, which was rescued by co-expressing shRNA-resistant full-length CaMKIIα, but not by a truncated monomeric CaMKIIα. Notably, overexpression of phospho-mimetic CaMKIIα-T286D, which exhibits activity-independent targeting to GluN2B, enhanced basal new spine survivorship in the absence of additional glutamatergic stimulation, even when kinase activity was disrupted. Together, our results support a model in which nascent dendritic spine stabilization requires structural and scaffolding interactions mediated by dodecameric CaMKIIα that are independent of its enzymatic activities.

Rapid, synapse-specific neurotransmission requires the precise alignment of presynaptic neurotransmitter release and postsynaptic receptors. How postsynaptic glutamate receptor accumulation is induced during maturation is not well understood. We find that in cultures of dissociated hippocampal neurons at 11 days in vitro (DIV) numerous synaptic contacts already exhibit pronounced accumulations of the pre- and postsynaptic markers synaptotagmin, synaptophysin, synapsin, bassoon, VGluT1, PSD-95, and Shank. The presence of an initial set of AMPARs and NMDARs is indicated by miniature excitatory postsynaptic currents (mEPSCs). However, AMPAR and NMDAR immunostainings reveal rather smooth distributions throughout dendrites and synaptic enrichment is not obvious. We found that brief periods of Ca2+ influx through NMDARs induced a surprisingly rapid accumulation of NMDARs within 1 min, followed by accumulation of CaMKII and then AMPARs within 2-5 min. Postsynaptic clustering of NMDARs and AMPARs was paralleled by an increase in their mEPSC amplitudes. A peptide that blocked the interaction of NMDAR subunits with PSD-95 prevented the NMDAR clustering. NMDAR clustering persisted for 3 days indicating that brief periods of elevated glutamate fosters permanent accumulation of NMDARs at postsynaptic sites in maturing synapses. These data support the model that strong glutamatergic stimulation of immature glutamatergic synapses results in a fast and substantial increase in postsynaptic NMDAR content that required NMDAR binding to PSD-95 or its homologues and is followed by recruitment of CaMKII and subsequently AMPARs.

Introduction: The TrkB receptor is known for its role in regulating excitatory neuronal plasticity. However, accumulating evidence over the past decade has highlighted the involvement of TrkB in regulating inhibitory synapse stability and plasticity, particularly through regulation of the inhibitory scaffold protein gephyrin, although with contradicting results. Methods: In this study, we extended on these findings by overexpressing rat TrkB mutants deficient in either Shc-or PLCγ-dependent signaling, as well as a kinase-dead mutant, to dissect the contributions of specific TrkB-dependent signaling pathways to gephyrin clustering. Results: Our results demonstrate that TrkB signaling is required for gephyrin clustering on the perisomatic area of granule cells in the dentate gyrus in vivo. To further investigate, we expressed TrkB wild-type and mutants in hippocampal neurons in vitro. Discussion: Under basal conditions, TrkB-Shc signaling was important for the reduction of gephyrin cluster size, while TrkB-PLCγ signaling accounts for gephyrin clustering specifically at synaptic sites. Concomitant, impaired PLCγ signaling was associated with disinhibition of transduced neurons. Moreover, chemically induced inhibitory long-term potentiation (chem iLTP) depended on TrkB signaling and the activation of both Shc and PLCγ pathways. Conclusion: Our findings suggest a complex, pathway-specific regulation of TrkB-dependent gephyrin clustering, both under basal conditions and during chem iLTP. [ABSTRACT FROM AUTHOR]

Background: Doxorubicin (DOX) is an effective anticancer drug, but it has a problem of cardiotoxicity that cannot be ignored. Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is tightly associated with the pathological progression of DOX‐induced cardiotoxicity. Ubiquitin‐specific protease 10 (USP10) plays an important role in many biological processes and cancers. However, its association with DOX‐induced cardiotoxicity and CaMKII remains unclear. Methods: H9C2 cells, HL‐1 cells and C57BL/6 mice were used to establish the DOX‐induced cardiotoxicity model, and the CaMKII‐specific inhibitor KN‐93 and USP10 specific inhibitor Spautin‐1 were used to observe the CaMKII and USP10 effect. In cell experiments, CCK‐8 method was used to assess cell viability, LDH kit was used to assess lactate dehydrogenase expression, DCFH‐DA staining was used to observe changes in active oxygen content, TUNEL staining was used to observe cell apoptosis, and Western blotting method was used to detect relevant protein markers. The expression of p‐CaMKII and USP10 was assessed by immunofluorescence staining. In animal experiments, mouse echocardiograph was used were used to evaluate cardiac function, and HE staining and Masson staining were used to evaluate myocardial injury. Cardiomyocyte apoptosis was detected by TUNEL staining. Western blotting method was used to detect relevant protein markers. Results: Our results demonstrated that activation of CaMKII and inhibition of USP10 pathway related to DOX‐induced cardiotoxicity. Inhibition of CaMKII with KN‐93 ameliorated DOX‐induced cardiac dysfunction and cytotoxicity. In addition, CaMKII inhibition prevented DOX‐induced apoptosis and ubiquitination. Furthermore, CaMKII inhibition increased USP10 expression in DOX‐treated mouse hearts, H9C2 cells and HL‐1 cells. At last, the USP10 inhibitor, Spautin‐1, blocked the regulatory effect of CaMKII inhibition on apoptosis and ubiquitination in DOX‐induced cardiotoxicity. Conclusion: Our findings revealed that DOX‐induced myocardial apoptosis and activated CaMKII through cellular and animal levels, while providing a novel probe into the mechanism of CaMKII action: promoting ubiquitination by inhibiting USP10 aggravated apoptosis. [ABSTRACT FROM AUTHOR]

A pervasive exposure to stressors and the consumption of fructose-containing beverages usually go hand-in-hand in everyday life. In contrast to their metabolic outcomes, their impact on the brain and behavior is still understudied. We examined the behavioral response to a novelty (open field test), the expression of biochemical indicators of neuronal activity (Egr1 and FosB/ΔFosB), the synaptic potentiation (CaMKIIα and pCaMKIIThr286), the synaptic plasticity (synaptophysin, PSD95, gephyrin, and drebrin), and the GABAergic system (parvalbumin and GAD67), along with the glucocorticoid receptor (GR) and AMPK, in the medial prefrontal cortex of female Wistar rats subjected to liquid fructose supplementation (F), chronic unpredictable stress (S), or both (SF) over 9 weeks. The only hallmark of the F group was an increased expression of pCaMKIIThr286, which was also observed in the S group, but not in the SF group. The SF group did not show hyperactivity, a decreased expression of FosB, or an increased expression of parvalbumin, as the S group did. The SF group, as with the S group, showed a decreased expression of the GR, although the basal level of corticosterone was unchanged. The SF group showed, as de novo marks, thigmotactic behavior, increased drebrin, and decreased gephyrin expression. These findings suggest that the long-term consumption of fructose, which itself has subtle neurobehavioral consequences, in combination with stress prevents some of its effects, but also contributes to novel outcomes not seen in single treatments. [ABSTRACT FROM AUTHOR]

