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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.

BackgroundNitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation.MethodsWe measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L).ResultsBoth WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events.ConclusionsWe conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

AimsAtrial fibrillation (AF), the most prevalent clinical arrhythmia, is associated with atrial remodelling manifesting as acute and chronic alterations in expression, function, and regulation of atrial electrophysiological and Ca2+-handling processes. These AF-induced modifications crosstalk and propagate across spatial scales creating a complex pathophysiological network, which renders AF resistant to existing pharmacotherapies that predominantly target transmembrane ion channels. Developing innovative therapeutic strategies requires a systems approach to disentangle quantitatively the pro-arrhythmic contributions of individual AF-induced alterations.Methods and resultsHere, we built a novel computational framework for simulating electrophysiology and Ca2+-handling in human atrial cardiomyocytes and tissues, and their regulation by key upstream signalling pathways [i.e. protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)] involved in AF-pathogenesis. Populations of atrial cardiomyocyte models were constructed to determine the influence of subcellular ionic processes, signalling components, and regulatory networks on atrial arrhythmogenesis. Our results reveal a novel synergistic crosstalk between PKA and CaMKII that promotes atrial cardiomyocyte electrical instability and arrhythmogenic triggered activity. Simulations of heterogeneous tissue demonstrate that this cellular triggered activity is further amplified by CaMKII- and PKA-dependent alterations of tissue properties, further exacerbating atrial arrhythmogenesis.ConclusionsOur analysis reveals potential mechanisms by which the stress-associated adaptive changes turn into maladaptive pro-arrhythmic triggers at the cellular and tissue levels and identifies potential anti-AF targets. Collectively, our integrative approach is powerful and instrumental to assemble and reconcile existing knowledge into a systems network for identifying novel anti-AF targets and innovative approaches moving beyond the traditional ion channel-based strategy.

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

Learning, memory, and cognition are thought to require synaptic plasticity, specifically including hippocampal long-term potentiation and depression (LTP and LTD). LTP versus LTD is induced by high-frequency stimulation versus low-frequency, but stimulating β-adrenergic receptors (βARs) enables LTP induction also by low-frequency stimulation (1 Hz) or theta frequencies (∼5 Hz) that do not cause plasticity by themselves. In contrast to high-frequency stimulation-LTP, such βAR-LTP requires Ca2+-flux through L-type voltage-gated Ca2+-channels, not N-methyl-D-aspartate-type glutamate receptors. Surprisingly, we found that βAR-LTP still required a nonionotropic scaffolding function of the N-methyl-D-aspartate-type glutamate receptor: the stimulus-induced binding of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to its GluN2B subunit that mediates CaMKII movement to excitatory synapses. In hippocampal neurons, β-adrenergic stimulation with isoproterenol (Iso) transformed LTD-type CaMKII movement to LTP-type movement, resulting in CaMKII movement to excitatory instead of inhibitory synapses. Additionally, Iso enabled induction of a major cell-biological feature of LTP in response to LTD stimuli: increased surface expression of GluA1 fused with super-ecliptic pHluorein. Like for βAR-LTP in hippocampal slices, the Iso effects on CaMKII movement and surface expression of GluA1 fused with super-ecliptic pHluorein involved L-type Ca2+-channels and specifically required β2-ARs. Taken together, these results indicate that Iso transforms LTD stimuli to LTP signals by switching CaMKII movement and GluN2B binding to LTP mode.

Yes-associated protein (YAP) and its homologue TAZ are transducers of several biochemical and biomechanical signals, integrating multiplexed inputs from the microenvironment into higher level cellular functions such as proliferation, differentiation and migration. Emerging evidence suggests that Ca2+ is a key second messenger that connects microenvironmental input signals and YAP/TAZ regulation. However, studies that directly modulate Ca2+ have reported contradictory YAP/TAZ responses: in some studies, a reduction in Ca2+ influx increases the activity of YAP/TAZ, while in others, an increase in Ca2+ influx activates YAP/TAZ. Importantly, Ca2+ and YAP/TAZ exhibit distinct spatiotemporal dynamics, making it difficult to unravel their connections from a purely experimental approach. In this study, we developed a network model of Ca2+ -mediated YAP/TAZ signalling to investigate how temporal dynamics and crosstalk of signalling pathways interacting with Ca2+ can alter the YAP/TAZ response, as observed in experiments. By including six signalling modules (e.g. GPCR, IP3-Ca2+ , kinases, RhoA, F-actin and Hippo-YAP/TAZ) that interact with Ca2+ , we investigated both transient and steady-state cell response to angiotensin II and thapsigargin stimuli. The model predicts that stimuli, Ca2+ transients and frequency-dependent relationships between Ca2+ and YAP/TAZ are primarily mediated by cPKC, DAG, CaMKII and F-actin. Simulation results illustrate the role of Ca2+ dynamics and CaMKII bistable response in switching the direction of changes in Ca2+ -induced YAP/TAZ activity. A frequency-dependent YAP/TAZ response revealed the competition between upstream regulators of LATS1/2, leading to the YAP/TAZ non-monotonic response to periodic GPCR stimulation. This study provides new insights into underlying mechanisms responsible for the controversial Ca2+ -YAP/TAZ relationship observed in experiments. KEY POINTS: YAP/TAZ integrates biochemical and biomechanical inputs to regulate cellular functions, and Ca2+ acts as a key second messenger linking cellular inputs to YAP/TAZ. Studies have reported contradictory Ca2+ -YAP/TAZ relationships for different cell types and stimuli. A network model of Ca2+ -mediated YAP/TAZ signalling was developed to investigate the underlying mechanisms of divergent Ca2+ -YAP/TAZ relationships. The model predicts context-dependent Ca2+ transient, CaMKII bistable response and frequency-dependent activation of LATS1/2 upstream regulators as mechanisms governing the Ca2+ -YAP/TAZ relationship. This study provides new insights into the underlying mechanisms of the controversial Ca2+ -YAP/TAZ relationship to better understand the dynamics of cellular functions controlled by YAP/TAZ activity.

The article presents a study on the tissue specificity of cardiac genome editing using adeno-associated virus (AAV) vectors. The researchers discovered that the commonly used AAV9-Tnnt2 system had some gene expression leakage in the liver. To address this issue, they incorporated microRNA-122 target sequences (miR122TS) into the AAV9-Tnnt2 vectors, which effectively reduced liver leakage without affecting cardiac gene expression. The study emphasizes the need to improve the tissue specificity of gene editing techniques for cardiovascular research and gene therapy applications. [Extracted from the article]

BackgroundThe sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR.MethodsA role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology.ResultsOur results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR.ConclusionsAKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

AimsDiabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors.Methods and resultsAction potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycaemia, consistent with elevated INaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs, or IK1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes.ConclusionWe conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

