CaMKII Activity Research Articles

Hyperbaric Oxygen Therapy Attenuated the Motor Coordination and Cognitive Impairment of Polyglutamine Spinocerebellar Ataxia SCA17 Mice

Spinocerebellar ataxias (SCAs) are a large and diverse group of autosomal-dominant neurodegenerative diseases. No drugs have been approved for these relentlessly progressive and fatal SCAs. Our previous studies indicate that oxidative stress, neuroinflammation, and neuronal apoptosis are elevated in the SCA17 mice, which are the main therapeutic targets of hyperbaric oxygen treatment (HBOT). HBOT is considered to be an alternative and less invasive therapy for SCAs. In this study, we evaluated the HBOT (2.2 ATA for 14 days) effect and the persistence for the management of SCA17 mice and their wild-type littermates. We found HBOT attenuated the motor coordination and cognitive impairment of SCA17 mice and which persisted for about 1 month after the treatment. The results of several biochemistry and liver/kidney hematoxylin and eosin staining show the HBOT condition has no obvious toxicity in the mice. Immunostaining analyses show that the neuroprotective effect of HBOT could be through the promotion of BDNF production and the amelioration of neuroinflammation. Surprisingly, HBOT executes different effects on the male and female SCA17 mice, including the reduction of neuroinflammation and activation of CaMKII and ERK. This study suggests HBOT is a potential alternative therapeutic treatment for SCA17. Accumulated findings have revealed the similarity in disease pathomechanisms and possible therapeutic strategies in polyQ diseases; therefore, HBOT could be an optional treatment as well as the other polyQ diseases.

Targeting Cellular Retinoic Acid Binding Protein 1 with Retinoic Acid-like Compounds to Mitigate Motor Neuron Degeneration.

All-trans-retinoic Acid (atRA) is the principal active metabolite of Vitamin A, essential for various biological processes. The activities of atRA are mediated by nuclear RA receptors (RARs) to alter gene expression (canonical activities) or by cellular retinoic acid binding protein 1 (CRABP1) to rapidly (minutes) modulate cytosolic kinase signaling, including calcium calmodulin-activated kinase 2 (CaMKII) (non-canonical activities). Clinically, atRA-like compounds have been extensively studied for therapeutic applications; however, RAR-mediated toxicity severely hindered the progress. It is highly desirable to identify CRABP1-binding ligands that lack RAR activity. Studies of CRABP1 knockout (CKO) mice revealed CRABP1 to be a new therapeutic target, especially for motor neuron (MN) degenerative diseases where CaMKII signaling in MN is critical. This study reports a P19-MN differentiation system, enabling studies of CRABP1 ligands in various stages of MN differentiation, and identifies a new CRABP1-binding ligand C32. Using the P19-MN differentiation system, the study establishes C32 and previously reported C4 as CRABP1 ligands that can modulate CaMKII activation in the P19-MN differentiation process. Further, in committed MN cells, elevating CRABP1 reduces excitotoxicity-triggered MN death, supporting a protective role for CRABP1 signaling in MN survival. C32 and C4 CRABP1 ligands were also protective against excitotoxicity-triggered MN death. The results provide insight into the potential of signaling pathway-selective, CRABP1-binding, atRA-like ligands in mitigating MN degenerative diseases.

S-nitrosylation of CaMKII cys273 suppresses CaMKII activation and translocation within cardiac myocytes.