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF. [ABSTRACT FROM AUTHOR]

α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (AMPARs) mediate rapid excitatory synaptic transmission in the mammalian brain, primarily driven by the neurotransmitter glutamate. The modulation of AMPAR activity, particularly calcium‐permeable AMPARs (CP‐AMPARs), is crucially influenced by various auxiliary subunits. These subunits are integral membrane proteins that bind to the receptor's core and modify its functional properties, including ion channel kinetics and receptor trafficking. This review comprehensively catalogs all known AMPAR auxiliary proteins, providing vital insights into the biochemical mechanisms governing synaptic modulation and the specific impact of CP‐AMPARs compared to their calcium‐impermeable AMPA receptor (CI‐AMPARs). Understanding the complex interplay between AMPARs and their auxiliary subunits in different brain regions is essential for elucidating their roles in cognitive functions such as learning and memory. Importantly, alterations in these auxiliary proteins' expression, function or interactions have been implicated in various neurological disorders. Aberrant signaling through CP‐AMPARs, in particular, is associated with severe synaptic dysfunctions across neurodevelopmental, neurodegenerative and psychiatric conditions. Targeting the distinct properties of AMPAR‐auxiliary subunit complexes, especially those involving CP‐AMPARs, could disclose new therapeutic strategies, potentially allowing for more precise interventions in treating complex neuronal disorders. [ABSTRACT FROM AUTHOR]

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. Synopsis: CaMKII activates the HRI/eIF2α signaling pathway and inhibits the proteotoxicity induced by proteasome inhibition through phosphorylating BAG3. CaMKII suppresses proteotoxicity and paraptosis during proteasome inhibition. Proteasome inhibition induces CaMKII to phosphorylate BAG3 at S173/377/386. Phosphorylated BAG3 activates the HRI/eIF2α signaling pathway and inhibits the production of aggregated and misfolded proteins. CamKII inhibition enhances the anti-tumor activity of the proteasome inhibitor Bortezimib. CaMKII activates the HRI/eIF2α signaling pathway and inhibits the proteotoxicity induced by proteasome inhibition through phosphorylating BAG3. [ABSTRACT FROM AUTHOR]

Introduction: The prevalence of heart failure with preserved ejection fraction (HFpEF) is continuously rising and predominantly affects older women often hypertensive and/or obese or diabetic. Indeed, there is evidence on sex differences in the development of HF. Hence, we studied cardiovascular performance dependent on sex and age as well as pathomechanisms on a cellular and molecular level. Methods: We studied 15-week- and 1-year-old female and male hypertensive transgenic rats carrying the mouse Ren-2 renin gene (TG) and compared them to wild-type (WT) controls at the same age. We tracked blood pressure and cardiac function via echocardiography. After sacrificing the 1-year survivors we studied vascular smooth muscle and endothelial function. Isolated single skinned cardiomyocytes were used to determine passive stiffness and Ca2+-dependent force. In addition, Western blots were applied to analyse the phosphorylation status of sarcomeric regulatory proteins, titin and of protein kinases AMPK, PKG, CaMKII as well as their expression. Protein kinase activity assays were used to measure activities of CaMKII, PKG and angiotensin-converting enzyme (ACE). Results: TG male rats showed significantly higher mortality at 1 year than females or WT male rats. Left ventricular (LV) ejection fraction was specifically reduced in male, but not in female TG rats, while LV diastolic dysfunction was evident in both TG sexes, but LV hypertrophy, increased LV ACE activity, and reduced AMPK activity as evident from AMPK hypophosphorylation were specific to male rats. Sex differences were also observed in vascular and cardiomyocyte function showing different response to acetylcholine and Ca2+-sensitivity of force production, respectively cardiomyocyte functional changes were associated with altered phosphorylation states of cardiac myosin binding protein C and cardiac troponin I phosphorylation in TG males only. Cardiomyocyte passive stiffness was increased in TG animals. On a molecular level titin phosphorylation pattern was altered, though alterations were sex-specific. Thus, also the reduction of PKG expression and activity was more pronounced in TG females. However, cardiomyocyte passive stiffness was restored by PKG and CaMKII treatments in both TG sexes. Conclusion: Here we demonstrated divergent sex-specific cardiovascular adaptation to the over-activation of the renin-angiotensin system in the rat. Higher mortality of male TG rats in contrast to female TG rats was observed as well as reduced LV systolic function, whereas females mainly developed HFpEF. Though both sexes developed increased myocardial stiffness to which an impaired titin function contributes to a sex-specific molecular mechanism. The functional derangements of titin are due to a sex-specific divergent regulation of PKG and CaMKII systems. [ABSTRACT FROM AUTHOR]