Persistent over-activation of CaMKII (Calcium/Calmodulin-dependent protein Kinase II) in the heart is implicated in arrhythmias, heart failure, pathological remodeling, and other cardiovascular diseases. Several post-translational modifications (PTMs)-including autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation-have been shown to trap CaMKII in an autonomously active state. The molecular mechanisms by which these PTMs regulate calmodulin (CaM) binding to CaMKIIδ-the primary cardiac isoform-has not been well-studied particularly in its native myocyte environment. Typically, CaMKII activates upon Ca-CaM binding during locally elevated [Ca]free and deactivates upon Ca-CaM dissociation when [Ca]free returns to basal levels. To assess the effects of CaMKIIδ PTMs on CaM binding, we developed a novel FRET (Förster resonance energy transfer) approach to directly measure CaM binding to and dissociation from CaMKIIδ in live cardiac myocytes. We demonstrate that autophosphorylation of CaMKIIδ increases affinity for CaM in its native environment and that this increase is dependent on [Ca]free. This leads to a 3-fold slowing of CaM dissociation from CaMKIIδ (time constant slows from ~0.5 to 1.5 s) when [Ca]free is reduced with physiological kinetics. Moreover, oxidation further slows CaM dissociation from CaMKIIδ T287D (phosphomimetic) upon rapid [Ca]free chelation and increases FRET between CaM and CaMKIIδ T287A (phosphoresistant). The CaM dissociation kinetics-measured here in myocytes-are similar to the interval between heartbeats, and integrative memory would be expected as a function of heart rate. Furthermore, the PTM-induced slowing of dissociation between beats would greatly promote persistent CaMKIIδ activity in the heart. Together, these findings suggest a significant role of PTM-induced changes in CaMKIIδ affinity for CaM and memory under physiological and pathophysiological processes in the heart.

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII-but not PKA, PKC or PKD-raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

Cardiomyocyte Na+ and Ca2+ mishandling, upregulated Ca2+/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibition and mitochondrial ROS scavenging, with a slight synergy with INaL inhibition. Inducing LQT by elevating INaL (by Anemone toxin II, ATX-II) caused markedly prolonged APD, increased STV, and early afterdepolarizations (EADs). Those proarrhythmic ATX-II effects were largely attenuated by mitochondrial ROS scavenging, and partially reduced by inhibition of CaMKII and pathological leaky RyRs using dantrolene. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) bearing LQT3 mutation SCN5A N406K, dantrolene significantly attenuated cell arrhythmias and APD prolongation. Targeting critical components of the Na+-Ca2+-CaMKII-ROS-INaL arrhythmogenic vicious cycle may exhibit important on-target and also trans-target effects (e.g., INaL and RyR inhibition can alter INaL-mediated LQT3 effects). Incorporating this vicious cycle into therapeutic strategies provides novel integrated insight for treating cardiac arrhythmias and diseases.

Myocardial infarction and revascularization cause cardiac ischemia/reperfusion (I/R) injury featuring cardiomyocyte death and inflammation. The Ca2+/calmodulin dependent protein kinase II (CaMKII) family are serine/ threonine protein kinases that are involved in I/R injury. CaMKII exists in four different isoforms, α, β, γ, and δ. In the heart, CaMKII-δ is the predominant isoform，with multiple splicing variants, such as δB, δC and δ9. During I/R, elevated intracellular Ca2+ concentrations and reactive oxygen species activate CaMKII. In this review, we summarized the regulation and function of CaMKII in multiple cell types including cardiomyocytes, endothelial cells, and macrophages during I/R. We conclude that CaMKII mediates inflammation in the microenvironment of the myocardium, resulting in cell dysfunction, elevated inflammation, and cell death. However, different CaMKII-δ variants exhibit distinct or even opposite functions. Therefore, reagents/approaches that selectively target specific CaMKII isoforms and variants are needed for evaluating and counteracting the exact role of CaMKII in I/R injury and developing effective treatments against I/R injury. Pathological functions of CaMKII in different cell types during ischemia/reperfusion (I/R) injury, activation of CaMKII in cardiomyocytes can cause inflammatory responses, cell death, increased infarct size, maladaptive hypertrophy, and arrhythmia. CaMKII activation can lead to endothelial cell inflammation, cell death, vascular hyperpermeability, and impaired vasodilation. Overactive CaMKII in macrophages can cause inflammation, impaired efferocytosis, and eventually lead to tissue fibrosis. [Display omitted] • We provides an overview of the inflammatory effect of CaMKII in cardiomyocytes, endothelial cells, macrophages, and other relevant cell types during I/R. • The review offers updates on recent advancements in CaMKII-targeted therapies, encompassing both pharmaceutical interventions and gene-editing approaches. • The potential of suppressing CaMKII as a therapeutic strategy for I/R treatment is emphasized, offering valuable insights for future research and clinical applications. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of curcumin (Cur) regulating N-methyl-D-aspartate receptor (NMDAR)/calcium ion (Ca2+ )/calmodulin dependent protein kinase Ⅱ (CaMKⅡ) signaling pathway on isoflurane (ISO) - induced postoperative cognitive dysfunction (POCD) in young mice. Methods Seventy-two C57BL/6J mice were separated into the control group, the ISO group, the low-dose Cur group (Cur-L group, 50 mg/kg), the medium-dose Cur group (Cur-M group, 100 mg/kg), the high-dose Cur group (Cur-H group, 200 mg/kg) and the Cur-H+NMDA (NMDAR activator) group (200 mg/kg+8 mg/kg), with 12 animals in each group. After 30 min of corresponding drug treatment, the mice in the control group inhaled a mixed gas containing 30% oxygen and air for 2 hours, and mice in the other groups inhaled 2% ISO for 2 hours, once a day for 14 days. Twenty-four hours after the last treatment, Morris water maze test was used to detect the learning and spatial memory abilities of mice. HE staining was used to detect the pathological changes in hippocampal CA1 area. Immunofluorescence staining was used to detect the positive expression of neuron specific nucleoprotein (NeuN) in hippocampus CA1 region of mice. TUNEL staining was used to detect neuronal apoptosis. Enzyme linked immunosorbent assay was used to detect levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in hippocampal CA1 tissue. Western blot assay was used to detect NMDAR1 and CaMK Ⅱ protein expression in mouse hippocampal CA1 tissue. Intracellular Ca2+ concentration in hippocampal CA1 region was detected by fluorescence probe. Results Compared with the control group, the pathological damage of hippocampal CA1 region of mice was severe in the ISO group, and the escape latency was prolonged. The apoptosis rate of neural cells, levels of IL-1β and TNF-α, expression levels of NMDAR1 and CaMKⅡ protein, and the concentration of Ca2+ in hippocampal CA1 region were increased (P＜0.05). Times of crossing platform and the number of NeuN positive cells were decreased in the ISO group (P＜0.05). Compared with the ISO group, pathological damages of hippocampal CA1 region of mice were alleviated in the Cur-L group, the Cur-M group and the CurH group, the escape latency was shortened, and the apoptosis rate of neural cells, levels of IL-1β and TNF-α, expression levels of NMDAR1 and CaMKⅡ protein, and the concentration of Ca2+ in hippocampal CA1 region were decreased (P＜ 0.05). Times of crossing platform and the number of NeuN positive cells were increased (P＜0.05), which was dosedependent. NMDA attenuated the improvement effect of high-dose Cur on ISO induced POCD in mice (P＜0.05). Conclusion Curcumin may improve ISO-induced POCD in mice by inhibiting NMDAR/Ca2+ /CaMKⅡ signaling pathway [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