In this study, we demonstrate that the Cys273 site on Calcium/Calmodulin-Dependent Protein Kinase II δ (CaMKII δ) is critical for nitric oxide (NO)-induced suppression of CaMKIIδ activation and mobility in adult ventricular myocytes. CaMKII is also regulated by nitrosylation in a potential positive feedback mechanism via S-Nitrosylation of Cys290 on CaMKIIδ which promotes autonomous kinase activity, versus S-Nitrosylation of Cys273 that suppresses activation by Ca-CaM. This suggests a complex interaction between NO and CaMKII in cells with fluctuating [Ca] - like cardiomyocytes. The (patho)physiological role of CaMKII nitrosylation in the cardiomyocyte, however, remains poorly understood. Here, using mutant CaMKIIδ variants of our FRET-based CaMKII activity sensor ‘Camui’ and GFP-tagged constructs of CaMKIIδ lacking the Cys273 S-nitrosylation site (CaMKIIδ-C273S), we assessed CaMKII activation and mobility after exposure to the NO donor S-nitrosoglutathione (GSNO) and Ca. We determined this by measuring Camui fluorescence lifetime values (FLIM) and assessing changes in CaMKII-GFP mobility using FRAP, in cardiomyocytes in the presence and absence of pre-treatment with GSNO. We show that in ventricular cardiomyocytes, nitrosylation of CaMKII at Cys273 prior to electrical field stimulation and activation by Ca2+ or β-adrenergic agonists suppresses CaMKIIδ activity and mobility, whereas mutation of the Cys273 to Serine prevents this effect. These findings support the suggestion that CaMKII nitrosylation at Cys273 could protect from the overactivation of CaMKIIδ in the heart, which has been implicated in numerous pathological states, including the progression from hypertrophy to heart failure, cardiac fibrosis, diabetic hyperglycemia, and upon reperfusion after ischemia.

Empagliflozin prevents oxidative stress induced by acute hyperglycemia in cardiac myocytes.

Empagliflozin, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, has shown to improve cardiovascular outcomes in patients with diabetes and heart failure. Empagliflozin has also been shown to provide advantageous effects in excised hearts and isolated cardiomyocytes despite SGLT-2 expression lacking in cardiac myocytes. This suggests empagliflozin may have important off-target effects. Previous studies suggested that empagliflozin effects may include attenuation of both reactive oxygen species (ROS) and Ca/calmodulin-dependent kinase II activation, but exact signaling details remained unclear. Using transgenic mice expressing glutaredoxin 1-coupled redox-sensitive green fluorescent protein 2 (Grx1-roGFP2) sensor localized to either the cytosol or mitochondria, we tested for empagliflozin effect on oxidative stress in myocytes bathed in high-glucose (30 mmol/L) containing Tyrode's solution. To assess the changes in redox potential, we exposed each cell to H2O2 and dithiothreitol to calibrate the sensor. We found acute hyperglycemia induced significant increases in ROS over a 3-5min of perfusion, specifically in the cytosolic compartment. This is in line with previous report showing that hyperglycemia activates CaMKII by intracellular O-GlcNAcylation, then the active CaMKII promotes cytosolic ROS production via NAPDH oxidase 2 (NOX2). Importantly, cells preincubated with empagliflozin were fully protected against the hyperglycemia-induced increase in ROS. We also extended our studies on angiotensin II (AngII)-dependent ROS production because AngII activates NOX2, and the excess ROS promotes oxidation- dependent CaMKII activation. Myocytes pretreated with the selective CaMKII inhibitor autocamtide-2-relative inhibitory peptide (cell-permeable myristoylated form) were used to test the role of CaMKII in ROS production by either hyperglycemia or Ang-II. These studies could reveal whether empagliflozin acts on glucose uptake, CaMKII activation, or via direct antioxidant mechanisms. Our findings may provide a mechanistic basis for the positive effects of empagliflozin in myocytes under conditions relevant to heart disease.

CaMKII-Dependent Contractile Dysfunction and Pro-Arrhythmic Activity in a Mouse Model of Obstructive Sleep Apnea.

Left ventricular contractile dysfunction and arrhythmias frequently occur in patients with sleep-disordered breathing (SDB). The CaMKII-dependent dysregulation of cellular Ca homeostasis has recently been described in SDB patients, but these studies only partly explain the mechanism and are limited by the patients' heterogeneity. Here, we analyzed contractile function and Ca homeostasis in a mouse model of obstructive sleep apnea (OSA) that is not limited by confounding comorbidities. OSA was induced by artificial tongue enlargement with polytetrafluorethylene (PTFE) injection into the tongue of wildtype mice and mice with a genetic ablation of the oxidative activation sites of CaMKII (MMVV knock-in). After eight weeks, cardiac function was assessed with echocardiography. Reactive oxygen species (ROS) and Ca transients were measured using confocal and epifluorescence microscopy, respectively. Wildtype PTFE mice exhibited an impaired ejection fraction, while MMVV PTFE mice were fully protected. As expected, isolated cardiomyocytes from PTFE mice showed increased ROS production. We further observed decreased levels of steady-state Ca transients, decreased levels of caffeine-induced Ca transients, and increased pro-arrhythmic activity (defined as deviations from the diastolic Ca baseline) only in wildtype but not in MMVV PTFE mice. In summary, in the absence of any comorbidities, OSA was associated with contractile dysfunction and pro-arrhythmic activity and the inhibition of the oxidative activation of CaMKII conveyed cardioprotection, which may have therapeutic implications.