Degenerative disc disease (DDD) represents a significant global health challenge, yet its underlying molecular mechanisms remain elusive. This study aimed to investigate the role of type 1 phosphatidylinositol 4‐phosphate 5‐kinase (Pip5k1) in intervertebral disc (IVD) homeostasis and disease. All three Pip5k1 isoforms, namely Pip5k1α, Pip5k1β, and Pip5k1γ, were detectable in mouse and human IVD tissues, with Pip5k1γ displaying a highest expression in nucleus pulposus (NP) cells. The expression of Pip5k1γ was significantly down‐regulated in the NP cells of aged mice and patients with severe DDD. To determine whether Pip5k1γ expression is required for disc homeostasis, we generated a Pip5k1γfl/fl; AggrecanCreERT2 mouse model for the conditional knockout of the Pip5k1γ gene in aggrecan‐expressing IVD cells. Our findings revealed that the conditional deletion of Pip5k1γ did not affect the disc structure or cellular composition in 5‐month‐old adult mice. However, in aged (15‐month‐old) mice, this deletion led to several severe degenerative disc defects, including decreased NP cellularity, spontaneous fibrosis and cleft formation, and a loss of the boundary between NP and annulus fibrosus. At the molecular level, the absence of Pip5k1γ reduced the anabolism of NP cells without markedly affecting their catabolic or anti‐catabolic activities. Moreover, the loss of Pip5k1γ significantly dampened the activation of the protective Ampk pathway in NP cells, thereby accelerating NP cell senescence. Notably, Pip5k1γ deficiency blunted the effectiveness of metformin, a potent Ampk activator, in activating the Ampk pathway and mitigating lumbar spine instability (LSI)‐induced disc lesions in mice. Overall, our study unveils a novel role for Pip5k1γ in promoting anabolism and maintaining disc homeostasis, suggesting it as a potential therapeutic target for DDD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptor (NMDAR) trafficking is a key process in the regulation of synaptic efficacy and brain function. However, the molecular mechanism underlying the surface transport of NMDARs is largely unknown. Here we identified myosin Va (MyoVa) as the specific motor protein that traffics NMDARs in hippocampal neurons. We found that MyoVa associates with NMDARs through its cargo binding domain. This association was increased during NMDAR surface transport. Knockdown of MyoVa suppressed NMDAR transport. We further demonstrated that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates NMDAR transport through its direct interaction with MyoVa. Furthermore, MyoVa employed Rab11 family-interacting protein 3 (Rab11/FIP3) as the adaptor proteins to couple themselves with NMDARs during their transport. Accordingly, the knockdown of FIP3 impairs hippocampal memory. Together, we conclude that in hippocampal neurons, MyoVa conducts active transport of NMDARs in a CaMKII-dependent manner. [ABSTRACT FROM AUTHOR]

Osteoblast differentiation and apoptosis are crucial in sustaining bone homeostasis, with deviations in these processes being implicated in a wide array of metabolic bone disorders. The calcium/calmodulin-dependent protein kinase II (CaMKII) family, a diverse and multifunctional group of protein kinases ubiquitous within the human body, plays a key role in the transducing intracellular Ca2+ signals into a broad range of cellular responses, both physiologically and pathologically. This transduction is pivotal in orchestrating the regulation of osteoblast differentiation and apoptosis. The influence of CaMKII in osteoblast differentiation is mediated predominantly via three signal transduction pathways: 1) modulation of osteoblast differentiation through the 1α-25(OH)2D3 membrane-mediated signaling pathway; 2) participation in the WNT/Ca2+ signaling cascade; and 3) exerting control over osteoblast differentiation via the RUNX2-OSX signaling axis. These intertwined pathways collaboratively contribute to the progression and functional efficacy of osteoblasts. Regarding osteoblast apoptosis, the role of CaMKII extends to adjusting the endoplasmic reticulum stress response and to managing the Bax/Bcl-2 expression ratio. Such regulatory mechanisms are essential for both the survival and programmed cell death of osteoblasts, thereby influencing the integrity and rejuvenation of bone tissue. In summary, CaMKII, as a versatile signaling entity, occupies a central position in the modulation of osteoblast differentiation and apoptosis. Advancing our understanding of CaMKII, especially its potential therapeutic applications in the realm of metabolic bone diseases, emerges as a paramount avenue for forthcoming research endeavors. [ABSTRACT FROM AUTHOR]

Brazilian green propolis (propolis) is a chemically complex resinous substance that is a potentially viable therapeutic agent for Alzheimer's disease. Herein, propolis induced a transient increase in intracellular Ca2+ concentration ([Ca2+] i) in Neuro-2A cells; moreover, propolis-induced [Ca2+] i elevations were suppressed prior to 24-h pretreatment with amyloid-β. To reveal the effect of [Ca2+] i elevation on impaired cognition, we performed memory-related behavioral tasks in APP-KI mice relative to WT mice at 4 and 12 months of age. Propolis, at 300–1000 mg/kg/d for 8 wk, significantly ameliorated cognitive deficits in APP-KI mice at 4 months, but not at 12 months of age. Consistent with behavioral observations, injured hippocampal long-term potentiation was markedly ameliorated in APP-KI mice at 4 months of age following repeated propolis administration. In addition, repeated administration of propolis significantly activated intracellular calcium signaling pathway in the CA1 region of APP-KI mice. These results suggest a preventive effect of propolis on cognitive decline through the activation of intracellular calcium signaling pathways in CA1 region of AD mice model. • Application of propolis induces a rise in [Ca2+] i levels. • Administration of propolis ameliorates the cognitive decline in APP-KI mice. • Impaired hippocampal LTP was ameliorated in propolis-treated APP-KI mice. • Propolis indicates preventive effects on early-onset of AD. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of target protein phosphorylation. Thus, auto-phosphorylation and binding to the GluN2B subunit are the only two requirements for CaMKII in synaptic memory. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Doxorubicin (DOX) is an effective anticancer drug, but it has a problem of cardiotoxicity that cannot be ignored. Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is tightly associated with the pathological progression of DOX‐induced cardiotoxicity. Ubiquitin‐specific protease 10 (USP10) plays an important role in many biological processes and cancers. However, its association with DOX‐induced cardiotoxicity and CaMKII remains unclear. Methods H9C2 cells, HL‐1 cells and C57BL/6 mice were used to establish the DOX‐induced cardiotoxicity model, and the CaMKII‐specific inhibitor KN‐93 and USP10 specific inhibitor Spautin‐1 were used to observe the CaMKII and USP10 effect. In cell experiments, CCK‐8 method was used to assess cell viability, LDH kit was used to assess lactate dehydrogenase expression, DCFH‐DA staining was used to observe changes in active oxygen content, TUNEL staining was used to observe cell apoptosis, and Western blotting method was used to detect relevant protein markers. The expression of p‐CaMKII and USP10 was assessed by immunofluorescence staining. In animal experiments, mouse echocardiograph was used were used to evaluate cardiac function, and HE staining and Masson staining were used to evaluate myocardial injury. Cardiomyocyte apoptosis was detected by TUNEL staining. Western blotting method was used to detect relevant protein markers. Results Our results demonstrated that activation of CaMKII and inhibition of USP10 pathway related to DOX‐induced cardiotoxicity. Inhibition of CaMKII with KN‐93 ameliorated DOX‐induced cardiac dysfunction and cytotoxicity. In addition, CaMKII inhibition prevented DOX‐induced apoptosis and ubiquitination. Furthermore, CaMKII inhibition increased USP10 expression in DOX‐treated mouse hearts, H9C2 cells and HL‐1 cells. At last, the USP10 inhibitor, Spautin‐1, blocked the regulatory effect of CaMKII inhibition on apoptosis and ubiquitination in DOX‐induced cardiotoxicity. Conclusion Our findings revealed that DOX‐induced myocardial apoptosis and activated CaMKII through cellular and animal levels, while providing a novel probe into the mechanism of CaMKII action: promoting ubiquitination by inhibiting USP10 aggravated apoptosis.