AimsIn mammalian ventricles, electrical gradients establish electrical heterogeneities as essential tissue mechanisms to optimize mechanical efficiency and safeguard electrical stability. Electrical gradients shape mammalian electrocardiographic patterns; disturbance of electrical gradients is proarrhythmic. The zebrafish heart is a popular surrogate model for human cardiac electrophysiology thanks to its remarkable recapitulation of human electrocardiogram and ventricular action potential features. Yet, zebrafish ventricular electrical gradients are largely unexplored. The goal of this study is to define the zebrafish ventricular electrical gradients that shape the QRS complex and T wave patterns at baseline and under oxidative stress.Methods and resultsWe performed in vivo electrocardiography and ex vivo voltage-sensitive fluorescent epicardial and transmural optical mapping of adult zebrafish hearts at baseline and during acute H2O2 exposure. At baseline, apicobasal activation and basoapical repolarization gradients accounted for the polarity concordance between the QRS complex and T wave. During H2O2 exposure, differential regional impairment of activation and repolarization at the apex and base disrupted prior to baseline electrical gradients, resulting in either reversal or loss of polarity concordance between the QRS complex and T wave. KN-93, a specific calcium/calmodulin-dependent protein kinase II inhibitor (CaMKII), protected zebrafish hearts from H2O2 disruption of electrical gradients. The protection was complete if administered prior to oxidative stress exposure.ConclusionsDespite remarkable apparent similarities, zebrafish and human ventricular electrocardiographic patterns are mirror images supported by opposite electrical gradients. Like mammalian ventricles, zebrafish ventricles are also susceptible to H2O2 proarrhythmic perturbation via CaMKII activation. Our findings suggest that the adult zebrafish heart may constitute a clinically relevant model to investigate ventricular arrhythmias induced by oxidative stress. However, the fundamental ventricular activation and repolarization differences between the two species that we demonstrated in this study highlight the potential limitations when extrapolating results from zebrafish experiments to human cardiac electrophysiology, arrhythmias, and drug toxicities.

[Figure: see text].

[Figure: see text].

RationaleThe mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca2+ regulates gene expression, but the nature and significance of nuclear Ca2+-changes in AF are largely unknown.ObjectiveTo elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca2+ ([Ca2+]Nuc) and CaMKII (Ca2+/calmodulin-dependent protein kinase-II)-related signaling.Methods and resultsAtrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca2+]Nuc and cytosolic [Ca2+] ([Ca2+]Cyto) were recorded via confocal microscopy. Diastolic [Ca2+]Nuc was greater than [Ca2+]Cyto under control conditions, while resting [Ca2+]Nuc was similar to [Ca2+]Cyto; both diastolic and resting [Ca2+]Nuc increased with AF. IP3R (Inositol-trisphosphate receptor) stimulation produced larger [Ca2+]Nuc increases in AF versus control cardiomyocytes, and IP3R-blockade suppressed the AF-related [Ca2+]Nuc differences. AF upregulated nuclear protein expression of IP3R1 (IP3R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca2+]Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP3R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP3R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP3R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca2+-loading.ConclusionsAF increases atrial-cardiomyocyte nucleoplasmic [Ca2+] by IP3R1-upregulation involving miR-26a, leading to enhanced IP3R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.

RationaleWe recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown.ObjectiveTo test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship.Methods and resultsWe used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts.ConclusionsWe have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Dopamine D1 receptor (D1R) function is regulated by membrane/lipid raft-resident protein caveolin-1 (Cav1). We examined whether altered expression of Cav1 in the dorsal striatum would affect self-administration of methamphetamine, an indirect agonist at the D1Rs. A lentiviral construct expressing Cav1 (LV-Cav1) or containing a short hairpin RNA against Cav1 (LV-shCav1) was used to overexpress or knock down Cav1 expression respectively, in the dorsal striatum. Under a fixed-ratio schedule, LV-Cav1 enhanced and LV-shCav1 reduced responding for methamphetamine in an extended access paradigm compared to LV-GFP controls. LV-Cav1 and LV-shCav1 also produced an upward and downward shift in a dose-response paradigm, generating a drug vulnerable/resistant phenotype. LV-Cav1 and LV-shCav1 did not alter responding for sucrose. Under a progressive-ratio schedule, LV-shCav1 generally reduced positive-reinforcing effects of methamphetamine and sucrose as seen by reduced breakpoints. Western blotting confirmed enhanced Cav1 expression in LV-Cav1 rats and reduced Cav1 expression in LV-shCav1 rats. Electrophysiological findings in LV-GFP rats demonstrated an absence of high-frequency stimulation (HFS)-induced long-term potentiation (LTP) in the dorsal striatum after extended access methamphetamine self-administration, indicating methamphetamine-induced occlusion of plasticity. LV-Cav1 prevented methamphetamine-induced plasticity via increasing phosphorylation of calcium calmodulin kinase II, suggesting a mechanism for addiction vulnerability. LV-shCav1 produced a marked deficit in the ability of HFS to produce LTP and, therefore, extended access methamphetamine was unable to alter striatal plasticity, indicating a mechanism for resistance to addiction-like behavior. Our results demonstrate that Cav1 expression and knockdown driven striatal plasticity assist with modulating addiction to drug and nondrug rewards, and inspire new strategies to reduce psychostimulant addiction.

Activation of Ca2+/calmodulin kinase II (CaMKII) and the N-Methyl D-aspartate receptor (NMDAR), particularly its GluN2B subunit, contribute to the central sensitization of nociceptive pathways and persistent pain. Using mutant mice wherein the activity-driven binding of CaMKII to S1303 in GluN2B is abrogated (GluN2BKI), this study investigated the importance of this interaction for acute and persistent inflammatory nociception. GluN2BKI, wild type and heterozygote mice did not differ in responses to acute noxious heat stimuli as measured with tail flick, paw flick, or hot plate assays, nor did they differ in their responses to mechanical stimulation with von Frey filaments. Surprisingly, the three genotypes exhibited similar spontaneous pain behaviors and hypersensitivity to heat or mechanical stimuli induced by intraplantar injection of capsaicin; however, GluN2BKI mice did not immediately attend to the paw. WT and GluN2BKI mice also did not differ in the nociceptive behaviors elicited by intraplantar injection of formalin, even though MK801 greatly reduced these behaviors in both genotypes concordant with NMDAR dependence. CaMKII binding to GluN2B at S1303 therefore does not appear to be critical for the development of inflammatory nociception. Finally, intrathecal KN93 reduced formalin-induced nociceptive behaviors in GluN2BKI mice. KN93 does not inhibit CaKMII, but rather binds Ca2+/calmodulin. It has multiple other targets including Ca2+-, Na+- and K+-channels, as well as various kinases. Therefore, the use of GluN2BKI mice provided genetic specificity in assessing the role of CaMKII in inflammatory pain signaling cascades. These results challenge current thinking on the involvement of the CaMKII-NMDAR interaction in inflammatory pain.