The beneficial effects of chick embryo extract preconditioning on hair follicle stem cells: A promising strategy to generate Schwann cells.

The beneficial effects of hair follicle stem cells in different animal models of nervous system conditions have been extensively studied. While chick embryo extract (CEE) has been used as a growth medium supplement for these stem cells, this is the first study to show the effect of CEE on them. The rat hair follicle stem cells were isolated and supplemented with 10% fetal bovine serum plus 10% CEE. The migration rate, proliferative capacity and multipotency were evaluated along with morphometric alteration and differentiation direction. The proteome analysis of CEE content identified effective factors of CEE that probably regulate fate and function of stem cells. The CEE enhances the migration rate of stem cells from explanted bulges as well as their proliferation, likely due to activation of AP-1 and translationally controlled tumour protein (TCTP) by thioredoxin found in CEE. The increased length of outgrowth may be the result of cyclic AMP response element binding protein (CREB) phosphorylation triggered by active CamKII contained in CEE. Further, CEE supplementation upregulates the expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. The elevated expression of target genes and proteins may be due to CREB, AP-1 and c-Myc activation in these stem cells. Given the increased transcript levels of neurotrophins, VEGF, and the expression of PDGFR-α, S100B, MBP and SOX-10 protein, it is possible that CEE promotes the fate of these stem cells towards Schwann cells.

The role of calcium, Akt and ERK signaling in cadmium-induced hair cell death

Exposure to heavy metals has been shown to cause damage to a variety of different tissues and cell types including hair cells, the sensory cells of our inner ears responsible for hearing and balance. Elevated levels of one such metal, cadmium, have been associated with hearing loss and shown to cause hair cell death in multiple experimental models. While the mechanisms of cadmium-induced cell death have been extensively studied in other cell types they remain relatively unknown in hair cells. We have found that calcium signaling, which is known to play a role in cadmium-induced cell death in other cell types through calmodulin and CaMKII activation as well as IP3 receptor and mitochondrial calcium uniporter mediated calcium flow, does not appear to play a significant role in cadmium-induced hair cell death. While calmodulin inhibition can partially protect hair cells this may be due to impacts on mechanotransduction activity. Removal of extracellular calcium, and inhibiting CaMKII, the IP3 receptor and the mitochondrial calcium uniporter all failed to protect against cadmium-induced hair cell death. We also found cadmium treatment increased pAkt levels in hair cells and pERK levels in supporting cells. This activation may be protective as inhibiting these pathways enhances cadmium-induced hair cell death rather than protecting cells. Thus cadmium-induced hair cell death appears distinct from cadmium-induced cell death in other cell types where calcium, Akt and ERK signaling all promote cell death.

Diminished α7 nicotinic acetylcholine receptor (α7nAChR) rescues amyloid-β induced atrial remodeling by oxi-CaMKII/MAPK/AP-1 axis-mediated mitochondrial oxidative stress

The potential coexistence of Alzheimer's disease (AD) and atrial fibrillation (AF) is increasingly common as aging-related diseases. However, little is known about mechanisms responsible for atrial remodeling in AD pathogenesis. α7 nicotinic acetylcholine receptors (α7nAChR) has been shown to have profound effects on mitochondrial oxidative stress in both organ diseases. Here, we investigate the role of α7nAChR in mediating the effects of amyloid-β (Aβ) in cultured mouse atrial cardiomyocytes (HL-1 cells) and AD model mice (APP/PS1). In vitro, apoptosis, oxidative stress and mitochondrial dysfunction induced by Aβ long-term (72h) in HL-1 cells were prevented by α-Bungarotoxin(α-BTX), an antagonist of α7nAChR. This cardioprotective effect was due to reinstating Ca2+ mishandling by decreasing the activation of CaMKII and MAPK signaling pathway, especially the oxidation of CaMKII (oxi-CaMKII). In vivo studies demonstrated that targeting knockdown of α7nAChR in cardiomyocytes could ameliorate AF progression in late-stage (12 months) APP/PS1 mice. Moreover, α7nAChR deficiency in cardiomyocytes attenuated APP/PS1-mutant induced atrial remodeling characterized by reducing fibrosis, atrial dilation, conduction dysfunction, and inflammatory mediator activities via suppressing oxi-CaMKII/MAPK/AP-1. Taken together, our findings suggest that diminished α7nAChR could rescue Aβ-induced atrial remodeling through oxi-CaMKII/MAPK/AP-1-mediated mitochondrial oxidative stress in atrial cells and AD mice.