IntroductionThe TrkB receptor is known for its role in regulating excitatory neuronal plasticity. However, accumulating evidence over the past decade has highlighted the involvement of TrkB in regulating inhibitory synapse stability and plasticity, particularly through regulation of the inhibitory scaffold protein gephyrin, although with contradicting results.MethodsIn this study, we extended on these findings by overexpressing rat TrkB mutants deficient in either Shc-or PLCγ-dependent signaling, as well as a kinase-dead mutant, to dissect the contributions of specific TrkB-dependent signaling pathways to gephyrin clustering.ResultsOur results demonstrate that TrkB signaling is required for gephyrin clustering on the perisomatic area of granule cells in the dentate gyrus in vivo. To further investigate, we expressed TrkB wild-type and mutants in hippocampal neurons in vitro.DiscussionUnder basal conditions, TrkB-Shc signaling was important for the reduction of gephyrin cluster size, while TrkB-PLCγ signaling accounts for gephyrin clustering specifically at synaptic sites. Concomitant, impaired PLCγ signaling was associated with disinhibition of transduced neurons. Moreover, chemically induced inhibitory long-term potentiation (chem iLTP) depended on TrkB signaling and the activation of both Shc and PLCγ pathways.ConclusionOur findings suggest a complex, pathway-specific regulation of TrkB-dependent gephyrin clustering, both under basal conditions and during chem iLTP.

Abstract Background Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. Methods We compared changes in NMJs and activity-dependent signalling pathways in HSA LR and Mbnl1 ΔE3/ΔE3 mice, two established mouse models of DM1. Results Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIβ/βM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1 ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. Conclusions Our results indicate that CaMKIIβ-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

Abstract Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a family of broad substrate specificity serine (Ser)/threonine (Thr) protein kinases that play a crucial role in the Ca2+-dependent signaling pathways. Its significance as an intracellular Ca2+ sensor has garnered abundant research interest in the domain of neurodegeneration. Accumulating evidences suggest that CaMKII is implicated in the pathology of degenerative retinopathies such as diabetic retinopathy (DR), age-related macular degeneration (AMD), retinitis pigmentosa (RP) and glaucoma optic neuropathy. CaMKII can induce the aberrant proliferation of retinal blood vessels, influence the synaptic signaling, and exert dual effects on the survival of retinal ganglion cells and pigment epithelial cells. Researchers have put forth multiple therapeutic agents, encompassing small molecules, peptides, and nucleotides that possess the capability to modulate CaMKII activity. Due to its broad range isoforms and splice variants therapeutic strategies seek to inhibit specifically the CaMKII are confronted with considerable challenges. Therefore, it becomes crucial to discern the detrimental and advantageous aspects of CaMKII, thereby facilitating the development of efficacious treatment. In this review, we summarize recent research findings on the cellular and molecular biology of CaMKII, with special emphasis on its metabolic and regulatory mechanisms. We delve into the involvement of CaMKII in the retinal signal transduction pathways and discuss the correlation between CaMKII and calcium overload. Furthermore, we elaborate the therapeutic trials targeting CaMKII, and introduce recent developments in the zone of CaMKII inhibitors. These findings would enrich our knowledge of CaMKII, and shed light on the development of a therapeutic target for degenerative retinopathy.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study ‘disease in a dish’. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia. [ABSTRACT FROM AUTHOR]

Background: Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. Methods: We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1ΔE3/ΔE3 mice, two established mouse models of DM1. Results: Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIβ/βM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. Conclusions: Our results indicate that CaMKIIβ-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a family of broad substrate specificity serine (Ser)/threonine (Thr) protein kinases that play a crucial role in the Ca2+-dependent signaling pathways. Its significance as an intracellular Ca2+ sensor has garnered abundant research interest in the domain of neurodegeneration. Accumulating evidences suggest that CaMKII is implicated in the pathology of degenerative retinopathies such as diabetic retinopathy (DR), age-related macular degeneration (AMD), retinitis pigmentosa (RP) and glaucoma optic neuropathy. CaMKII can induce the aberrant proliferation of retinal blood vessels, influence the synaptic signaling, and exert dual effects on the survival of retinal ganglion cells and pigment epithelial cells. Researchers have put forth multiple therapeutic agents, encompassing small molecules, peptides, and nucleotides that possess the capability to modulate CaMKII activity. Due to its broad range isoforms and splice variants therapeutic strategies seek to inhibit specifically the CaMKII are confronted with considerable challenges. Therefore, it becomes crucial to discern the detrimental and advantageous aspects of CaMKII, thereby facilitating the development of efficacious treatment. In this review, we summarize recent research findings on the cellular and molecular biology of CaMKII, with special emphasis on its metabolic and regulatory mechanisms. We delve into the involvement of CaMKII in the retinal signal transduction pathways and discuss the correlation between CaMKII and calcium overload. Furthermore, we elaborate the therapeutic trials targeting CaMKII, and introduce recent developments in the zone of CaMKII inhibitors. These findings would enrich our knowledge of CaMKII, and shed light on the development of a therapeutic target for degenerative retinopathy. [ABSTRACT FROM AUTHOR]