Chronic hyperglycemia and diabetes lead to impaired cardiac repolarization, K+ channel remodeling and increased arrhythmia risk. However, the exact signaling mechanism by which diabetic hyperglycemia regulates cardiac K+ channels remains elusive. Here, we show that acute hyperglycemia increases inward rectifier K+ current (IK1), but reduces the amplitude and inactivation recovery time of the transient outward K+ current (Ito) in mouse, rat, and rabbit myocytes. These changes were all critically dependent on intracellular O-GlcNAcylation. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor autocamtide-2-related inhibitory peptide, CaMKIIδ-knockout, and O-GlcNAc-resistant CaMKIIδ-S280A knock-in. Ito reduction was prevented by inhibition of protein kinase C (PKC) and NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). In mouse models of chronic diabetes (streptozotocin, db/db, and high-fat diet), heart failure, and CaMKIIδ overexpression, both Ito and IK1 were reduced in line with the downregulated K+ channel expression. However, IK1 downregulation in diabetes was markedly attenuated in CaMKIIδ-S280A. We conclude that acute hyperglycemia enhances IK1 and Ito recovery via CaMKIIδ-S280 O-GlcNAcylation, but reduces Ito amplitude via a NOX2-ROS-PKC pathway. Moreover, chronic hyperglycemia during diabetes and CaMKII activation downregulate K+ channel expression and function, which may further increase arrhythmia susceptibility.

RationaleCaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF.ObjectiveTo measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice.Methods and resultsWe used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions.ConclusionsWe conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.

Mutations in CAPN3 cause limb girdle muscular dystrophy R1 (LGMDR1, formerly LGMD2A) and lead to progressive and debilitating muscle wasting. Calpain 3 deficiency is associated with impaired CaMKIIβ signaling and blunted transcriptional programs that encode the slow-oxidative muscle phenotype. We conducted a high-throughput screen on a target of CaMKII (Myl2) to identify compounds to override this signaling defect; 4 were tested in vivo in the Capn3 knockout (C3KO) model of LGMDR1. The leading compound, AMBMP, showed good exposure and was able to reverse the LGMDR1 phenotype in vivo, including improved oxidative properties, increased slow fiber size, and enhanced exercise performance. AMBMP also activated CaMKIIβ signaling, but it did not alter other pathways known to be associated with muscle growth. Thus, AMBMP treatment activates CaMKII and metabolically reprograms skeletal muscle toward a slow muscle phenotype. These proof-of-concept studies lend support for an approach to the development of therapeutics for LGMDR1.

Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. Here, we report increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. By contrast, mice with genetic mitochondrial CaMKII inhibition are protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII overexpression (mtCaMKII) have severe dilated cardiomyopathy and decreased ATP that causes elevated cytoplasmic resting (diastolic) Ca2+ concentration and reduced mechanical performance. We map a metabolic pathway that rescues disease phenotypes in mtCaMKII mice, providing insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase or mitochondrial-targeted CaMKII inhibition.

Increased pulmonary vascular permeability due to endothelial cell (EC) barrier dysfunction is a major pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI), which is a devastating critical illness with high incidence and excessive mortality. Activation of NADPH oxidase (NOX) induces EC dysfunction via production of reactive oxygen species (ROS). However, the role(s) of NOX isoform(s), and their downstream signaling events, in the development of ARDS/ALI have remained unclear. Cecal Ligation Puncture (CLP) was used to induce preclinical septic ALI in wild-type mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA in vivo. The survival rate of the CLP group at 24 h (26.6%, control siRNA treated) was substantially improved by NOX4 knockdown (52.9%). Mice lacking NOX2 or p47phox, however, had worse outcomes after CLP (survival rates at 0% and 8.3% respectively), whereas NOX1-silenced mice had similar survival rate (30%). NOX4 knockdown attenuated lung ROS production in septic mice, whereas NOX1 knockdown, NOX2 knockout, or p47phox knockout in mice had no effects. In addition, NOX4 knockdown attenuated redox-sensitive activation of the CaMKII/ERK1/2/MLCK pathway, and restored expression of EC tight junction proteins ZO-1 and Occludin to maintain EC barrier integrity. Correspondingly, NOX4 knockdown in cultured human lung microvascular ECs also reduced LPS-induced ROS production, CaMKII/ERK1/2/MLCK activation and EC barrier dysfunction. Scavenging superoxide in vitro and in vivo with TEMPO, or inhibiting CaMKII activation with KN93, had similar effects as NOX4 knockdown in preserving EC barrier dysfunction. In summary, we have identified a novel, selective and causal role of NOX4 (versus other NOX isoforms) in inducing lung EC barrier dysfunction and injury/mortality in a preclinical CLP-induced septic model, which involves redox-sensitive activation of CaMKII/ERK1/2/MLCK pathway. Targeting NOX4 may therefore prove to an innovative therapeutic option that is markedly effective in treating ALI/ARDS.

RationaleCardiotoxic β1 adrenergic receptor (β1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome.ObjectiveWe aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure.Methods and resultsThe integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts.ConclusionsThese data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Prevalence of left ventricular (LV) systolic and diastolic dysfunction increases with aging. We previously reported that urocortin 2 (Ucn2) gene transfer increases heart function in mice with heart failure with reduced ejection fraction. Here, we test the hypotheses that (1) Ucn2 gene transfer will increase LV function in aged mice and that (2) Ucn2 gene transfer given in early life will prevent age-related LV dysfunction. Nineteen-month-old (treatment study) and 3-month-old (prevention study) mice received Ucn2 gene transfer or saline. LV function was examined 3-4 months (treatment study) or 20 months (prevention study) after Ucn2 gene transfer or saline injection. In both the treatment and prevention strategies, Ucn2 gene transfer increased ejection fraction, reduced LV volume, increased LV peak -dP/dt and peak +dP/dt, and reduced global longitudinal strain. Ucn2 gene transfer-in both treatment and prevention strategies-was associated with higher levels of LV SERCA2a protein, reduced phosphorylation of LV CaMKIIa, and reduced LV α-skeletal actin mRNA expression (reflecting reduced cardiac stress). In conclusion, Ucn2 gene transfer restores normal cardiac function in mice with age-related LV dysfunction and prevents development of LV dysfunction.

Calmodulin-dependent kinase II (CaMKII) has long been known to play an important role in learning and memory as well as long term potentiation (LTP). More recently it has been suggested that it might be involved in the time averaging of synaptic signals, which can then lead to the high precision of information stored at a single synapse. However, the role of the scaffolding molecule, neurogranin (Ng), in governing the dynamics of CaMKII is not yet fully understood. In this work, we adopt a rule-based modeling approach through the Monte Carlo method to study the effect of Ca2+ signals on the dynamics of CaMKII phosphorylation in the postsynaptic density (PSD). Calcium surges are observed in synaptic spines during an EPSP and back-propagating action potential due to the opening of NMDA receptors and voltage dependent calcium channels. Using agent-based models, we computationally investigate the dynamics of phosphorylation of CaMKII monomers and dodecameric holoenzymes. The scaffolding molecule, Ng, when present in significant concentration, limits the availability of free calmodulin (CaM), the protein which activates CaMKII in the presence of calcium. We show that Ng plays an important modulatory role in CaMKII phosphorylation following a surge of high calcium concentration. We find a non-intuitive dependence of this effect on CaM concentration that results from the different affinities of CaM for CaMKII depending on the number of calcium ions bound to the former. It has been shown previously that in the absence of phosphatase, CaMKII monomers integrate over Ca2+ signals of certain frequencies through autophosphorylation (Pepke et al, Plos Comp. Bio., 2010). We also study the effect of multiple calcium spikes on CaMKII holoenzyme autophosphorylation, and show that in the presence of phosphatase, CaMKII behaves as a leaky integrator of calcium signals, a result that has been recently observed in vivo. Our models predict that the parameters of this leaky integrator are finely tuned through the interactions of Ng, CaM, CaMKII, and PP1, providing a mechanism to precisely control the sensitivity of synapses to calcium signals. Author Summary not valid for PLOS ONE submissions.

Background: Synaptic plasticity contributes to nociceptive signal transmission and modulation, with calcium/ calmodulin-dependent protein kinase II (CaMK II) playing a fundamental role in neural plasticity. This research was conducted to investigate the role of CaMK II in the transmission and regulation of nociceptive information within the nucleus accumbens (NAc) of naïve and morphine-tolerant rats. Methods: Randall Selitto and hot-plate tests were utilized to measure the hindpaw withdrawal latencies (HWLs) in response to noxious mechanical and thermal stimuli. To induce chronic morphine tolerance, rats received intraperitoneal morphine injection twice per day for seven days. CaMK II expression and activity were assessed using western blotting. Results: Intra-NAc microinjection of autocamtide-2-related inhibitory peptide (AIP) induced an increase in HWLs in naïve rats in response to noxious thermal and mechanical stimuli. Moreover, the expression of the phosphorylated CaMK II (p-CaMK II) was significantly decreased as determined by western blotting. Chronic intraperitoneal injection of morphine resulted in significant morphine tolerance in rats on Day 7, and an increase of p-CaMK II expression in NAc in morphine-tolerant rats was observed. Furthermore, intra-NAc administration of AIP elicited significant antinociceptive responses in morphine-tolerant rats. In addition, compared with naïve rats, AIP induced stronger thermal antinociceptive effects of the same dose in rats exhibiting morphine tolerance. Conclusions: This study shows that CaMK II in the NAc is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rats. [ABSTRACT FROM AUTHOR]

Temporal lobe epilepsy (TLE) is a prevalent neurological disorder with many patients experiencing poor seizure control with existing anti-epileptic drugs. Thus, novel insights into the mechanisms of epileptogenesis and identification of new drug targets can be transformative. Changes in ion channel function have been shown to play a role in generating the aberrant neuronal activity observed in TLE. Previous work demonstrates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are mislocalized within CA1 pyramidal cells in a rodent model of TLE. The subcellular distribution of HCN channels is regulated by an auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), and disruption of this interaction correlates with channel mislocalization. However, the molecular mechanisms responsible for HCN channel dysregulation in TLE are unclear. Here we investigated whether changes in TRIP8b phosphorylation are sufficient to alter HCN channel function. We identified a phosphorylation site at residue Ser237 of TRIP8b that enhances binding to HCN channels and influences channel gating by altering the affinity of TRIP8b for the HCN cytoplasmic domain. Using a phosphospecific antibody, we demonstrate that TRIP8b phosphorylated at Ser237 is enriched in CA1 distal dendrites and that phosphorylation is reduced in the kainic acid model of TLE. Overall, our findings indicate that the TRIP8b-HCN interaction can be modulated by changes in phosphorylation and suggest that loss of TRIP8b phosphorylation may affect HCN channel properties during epileptogenesis. These results highlight the potential of drugs targeting posttranslational modifications to restore TRIP8b phosphorylation to reduce excitability in TLE.

ObjectiveEndurance exercise training remodels skeletal muscle, leading to increased mitochondrial content and oxidative capacity. How exercise entrains skeletal muscle signaling pathways to induce adaptive responses remains unclear. In past studies, we identified Perm1 (PGC-1 and ERR induced regulator, muscle 1) as an exercise-induced gene and showed that Perm1 overexpression elicits similar muscle adaptations as endurance exercise training. The mechanism of action and the role of Perm1 in exercise-induced responses are not known. In this study, we aimed to determine the pathway by which Perm1 acts as well as the importance of Perm1 for acute and long-term responses to exercise.MethodsWe performed immunoprecipitation and mass spectrometry to identify Perm1 associated proteins, and validated Perm1 interactions with the Ca2+/calmodulin-dependent protein kinase II (CaMKII). We also knocked down Perm1 expression in gastrocnemius muscles of mice via AAV-mediated delivery of shRNA and assessed the impact of reduced Perm1 expression on both acute molecular responses to a single treadmill exercise bout and long-term adaptive responses to four weeks of voluntary wheel running training. Finally, we asked whether Perm1 levels are modulated by diet or diseases affecting skeletal muscle function.ResultsWe show that Perm1 associates with skeletal muscle CaMKII and promotes CaMKII activation. In response to an acute exercise bout, muscles with a knock down of Perm1 showed defects in the activation of CaMKII and p38 MAPK and blunted induction of regulators of oxidative metabolism. Following four weeks of voluntary training, Perm1 knockdown muscles had attenuated mitochondrial biogenesis. Finally, we found that Perm1 expression is reduced in diet-induced obese mice and in muscular dystrophy patients and mouse models.ConclusionsOur findings identify Perm1 as a muscle-specific regulator of exercise-induced signaling and Perm1 levels as tuners of the skeletal muscle response to exercise. The decreased Perm1 levels in states of obesity or muscle disease suggest that Perm1 may link pathological states to inefficient exercise responses.

BackgroundHeart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na+ current (INaL), L-type Ca2+ current, and NCX (Na+/Ca2+ exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca2+ buffers).MethodsWe measured the major inward currents and their Ca2+- and β-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control).ResultsAP duration and short-term variability of AP repolarization were increased in HF, and importantly, inhibition of INaL decreased both parameters to the control level. INaL was slightly increased in HF versus control even when intracellular Ca2+ was strongly buffered. But under physiological AP clamp with normal Ca2+ cycling, INaL was markedly upregulated in HF versus control (dependent largely on CaMKII [Ca2+/calmodulin-dependent protein kinase II] activity). β-Adrenergic stimulation (often elevated in HF) further enhanced INaL. L-type Ca2+ current was decreased in HF when Ca2+ was buffered, but CaMKII-mediated Ca2+-dependent facilitation upregulated physiological L-type Ca2+ current to the control level. Furthermore, L-type Ca2+ current response to β-adrenergic stimulation was significantly attenuated in HF. Inward NCX current was upregulated at phase 3 of AP in HF when assessed by combining experimental data and computational modeling.ConclusionsOur results suggest that CaMKII-dependent upregulation of INaL in HF significantly contributes to AP prolongation and increased short-term variability of AP repolarization, which may lead to increased arrhythmia propensity, and is further exacerbated by adrenergic stress.