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

Background: Noncanonical Wnts are morphogens that can elevate intracellular Ca2+, activate the Ca2+/calmodulin‐dependent protein kinase, CaMKII, and promote cell movements during vertebrate gastrulation. Results: Zebrafish express seven CaMKII genes during embryogenesis; two of these, camk2b1 and camk2g1, are necessary for convergent extension (CE) cell movements. CaMKII morphant phenotypes were observed as early as epiboly. At the 1–3 somite stage, neuroectoderm and paraxial cells remained unconverged in both morphants. Later, somites lacked their stereotypical shape and were wider, more closely spaced, and body gap angles increased. At 24hpf, somite compression and notochord undulation coincided with a shorter and broader body axis. A camk2b1 crispant was generated which phenocopied the camk2b1 morphant. The levels of cell proliferation, apoptosis and paraxial and neuroectodermal markers were unchanged in morphants. Hyperactivation of CaMKII during gastrulation by transient pharmacological intervention (thapsigargin) also caused CE defects. Mosaically expressed dominant‐negative CaMKII recapitulated these phenotypes and showed significant midline bifurcation. Finally, the introduction of CaMKII partially rescued Wnt11 morphant phenotypes. Conclusions: Overall, these data support a model whereby cyclically activated CaMKII encoded from two genes enables cell migration during the process of CE. Key Findings: CaMKII morphants phenocopy Wnt11 CE defects.CaMKII rescues Wnt11 morphants.Hyperactivated CaMKII induces CE defects.Dominant negative CaMKII causes midline bifurcation.CaMKII crispant phenocopies morphant. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a multidomain serine/threonine kinase that plays important roles in the brain, heart, muscle tissue, and eggs/sperm. The N‐terminal kinase and regulatory domain is connected by a flexible linker to the C‐terminal hub domain. The hub domain drives the oligomeric organization of CaMKII, assembling the kinase domains into high local concentration. Previous structural studies have shown multiple stoichiometries of the holoenzyme as well as the hub domain alone. Here, we report a comprehensive study of the hub domain stoichiometry and stability in solution. We solved two crystal structures of the CaMKIIβ hub domain that show 14‐mer (3.1 Å) and 16‐mer (3.4 Å) assemblies. Both crystal structures were determined from crystals grown in the same drop, which suggests that CaMKII oligomers with different stoichiometries likely coexist. To further interrogate hub stability, we employed mass photometry and temperature denaturation studies of CaMKIIβ and CaMKIIα hubs, which highlight major differences between these highly similar domains. We created a dimeric CaMKIIβ hub unit using rational mutagenesis, which is significantly less stable than the oligomer. Both hub domains populate an intermediate during unfolding. We found that multiple CaMKIIβ hub stoichiometries are present in solution and that larger oligomers are more stable. CaMKIIα had a narrower distribution of molecular weight and was distinctly more stable than CaMKIIβ. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a metabolic disorder denoted by chronic hyperglycemia that drives maladaptive structural changes and functional damage to the vasculature. Attenuation of this pathological remodeling of blood vessels remains an unmet target owing to paucity of information on the metabolic signatures of this process. Ca2+/calmodulin-dependent kinase II (CaMKII) is expressed in the vasculature and is implicated in the control of blood vessels homeostasis. Recently, CaMKII has attracted a special attention in view of its chronic upregulated activity in diabetic tissues, yet its role in the diabetic vasculature remains under investigation. This review highlights the physiological and pathological actions of CaMKII in the diabetic vasculature, with focus on the control of the dialogue between endothelial (EC) and vascular smooth muscle cells (VSMC). Activation of CaMKII enhances EC and VSMC proliferation and migration, and increases the production of extracellular matrix which leads to maladaptive remodeling of vessels. This is manifested by activation of genes/proteins implicated in the control of the cell cycle, cytoskeleton organization, proliferation, migration, and inflammation. Endothelial dysfunction is paralleled by impaired nitric oxide signaling, which is also influenced by CaMKII signaling (activation/oxidation). The efficiency of CaMKII inhibitors is currently being tested in animal models, with a focus on the genetic pathways involved in the regulation of CaMKII expression (microRNAs and single nucleotide polymorphisms). Interestingly, studies highlight an interaction between the anti-diabetic drugs and CaMKII expression/activity which requires further investigation. Together, the studies reviewed herein may guide pharmacological approaches to improve health-related outcomes in patients with diabetes. [ABSTRACT FROM AUTHOR]

Early life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. Male Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. We report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. The study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. Double-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone. [ABSTRACT FROM AUTHOR]

Purpose: It is well known that inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) provides cardiac protection in cases of myocardial ischemia–reperfusion injury. However, there are currently no cytoplasm-impermeable drugs that target CaMKII. The aim of this study was to develop curcumin albumin nanoparticles (HSA-CCM NPs) containing AC3-I and investigate their protective effects on hypoxia–reoxygenation (H/R)-induced injuries in adult rat cardiomyocytes and ischemia–reperfusion (I/R) injuries in isolated rat hearts. Methods: HSA-CCM NPs were synthesized using β-ME methods, while the membrane-impermeable peptide AC3-I was covalently linked via a disulfide bond to synthesize AC3-I@HSA-CCM NPs (AC3-I@NPs). Nanoparticle stability and drug release were characterized. To assess the cardiomyocyte uptake of AC3-I@NPs, AC3-I@NPs were incubated with cardiomyocytes under normoxia and hypoxia, respectively. The cardioprotective effect of AC3-I@NPs was determined by using a lactate dehydrogenase kit (LDH) and PI/Hoechst staining. The phosphorylation of phospholamban (p-PLB) was detected by Western blotting in hypoxia–reoxygenation and electric field stimulation models. To further investigate the protective role of AC3-I@NPs against myocardial ischemia–reperfusion injury, we collected coronary effluents and measured creatine kinase (CK) and LDH release in Langendorff rat hearts. Results:AC3-I@NPs were successfully prepared and characterized. Both HSA-CCM NPs and AC3-I@NPs were taken up by cardiomyocytes. AC3-I@NPs protected cardiomyocytes from injury caused by hypoxia–reoxygenation, as demonstrated by decreased cardiomyocyte death and LDH release. AC3-I@NPs reduced p-PLB levels evoked by hypoxia–reoxygenation and electrical field stimulation in adult rat cardiac myocytes. AC3-I@NPs decreased the release of LDH and CK from coronary effluents. Conclusions: AC3-I@NPs showed protective effects against myocardial injuries induced by hypoxia–reoxygenation in cardiomyocytes and ischemia–reperfusion in isolated hearts. [ABSTRACT FROM AUTHOR]

Aims: Major depressive disorder is a severe psychiatric disorder that afflicts ~17% of the world population. Neuroimaging investigations of depressed patients have consistently reported the dysfunction of the basolateral amygdala in the pathophysiology of depression. However, how the BLA and related circuits are implicated in the pathogenesis of depression is poorly understood. Methods: Here, we combined fiber photometry, immediate early gene expression (c‐fos), optogenetics, chemogenetics, behavioral analysis, and viral tracing techniques to provide multiple lines of evidence of how the BLA neurons mediate depressive‐like behavior. Results: We demonstrated that the aversive stimuli elevated the neuronal activity of the excitatory BLA neurons (BLACAMKII neurons). Optogenetic activation of CAMKII neurons facilitates the induction of depressive‐like behavior while inhibition of these neurons alleviates the depressive‐like behavior. Next, we found that the chemogenetic inhibition of GABAergic neurons in the BLA (BLAGABA) increased the firing frequency of CAMKII neurons and mediates the depressive‐like phenotypes. Finally, through fiber photometry recording and chemogenetic manipulation, we proved that the activation of BLAGABA neurons inhibits BLACAMKII neuronal activity and alleviates depressive‐like behavior in the mice. Conclusion: Thus, through evaluating BLAGABA and BLACAMKII neurons by distinct interaction, the BLA regulates depressive‐like behavior. [ABSTRACT FROM AUTHOR]