Quantitative systems modeling aims to integrate knowledge in different research areas with models describing biological mechanisms and dynamics to gain a better understanding of complex clinical syndromes. Heart failure (HF) is a chronic complex cardiac disease that results from structural or functional disorders impairing the ability of the ventricle to fill with or eject blood. Highly interactive and dynamic changes in mechanical, structural, neurohumoral, metabolic, and electrophysiological properties collectively predispose the failing heart to cardiac arrhythmias, which are responsible for about a half of HF deaths. Multiscale cardiac modeling and simulation integrate structural and functional data from HF experimental models and patients to improve our mechanistic understanding of this complex arrhythmia syndrome. In particular, they allow investigating how disease-induced remodeling alters the coupling of electrophysiology, Ca2+ and Na+ handling, contraction, and energetics that lead to rhythm derangements. The Ca2+ /calmodulin-dependent protein kinase II, which expression and activity are enhanced in HF, emerges as a critical hub that modulates the feedbacks between these various subsystems and promotes arrhythmogenesis. This article is categorized under: Physiology > Mammalian Physiology in Health and Disease Models of Systems Properties and Processes > Mechanistic Models Models of Systems Properties and Processes > Cellular Models Models of Systems Properties and Processes > Organ, Tissue, and Physiological Models.

Pressure overload-induced cardiac hypertrophy occurs in response to chronic blood pressure increase, and dysfunction of CaV1.2 calcium channel involves in cardiac hypertrophic processes by perturbing intracellular calcium concentration ([Ca2+]i) and calcium-dependent signaling. As a carbohydrate-binding protein, galectin-1 (Gal-1) is found to bind with CaV1.2 channel, which regulates vascular CaV1.2 channel functions and blood pressure. However, the potential roles of Gal-1 in cardiac CaV1.2 channel (CaV1.2CM) and cardiomyocyte hypertrophy remain elusive. By whole-cell patch clamp, we find Gal-1 decreases the ICa,L with or without isoproterenol (ISO) application by reducing the channel membrane expression in neonatal rat ventricular myocytes (NRVMs). Moreover, Gal-1 could inhibit the current densities of CaV1.2CM by an alternative exon 9*-dependent manner in heterologously expressed HEK293 cells. Of significance, overexpression of Gal-1 diminishes ISO or KCl-induced [Ca2+]i elevation and attenuates ISO-induced hypertrophy in NRVMs. Mechanistically, Gal-1 decreases the ISO or Bay K8644-induced phosphorylation of intracellular calcium-dependent signaling proteins δCaMKII and HDAC4, and inhibits ISO-triggered translocation of HDAC4 in NRVMs. Pathologically, we observe that the expressions of Gal-1 and CaV1.2E9* channels are synchronously increased in rat hypertrophic cardiomyocytes and hearts. Taken together, our study indicates that Gal-1 reduces the channel membrane expression to inhibit the currents of CaV1.2CM in a splice-variant specific manner, which diminishes [Ca2+]i elevation, and attenuates cardiomyocyte hypertrophy by inhibiting the phosphorylation of δCaMKII and HDAC4. Furthermore, our work suggests that dysregulated Gal-1 and CaV1.2 alternative exon 9* might be attributed to the pathological processes of cardiac hypertrophy, and provides a potential anti-hypertrophic target in the heart.

Calcium-calmodulin dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac electrophysiology, calcium (Ca) balance, contraction, transcription, arrhythmias and progression to heart failure. CaMKII is readily activated at mouths of dyadic cleft Ca channels, but because of its low Ca-calmodulin affinity and presumed immobility it is less clear how CaMKII gets activated near other known, extra-dyad targets. CaMKII is typically considered to be anchored in cardiomyocytes, but while untested, mobility of active CaMKII could provide a mechanism for broader target phosphorylation in cardiomyocytes. We therefore tested CaMKII mobility and how this is affected by kinase activation in adult rabbit cardiomyocytes. We measured translocation of both endogenous and fluorescence-tagged CaMKII using immunocytochemistry, fluorescence recovery after photobleach (FRAP) and photoactivation of fluorescence. In contrast to the prevailing view that CaMKII is anchored near its myocyte targets, we found CaMKII to be highly mobile in resting myocytes, which was slowed by Ca chelation and accelerated by pacing. At low [Ca], CaMKII was concentrated at Z-lines near the dyad but spread throughout the sarcomere upon pacing. Nuclear exchange of CaMKII was also enhanced upon pacing- and heart failure-induced chronic activation. This mobilization of active CaMKII and its intrinsic memory may allow CaMKII to be activated in high [Ca] regions and then move towards more distant myocyte target sites.

Ischemic stroke is the most common, fatal and disabling disease of the central nervous system. Despite considerable progress in primary prevention, diagnostic testing, and treatment, the increased incidence of stroke has created a serious public health problem worldwide, with a significant impact on the families and societies of those affected. The study of ketogenic (KD) and calorie-restricted (CR) diets has become a hot topic of research and is gradually penetrating into the daily diet. Although there are many studies on the neuroprotective mechanisms of KD and CR, the duration of the interventions is relatively short and the mechanisms are not well understood. Therefore, whether long-term ketogenic diet and calorie-restricted diet treatment can improve the resistance to ischemic-hypoxic injury in healthy aged C57 mice, and the mechanism still deserves further investigation. Young 6-month-old mice were randomly divided into KD, CR and control groups, and an ischemic stroke model of endothelin-1 was established on the basis of dietary intervention for 15 months, and the mechanism was explored based on bioinformatics method. Our results confirm that long-term dietary treatment of KD and CR reduces neuronal death in infarct foci and improves neuronal resistance to ischemia and hypoxia by upregulating UBR4 to inhibit the CamkⅡ/TAK1/JNK signaling pathway, then suppress oxidative stress, thus acting as a neuroprotective agent. To be conclusive, ketogenic and calorie-restricted diets have a significant effect on ischemic stroke, but well-designed future clinical studies are necessary to provide solid evidence on this topic.