Background Doxorubicin (Dox) is effective against different types of cancers, but it poses cardiotoxic side effects, frequently resulting in irreversible heart failure. However, the complexities surrounding this cardiotoxicity, especially at sublethal dosages, remain to be fully elucidated. We investigated early cellular disruptions in response to sublethal Dox, with a specific emphasis on the role of phosphorylated calcium/calmodulindependent protein kinase II (CaMKII) in initiating mitochondrial dysfunction. Methods This study utilized the H9c2 cardiomyocyte model to identify a sublethal concentration of Dox and investigate its impact on mitochondrial health using markers such as mitochondrial membrane potential (MMP), mitophagy initiation, and mitochondrial calcium dynamics. We examined the roles of and interactions between CaMKII, dynamin-related protein 1 (Drp1), and the mitochondrial calcium uniporter (MCU) in Dox-induced mitochondrial disruption using specific inhibitors, such as KN-93, Mdivi-1, and Ru360, respectively. Results Exposure to a sublethal dose of Dox reduced the MMP red-to-green fluorescence ratio in H9c2 cells by 40.6% compared with vehicle, and increased the proportion of cells undergoing mitophagy from negligible levels compared with vehicle to 62.2%. Mitochondrial calcium levels also increased by 8.7-fold compared with the vehicle group. Notably, the activation of CaMKII, particularly its phosphorylated form, was pivotal in driving these mitochondrial changes, as inhibition using KN-93 restored MMP and decreased mitophagy. However, inhibition of Drp1 and MCU functions had a limited impact on the observed mitochondrial disruptions. Conclusion Sublethal administration of Dox is closely linked to CaMKII activation through phosphorylation, emphasizing its pivotal role in early mitochondrial disruption. These findings present a promising direction for developing therapeutic strategies that may alleviate the cardiotoxic effects of Dox, potentially increasing its clinical efficacy. [ABSTRACT FROM AUTHOR]

BackgroundAcute stimulation of the late sodium current (INaL) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient upon oxidative stress.ObjectivesWe aimed to investigate whether the increase of the systolic Ca2+ transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa through oxidized PKARIα.MethodsWe used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca2+ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and PKA target structures was studied using Western blot analysis.ResultsATX-II increased INaL to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations during Ca2+ transient decay and by diastolic SR Ca2+-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of ICa in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment.ConclusionsThe inotropic and arrhythmogenic effects of acutely increased INaL are associated with elevated ROS, but do not involve oxidation of PKARIα.

Metabolic syndrome (MetS) is characterized by insulin resistance, hyperglycemia, excessive fat accumulation and dyslipidemia, and is known to be accompanied by neuropathological symptoms such as memory loss, anxiety, and depression. As the number of MetS patients is rapidly increasing globally, studies on the mechanisms of metabolic imbalance-related neuropathology are emerging as an important issue. Ca2+/calmodulin-dependent kinase II (CaMKII) is the main Ca2+ sensor and contributes to diverse intracellular signaling in peripheral organs and the central nervous system (CNS). CaMKII exerts diverse functions in cells, related to mechanisms such as RNA splicing, reactive oxygen species (ROS) generation, cytoskeleton, and protein-protein interactions. In the CNS, CaMKII regulates vascular function, neuronal circuits, neurotransmission, synaptic plasticity, amyloid beta toxicity, lipid metabolism, and mitochondrial function. Here, we review recent evidence for the role of CaMKII in neuropathologic issues associated with metabolic disorders.

Dendritic spines are unique postsynaptic structures that emerge from the dendrites of neurons. They undergo activity-dependent morphological changes known as structural plasticity. The changes involve actin cytoskeletal remodeling, which is regulated by actin-binding proteins. CaMKII is a crucial molecule in synaptic plasticity. Notably, CaMKIIβ subtype is known to bind to filamentous-actin and is closely involved in structural plasticity. We have shown that CaMKIIβ binds to drebrin, and is localized in spines as both drebrin-dependent and drebrin-independent pools. However, the nanoscale relationship between drebrin and CaMKIIβ within dendritic spines has not been clarified. In this study, we used stochastic optical reconstruction microscopy (STORM) to examine the detailed localization of these proteins. STORM imaging showed that CaMKIIβ co-localized with drebrin in the core region of spines, and localized in the submembrane region of spines without drebrin. Interestingly, the dissociation of CaMKIIβ and drebrin in the core region was induced by NMDA receptor activation. In drebrin knockdown neurons, CaMKIIβ was decreased in the core region but not in the submembrane region. Together it indicates that the clustering of CaMKIIβ in the spine core region is dependent on drebrin. These findings suggest that drebrin-dependent CaMKIIβ is in a standby state before its activation.

Objective To investigate the relativity between levels of activated transcription factor 6 (ATF6) or calcium / calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), and coronary heart disease (CHD) and the degree of coronary artery stenosis. To study their predictive value on CHD. Methods We selected 265 patients with CHD who had undergone coronary angiography, and collected their clinical data. According to the coronary angiographic results and the median of Gensini Score (38 points), these patients were divided into mild stenosis group (Gensini Score≤38 points, n = 105), moderate and severe stenosis group (Gensini Score>38 points, n = 100); those with normal coronary angiographic results were enrolled into control group (n = 60). The expression levels of ATF6 and CaMKⅡ were observed and statistically analyzed among the three groups. Results Compared with the control group, the levels of serum ATF6 and CaMKⅡ in the mild stenosis group, and moderate and severe stenosis group were both significantly higher; the levels increased with the increasing degree of coronary artery stenosis, with statistically significant differences (P<0. 05). According to Logistic regression analysis, ATF6 and CaMKⅡ may be independent risk factors for coronary heart disease. ROC curve analysis showed that the AUC value of the combination of serum ATF6 and CaMK Ⅱ in predicting CHD was 0. 749 ( 95% CI 0. 676 0. 822), with a sensitivity of 0. 663 and a specificity of 0. 717. Its diagnostic efficiency was higher than that of either of ATF6 or CaMKⅡ alone. Conclusion The levels of serum ATF6 and CaMKⅡ are positively correlated with the degree of coronary artery stenosis. The levels of ATF6 and CaMKⅡ have predictive value for CHD, which are expected to provide more references for diagnosing CHD and evaluating its severity [ABSTRACT FROM AUTHOR]

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF.