BackgroundInflammation is associated with cardiac remodeling and heart failure, but how it is initiated in response to nonischemic interventions in the absence of cell death is not known. We tested the hypothesis that activation of Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) in cardiomyocytes (CMs) in response to pressure overload elicits inflammatory responses leading to adverse remodeling.MethodsMice in which CaMKIIδ was selectively deleted from CMs (cardiac-specific knockout [CKO]) and floxed control mice were subjected to transverse aortic constriction (TAC). The effects of CM-specific CaMKIIδ deletion on inflammatory gene expression, inflammasome activation, macrophage accumulation, and fibrosis were assessed by quantitative polymerase chain reaction, histochemistry, and ventricular remodeling by echocardiography.ResultsTAC induced increases in cardiac mRNA levels for proinflammatory chemokines and cytokines in ≤3 days, and these responses were significantly blunted when CM CaMKIIδ was deleted. Apoptotic and necrotic cell death were absent at this time. CMs isolated from TAC hearts mirrored these robust increases in gene expression, which were markedly attenuated in CKO. Priming and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome, assessed by measuring interleukin-1β and NOD-like receptor pyrin domain-containing protein 3 mRNA levels, caspase-1 activity, and interleukin-18 cleavage, were increased at day 3 after TAC in control hearts and in CMs isolated from these hearts. These responses were dependent on CaMKIIδ and associated with activation of Nuclear Factor-kappa B and reactive oxygen species. Accumulation of macrophages observed at days 7 to 14 after TAC was diminished in CKO and, by blocking Monocyte Chemotactic Protein-1 signaling, deletion of CM Monocyte Chemotactic Protein-1 or inhibition of inflammasome activation. Fibrosis was also attenuated by these interventions and in the CKO heart. Ventricular dilation and contractile dysfunction observed at day 42 after TAC were diminished in the CKO. Inhibition of CaMKII, Nuclear Factor-kappa B, inflammasome, or Monocyte Chemotactic Protein-1 signaling in the first 1 or 2 weeks after TAC decreased remodeling, but inhibition of CaMKII after 2 weeks did not.ConclusionsActivation of CaMKIIδ in response to pressure overload triggers inflammatory gene expression and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome in CMs. These responses provide signals for macrophage recruitment, fibrosis, and myocardial dysfunction in the heart. Our work suggests the importance of targeting early inflammatory responses induced by CM CaMKIIδ signaling to prevent progression to heart failure.

The synapse transmits, processes, and stores data within its tiny space. Effective and specific signaling requires precise alignment of the relevant components. This review examines current insights into mechanisms of AMPAR and NMDAR localization by PSD-95 and their spatial distribution at postsynaptic sites to illuminate the structural and functional framework of postsynaptic signaling. It subsequently delineates how β2 adrenergic receptor (β2 AR) signaling via adenylyl cyclase and the cAMP-dependent protein kinase PKA is organized within nanodomains. Here, we discuss targeting of β2 AR, adenylyl cyclase, and PKA to defined signaling complexes at postsynaptic sites, i.e., AMPARs and the L-type Ca2+ channel Cav1.2, and other subcellular surface localizations, the role of A kinase anchor proteins, the physiological relevance of the spatial restriction of corresponding signaling, and their interplay with signal transduction by the Ca2+- and calmodulin-dependent kinase CaMKII How localized and specific signaling by cAMP occurs is a central cellular question. The dendritic spine constitutes an ideal paradigm for elucidating the dimensions of spatially restricted signaling because of their small size and defined protein composition.

To uncover the circuit-level alterations that underlie atypical sensory processing associated with autism, we adopted a symptom-to-circuit approach in the Fmr1-knockout (Fmr1-/-) mouse model of Fragile X syndrome. Using a go/no-go task and in vivo two-photon calcium imaging, we find that impaired visual discrimination in Fmr1-/- mice correlates with marked deficits in orientation tuning of principal neurons and with a decrease in the activity of parvalbumin interneurons in primary visual cortex. Restoring visually evoked activity in parvalbumin cells in Fmr1-/- mice with a chemogenetic strategy using designer receptors exclusively activated by designer drugs was sufficient to rescue their behavioral performance. Strikingly, human subjects with Fragile X syndrome exhibit impairments in visual discrimination similar to those in Fmr1-/- mice. These results suggest that manipulating inhibition may help sensory processing in Fragile X syndrome.

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. β-adrenergic receptor (βAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate INaL. However, it remains unclear how each of these pathways regulates INaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on INaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that INaL had a basal level current independent of PKA, but partially dependent on CaMKII. βAR activation (10 nM isoproterenol, ISO) further enhanced INaL via both PKA and CaMKII pathways. However, PKA predominantly increased INaL early during the AP plateau, whereas CaMKII mainly increased INaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced INaL similar to the βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-induced CaMKII-dependent INaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced INaL activation early in the AP. Taken together, our data reveal differential modulations of INaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in INaL during AP deepens our understanding of the important role of INaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

Repeated spike firing can transmit information at synapses and modulate spike timing, shape, and conduction velocity. These latter effects have been found to result from voltage-induced changes in ion currents and could alter the signals carried by axons. Here, we test whether Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates spike propagation in adult rat optic nerve. We find that small-, medium-, and large-diameter axons bind anti-Thr286-phosphorylated CaMKII (pT286) antibodies and that, in isolated optic nerves, electrical stimulation reduces pT286 levels, spike propagation is hastened by CaMKII autophosphorylation and slowed by CaMKII dephosphorylation, single and multiple spikes slow propagation of subsequently activated spikes, and more frequent stimulation produces greater slowing. Likewise, exposing freely moving animals to flickering illumination reduces pT286 levels in optic nerves and electrically eliciting spikes in vivo in either the optic nerve or optic chiasm slows subsequent spike propagation in the optic nerve. By increasing the time that elapses between successive spikes as they propagate, pT286 dephosphorylation and activity-induced spike slowing reduce the frequency of propagated spikes below the frequency at which they were elicited and would thus limit the frequency at which axons synaptically drive target neurons. Consistent with this, the ability of retinal ganglion cells to drive at least some lateral geniculate neurons has been found to increase when presented with light flashes at low and moderate temporal frequencies but less so at high frequencies. Activity-induced decreases in spike frequency may also reduce the energy required to maintain normal intracellular Na+ and Ca2+ levels.SIGNIFICANCE STATEMENT By propagating along axons at constant velocities, spikes could drive synapses as frequently as they are initiated. However, the onset of spiking has been found to alter the conduction velocity of subsequent ("follower") spikes in various preparations. Here, we find that spikes reduce spike frequency in rat optic nerve by slowing follower spike propagation and that electrically stimulated spiking ex vivo and spike-generating flickering illumination in vivo produce net decreases in axonal Ca2+/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation. Consistent with these effects, propagation speed increases and decreases, respectively, with CaMKII autophosphorylation and dephosphorylation. Lowering spike frequency by CaMKII dephosphorylation is a novel consequence of axonal spiking and light adaptation that could decrease synaptic gain as stimulus frequency increases and may also reduce energy use.

The Ca2+-calmodulin dependent protein kinase II (CaMKII) is an established central mediator of electrophysiological and contractile responses to cardiac stress, and its hyper-activation in cardiac diseases has been linked to heart failure (HF) and arrhythmia. Here we summarize the evidence supporting the role of CaMKII as a critical nodal point for therapeutic intervention against HF and atrial and ventricular tachyarrhythmias. Targeting of CaMKII in heart with inhibitors possessing appropriate selectivity might represent a novel therapeutic approach for HF and arrhythmias.

Inflammation accompanies heart failure and is a mediator of cardiac fibrosis. CaMKIIδ plays an essential role in adverse remodeling and decompensation to heart failure. We postulated that inflammation is the mechanism by which CaMKIIδ contributes to adverse remodeling in response to nonischemic interventions. We demonstrate that deletion of CaMKIIδ in the cardiomyocyte (CKO) significantly attenuates activation of NF-κB, expression of inflammatory chemokines and cytokines, and macrophage accumulation induced by angiotensin II (Ang II) infusion. The inflammasome was activated by Ang II, and this response was also diminished in CKO mice. These events occurred prior to any evidence of Ang II-induced cell death. In addition, CaMKII-dependent inflammatory gene expression and inflammasome priming were observed as early as the third hour of infusion, a time point at which macrophage recruitment was not evident. Inhibition of either the inflammasome or monocyte chemoattractant protein 1 (MCP1) signaling attenuated macrophage accumulation, and these interventions, like cardiomyocyte CaMKIIδ deletion, diminished the fibrotic response to Ang II. Thus, activation of CaMKIIδ in the cardiomyocyte represents what we believe to be a novel mechanism for initiating inflammasome activation and an inflammatory gene program that leads to macrophage recruitment and ultimately to development of fibrosis.

CaMKII is one of the most studied synaptic proteins, but many critical issues regarding its role in synaptic function remain unresolved. Using a CRISPR-based system to delete CaMKII and replace it with mutated forms in single neurons, we have rigorously addressed its various synaptic roles. In brief, basal AMPAR and NMDAR synaptic transmission both require CaMKIIα, but not CaMKIIβ, indicating that, even in the adult, synaptic transmission is determined by the ongoing action of CaMKIIα. While AMPAR transmission requires kinase activity, NMDAR transmission does not, implying a scaffolding role for the CaMKII protein instead. LTP is abolished in the absence of CaMKIIα and/or CaMKIIβ and with an autophosphorylation impaired CaMKIIα (T286A). With the exception of NMDAR synaptic currents, all aspects of CaMKIIα signaling examined require binding to the NMDAR, emphasizing the essential role of this receptor as a master synaptic signaling hub.

In human drug users, cue-induced drug craving progressively intensifies after drug abstinence, promoting drug relapse. This time-dependent progression of drug craving is recapitulated in rodent models, in which rats exhibit progressive intensification of cue-induced drug seeking after withdrawal from drug self-administration, a phenomenon termed incubation of drug craving. Although recent results suggest that functional alterations of the nucleus accumbens (NAc) contribute to incubation of drug craving, it remains poorly understood how NAc function evolves after drug withdrawal to progressively intensify drug seeking. The functional output of NAc relies on how the membrane excitability of its principal medium spiny neurons (MSNs) translates excitatory synaptic inputs into action potential firing. Here, we report a synapse-membrane homeostatic crosstalk (SMHC) in male rats, through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the intrinsic membrane excitability of NAc MSNs, and vice versa. After short-term withdrawal from cocaine self-administration, despite no actual change in the AMPA receptor-mediated excitatory synaptic strength, GluN2B NMDA receptors, the SMHC sensors of synaptic strength, are upregulated. This may create false SMHC signals, leading to a decrease in the membrane excitability of NAc MSNs. The decreased membrane excitability subsequently induces another round of SMHC, leading to synaptic accumulation of calcium-permeable AMPA receptors and upregulation of excitatory synaptic strength after long-term withdrawal from cocaine. Disrupting SMHC-based dysregulation cascades after cocaine exposure prevents incubation of cocaine craving. Thus, cocaine triggers cascades of SMHC-based dysregulation in NAc MSNs, promoting incubated cocaine seeking after drug withdrawal.SIGNIFICANCE STATEMENT Here, we report a bidirectional homeostatic plasticity between the excitatory synaptic input and membrane excitability of nucleus accumbens (NAc) medium spiny neurons (MSNs), through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the membrane excitability, and vice versa. Cocaine self-administration creates a false homeostatic signal that engages this synapse-membrane homeostatic crosstalk mechanism, and produces cascades of alterations in excitatory synapses and membrane properties of NAc MSNs after withdrawal from cocaine. Experimentally preventing this homeostatic dysregulation cascade prevents the progressive intensification of cocaine seeking after drug withdrawal. These results provide a novel mechanism through which drug-induced homeostatic dysregulation cascades progressively alter the functional output of NAc MSNs and promote drug relapse.

Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKIIβ signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKIIβ signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKIIβ regulates remodeling in multiple contexts: endurance exercise and growth after atrophy.

Treatment for Angelman syndrome (AS) is currently limited to symptomatic interventions. A mouse model of AS has reduced calcium/calmodulin-dependent kinase II activity due to excessive phosphorylation of specific threonine residues, leading to diminished long-term potentiation. In a rat model of Parkinson disease, levodopa reduced phosphorylation of various proteins, including calcium/calmodulin-dependent kinase II. Further studies demonstrated that AS mice treated with levodopa performed better on rotarod testing than untreated AS mice. We conducted a multi-center double-blind randomized placebo-controlled 1-year trial of levodopa / carbidopa with either 10 or 15 mg/kg/day of levodopa in children with AS. The outcome of this intervention was assessed using either the Bayley Scales of Infant Development or the Mullen Scales of Early Learning, as well as the Vineland Adaptive Behavior Scales, and the Aberrant Behavior Checklist. Of the 78 participants enrolled, 67 participants received study medication (33 on levodopa, 34 on placebo), and 55 participants (29 on levodopa, 26 on placebo) completed the 1-year study. There were no clinically or statistically significant changes in any of the outcome measures over a 1-year period comparing the levodopa and placebo groups. The number of adverse events reported, including the more serious adverse events, was similar in both groups, but none were related to treatment with levodopa. Our data demonstrate that levodopa is well-tolerated by children with AS. However, in the doses used in this study, it failed to improve their neurodevelopment or behavioral outcome.

RationaleAtrial fibrillation (AF) is the most common arrhythmia, and advanced age is an inevitable and predominant AF risk factor. However, the mechanisms that couple aging and AF propensity remain unclear, making targeted therapeutic interventions unattainable.ObjectiveTo explore the functional role of an important stress response JNK (c-Jun N-terminal kinase) in sarcoplasmic reticulum Ca2+ handling and consequently Ca2+-mediated atrial arrhythmias.Methods and resultsWe used a series of cutting-edge electrophysiological and molecular techniques, exploited the power of transgenic mouse models to detail the molecular mechanism, and verified its clinical applicability in parallel studies on donor human hearts. We discovered that significantly increased activity of the stress response kinase JNK2 (JNK isoform 2) in the aged atria is involved in arrhythmic remodeling. The JNK-driven atrial proarrhythmic mechanism is supported by a pathway linking JNK, CaMKII (Ca2+/calmodulin-dependent kinase II), and sarcoplasmic reticulum Ca2+ release RyR2 (ryanodine receptor) channels. JNK2 activates CaMKII, a critical proarrhythmic molecule in cardiac muscle. In turn, activated CaMKII upregulates diastolic sarcoplasmic reticulum Ca2+ leak mediated by RyR2 channels. This leads to aberrant intracellular Ca2+ waves and enhanced AF propensity. In contrast, this mechanism is absent in young atria. In JNK challenged animal models, this is eliminated by JNK2 ablation or CaMKII inhibition.ConclusionsWe have identified JNK2-driven CaMKII activation as a novel mode of kinase crosstalk and a causal factor in atrial arrhythmic remodeling, making JNK2 a compelling new therapeutic target for AF prevention and treatment.