Dendritic spines are unique postsynaptic structures that emerge from the dendrites of neurons. They undergo activity-dependent morphological changes known as structural plasticity. The changes involve actin cytoskeletal remodeling, which is regulated by actin-binding proteins. CaMKII is a crucial molecule in synaptic plasticity. Notably, CaMKIIβ subtype is known to bind to filamentous-actin and is closely involved in structural plasticity. We have shown that CaMKIIβ binds to drebrin, and is localized in spines as both drebrin-dependent and drebrin-independent pools. However, the nanoscale relationship between drebrin and CaMKIIβ within dendritic spines has not been clarified. In this study, we used stochastic optical reconstruction microscopy (STORM) to examine the detailed localization of these proteins. STORM imaging showed that CaMKIIβ co-localized with drebrin in the core region of spines, and localized in the submembrane region of spines without drebrin. Interestingly, the dissociation of CaMKIIβ and drebrin in the core region was induced by NMDA receptor activation. In drebrin knockdown neurons, CaMKIIβ was decreased in the core region but not in the submembrane region. Together it indicates that the clustering of CaMKIIβ in the spine core region is dependent on drebrin. These findings suggest that drebrin-dependent CaMKIIβ is in a standby state before its activation. • CaMKIIβ localized in the core of dendritic spines in a drebrin-dependent manner. • CaMKIIβ was not colocalized with drebrin in submembrane region. • The interaction between CaMKIIβ and drebrin was abolished by NMDA receptor activation. [ABSTRACT FROM AUTHOR]

Evidence has suggested that addiction and memory systems are related, but the signaling cascades underlying this interaction have not been completelyealed yet. The importance of calcium-calmodulin-dependent protein kinase II (CaMKII) and brain-derived neurotrophic factor (BDNF) in the memory processes and also in drug addiction has been previously established. In this present investigation, we examined the effects of repeated morphine pretreatment on impairment of spatial learning and memory acquisition induced by systemic ethanol administration in adult male rats. Also, we assessed how these drug exposures influence the expression level of CaMKII and BDNF in the hippocampus and amygdala. Animals were trained by a single training session of 8 trials, and a probe test containing a 60-s free-swim without a platform was administered 24 h later. Before training trials, rats were treated with a once-daily subcutaneous morphine injection for 3 days followed by a 5-day washout period. The results showed that pre-training ethanol (1 g/kg) impaired spatial learning and memory acquisition and down-regulated the mRNA expression of CaMKII and BDNF. The amnesic effect of ethanol was suppressed in morphine- (15 mg/kg/day) pretreated animals. Furthermore, the mRNA expression level of CaMKII and BDNF increased significantly following ethanol administration in morphine-pretreated rats. Conversely, this improvement in spatial memory acquisition was prevented by daily subcutaneous administration of naloxone (2 mg/kg) 15 min prior to morphine administration. Our findings suggest that sub-chronic morphine treatment reverses ethanol-induced spatial memory impairment, which could be explained by modulating CaMKII and BDNF mRNA expressions in the hippocampus and amygdala. [Display omitted] • Pre-training acute administration of ethanol impaired spatial memory. • Amnesia induced by ethanol was decreased by morphine pre-exposure. • Ethanol increased CaMKII in the hippocampus and amygdala in morphine-pretreated rats. • Ethanol increased BDNF in the hippocampus and amygdala in morphine-pretreated rats. [ABSTRACT FROM AUTHOR]

• The phosphorylation of 4.1N is regulated by activated CaMKII during TBS-LTP. • The binding of GluA1 and 4.1N is critical to TBS-LTP. • The formation of p-CaMKII-4.1N-GluA1 results in GluA1 trafficking to the postsynaptic membrane of neurons during TBS-LTP. GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) inserted into postsynaptic membranes are key to the process of long-term potentiation (LTP). Some evidence has shown that 4.1N plays a critical role in the membrane trafficking of AMPARs. However, the underlying mechanism behind this is still unclear. We investigated the role of 4.1N-mediated membrane trafficking of AMPARs during theta-burst stimulation long-term potentiation (TBS-LTP), to illustrate the molecular mechanism behind LTP. LTP was induced by TBS in rat hippocampal CA1 neuron. Tat-GluA1 (MPR), which disrupts the association of 4.1N-GluA1, and autocamtide-2-inhibitory peptide, myristoylated (Myr-AIP), a CaMKII antagonist, were used to explore the role of 4.1N in the AMPARs trafficking during TBS-induced LTP. Immunoprecipitation (IP) and immunoblotting (IB)were used to detect protein expression, phosphorylation, and the interaction of p-CaMKII-4.1N-GluA1. We found that Myr-AIP attenuated increases of p-CaMKII (T286), p-GluA1 (ser831), and 4.1N phosphorylation after TBS-LTP, and decreased the association of p-CaMKII-4.1N-GluA1, along with the expression of GluA1, at postsynaptic densities during TBS-LTP. We also designed interfering peptides to disrupt the interaction between 4.1N and GluA1, which showed that Tat-GluA1 (MPR) or Myr-AIP inhibited TBS-LTP and attenuated increases of GluA1 at postsynaptic sites, while Tat-GluA1 (MPR) or Myr-AIP had no effects on miniature excitatory postsynaptic currents (mEPSCs) in non-stimulated hippocampal CA1 neurons. Active CaMKII enhanced the phosphorylation of 4.1N and facilitated the association of p-CaMKII with 4.1N-GluA1, which in turn resulted in GluA1 trafficking during TBS-LTP. The association of 4.1N-GluA1 is required for LTP, but not for basal synaptic transmission. [ABSTRACT FROM AUTHOR]

The deposition of cholesterol-rich lipoproteins in the arterial wall triggers macrophage inflammatory responses, which promote atherosclerosis. The NLRP3 inflammasome aggravates atherosclerosis; however, cellular mechanisms connecting macrophage cholesterol accumulation to inflammasome activation are poorly understood. We investigated the mechanisms of NLRP3 inflammasome activation in cholesterol-loaded macrophages and in atherosclerosis-prone Ldlr−/− mice with defects in macrophage cholesterol efflux. We found that accumulation of cholesterol in macrophages treated with modified LDL or cholesterol crystals, or in macrophages defective in the cholesterol efflux promoting transporters ABCA1 and ABCG1, leads to activation of NLRP3 inflammasomes as a result of increased cholesterol trafficking from the plasma membrane to the ER, via Aster-B. In turn, the accumulation of cholesterol in the ER activates the inositol triphosphate-3 receptor, CaMKII/JNK, and induces NLRP3 deubiquitylation by BRCC3. An NLRP3 deubiquitylation inhibitor or deficiency of Abro1, an essential scaffolding protein in the BRCC3-containing cytosolic complex, suppressed inflammasome activation, neutrophil extracellular trap formation (NETosis), and atherosclerosis in vivo. These results identify a link between the trafficking of cholesterol to the ER, NLRP3 deubiquitylation, inflammasome activation, and atherosclerosis.

Rapid, synapse-specific neurotransmission requires the precise alignment of presynaptic neurotransmitter release and postsynaptic receptors. How postsynaptic glutamate receptor accumulation is induced during maturation is not well understood. We find that in cultures of dissociated hippocampal neurons at 11 days in vitro (DIV) numerous synaptic contacts already exhibit pronounced accumulations of the pre- and postsynaptic markers synaptotagmin, synaptophysin, synapsin, bassoon, VGluT1, PSD-95, and Shank. The presence of an initial set of AMPARs and NMDARs is indicated by miniature excitatory postsynaptic currents (mEPSCs). However, AMPAR and NMDAR immunostainings reveal rather smooth distributions throughout dendrites and synaptic enrichment is not obvious. We found that brief periods of Ca2+ influx through NMDARs induced a surprisingly rapid accumulation of NMDARs within 1 min, followed by accumulation of CaMKII and then AMPARs within 2–5 min. Postsynaptic clustering of NMDARs and AMPARs was paralleled by an increase in their mEPSC amplitudes. A peptide that blocked the interaction of NMDAR subunits with PSD-95 prevented the NMDAR clustering. NMDAR clustering persisted for 3 days indicating that brief periods of elevated glutamate fosters permanent accumulation of NMDARs at postsynaptic sites in maturing synapses. These data support the model that strong glutamatergic stimulation of immature glutamatergic synapses results in a fast and substantial increase in postsynaptic NMDAR content that required NMDAR binding to PSD-95 or its homologues and is followed by recruitment of CaMKII and subsequently AMPARs.

Background: Diabetic cardiomyopathy is one common cardiovascular complication without effective treatments. Dihydromyricetin (DHY), a natural dihydroflavonol compound extracted from Ampelopsis grossedentata, possesses versatile pharmacologically important effects. In our current research, we planned to evaluate the impact and probable DHY mechanisms in high glucose (HG)-induced cardiomyocytes. Methods: Primary cardiomyocytes were pretreated with different concentrations of DHY (0, 20, 40, 80, 160, and 320 μM) for various time (0, 1, 2, 4, 12, and 24 h). They were then stimulated for 48 h with 5.5 mmol/L normal glucose (NG) and 33.3 mmol/L high glucose (HG). Cell viability, adenosine-triphosphate (ATP) levels, and lactate dehydrogenase (LDH) release of cardiomyocytes were detected. JC-1 staining was employed to measure the mitochondrial membrane potential. MitoSOX staining and dihydroethidium (DHE) staining were applied to evaluate the oxidative stress levels. TDT mediated dUTP nick end labeling (TUNEL) was used to measure apoptotic levels. Expressions of calcium/calmodulin-dependent protein kinase II (CaMKII), phospholamban (PLB), optic atrophy 1 (OPA1), dynamin-related protein 1 (DRP1), caspase 3, mixed kinase lineage domain like protein (MLKL), receptor interacting protein kinase 3 (RIPK3), and receptor interacting protein kinase 1 (RIPK1) were detected by immunofluorescence and/or Western blot. Results: DHY improved cell viability, enhanced ATP level, and decreased LDH content in HG-stimulated cardiomyocytes, suggesting DHY attenuating cell injury. DHY reduced number of TUNEL positive cells, inhibited RIPK3 and cleaved-caspase 3 expression, implying DHY alleviated necroptosis in HG-stimulated cardiomyocytes. DHY diminished JC-1 monomers, DHE and MitoSOX fluorescence intensity as well as DRP1 expression but increased JC-1 aggregates intensity and OPA1 expression, indicating that DHY attenuated oxidative stress in HG-stimulated cardiomyocytes. DHY also attenuated CaMKII activity by suppressed PLB phosphorylation and inhibited CaMKII oxidation in HG-stimulated cardiomyocytes. Conclusions: HG-induced cardiomyocytes injury was alleviated wherein DHY attenuated necroptosis, repressed ROS production, and inhibited CaMKII oxidation, suggesting that DHY may serve as potential agent to prevent and treat diabetic cardiomyopathy.

Abstract Background Glucose fluctuations (GF) are a risk factor for cardiovascular complications associated with type 2 diabetes. However, there is a lack of adequate research on the effect of GF on myocardial fibrosis and the underlying mechanisms in type 2 diabetes. This study aimed to investigate the impact of glucose fluctuations on myocardial fibrosis and explore the potential mechanisms in type 2 diabetes. Methods Sprague Dawley (SD) rats were randomly divided into three groups: the control (Con) group, the type 2 diabetic (DM) group and the glucose fluctuations (GF) group. The type 2 diabetic rat model was established using a high-fat diet combined with low-dose streptozotocin injection and the GF model was induced by using staggered glucose and insulin injections daily. After eight weeks, echocardiography was used to assess the cardiac function of the three groups. Hematoxylin-eosin and Masson staining were utilized to evaluate the degree of pathological damage and fibrosis. Meanwhile, a neonatal rat cardiac fibroblast model with GF was established. Western and immunofluorescence were used to find the specific mechanism of myocardial fibrosis caused by GF. Results Compared with rats in the Con and the DM group, cardiac function in the GF group showed significant impairments. Additionally, the results showed that GF aggravated myocardial fibrosis in vitro and in vivo. Moreover, Ca2+/calmodulin‑dependent protein kinase II (CaMKII) was activated by phosphorylation, prompting an increase in phosphorylation of signal transducer and activator of transcription 3 (Stat3) and induced nuclear translocation. Pretreatment with KN-93 (a CaMKII inhibitor) blocked GF-induced Stat3 activation and significantly suppressed myocardial fibrosis. Conclusions Glucose fluctuations exacerbate myocardial fibrosis by triggering the CaMKII/Stat3 pathway in type 2 diabetes.