Calcium/Calmodulin-Dependent Kinases in the Hypothalamus, Pituitary, and Pineal Gland: An Overview.

We review the literature on the little-known roles of specific CaMKs in regulating endocrine functions of the pineal gland, the pituitary gland, and the hypothalamus. Melatonin activates hippocampal CaMKII, which then influences dendritogenesis. In the pituitary gland, the signal pathways activated by the CaMK in lower vertebrates, such as fishes, differ from those of mammals. In the teleost anterior pituitary, the activation of CaMKII induces the expression of somatolactin by glucagon b. In rats and humans, CaMKIVs have been associated with gonadotropes and thyrotropes and CaMKII with several types of human tumor cells and with a specific signaling pathway. Neuropeptides such as vasopressin and endothelin are also involved in the CaMKII signaling chain, as is the CaMKIIδ isoform which participates in generating the circadian rhythms of the suprachiasmatic nucleus. What arises from this review is that most of the hypothalamic CaMKs are involved in activities of the endocrine brain. Furthermore, among the CaMKs, type II occurs with the highest frequency followed by CaMKIV and CaMKI.

A glutamate receptor C-tail recruits CaMKII to suppress retrograde homeostatic signaling.

Presynaptic homeostatic plasticity (PHP) adaptively enhances neurotransmitter release following diminished postsynaptic glutamate receptor (GluR) functionality to maintain synaptic strength. While much is known about PHP expression mechanisms, postsynaptic induction remains enigmatic. For over 20 years, diminished postsynaptic Ca2+ influx was hypothesized to reduce CaMKII activity and enable retrograde PHP signaling at the Drosophila neuromuscular junction. Here, we have interrogated inductive signaling and find that active CaMKII colocalizes with and requires the GluRIIA receptor subunit. Next, we generated Ca2+-impermeable GluRs to reveal that both CaMKII activity and PHP induction are Ca2+-insensitive. Rather, a GluRIIA C-tail domain is necessary and sufficient to recruit active CaMKII. Finally, chimeric receptors demonstrate that the GluRIIA tail constitutively occludes retrograde homeostatic signaling by stabilizing active CaMKII. Thus, the physical loss of the GluRIIA tail is sensed, rather than reduced Ca2+, to enable retrograde PHP signaling, highlighting a unique, Ca2+-independent control mechanism for CaMKII in gating homeostatic plasticity.

Subcortical glutamatergic inputs exhibit a Hebbian form of long-term potentiation in the dentate gyrus

The hippocampus receives glutamatergic and GABAergic inputs from subcortical regions. Despite the important roles of these subcortical inputs in the regulation of hippocampal circuit, it has not been explored whether associative activation of the subcorticohippocampal pathway induces Hebbian plasticity of subcortical inputs. Here, we demonstrate that the hypothalamic supramammillary nucleus (SuM) to the dentate granule cell (GC) synapses, which co-release glutamate and GABA, undergo associative long-term potentiation (LTP) of glutamatergic, but not GABAergic, co-transmission. This LTP is induced by pairing of SuM inputs with GC spikes. We found that this Hebbian LTP is input-specific, requires NMDA receptors and CaMKII activation, and is expressed postsynaptically. By the net increase in excitatory drive of SuM inputs following LTP induction, associative inputs of SuM and the perforant path effectively discharge GCs. Our results highlight the important role of associative plasticity at SuM-GC synapses in the regulation of dentate gyrus activity and for the encoding of SuM-related information.

Ablation of Shank3 alleviates cardiac dysfunction in aging mice by promoting CaMKII activation and Parkin-mediated mitophagy

Compromised mitophagy and mitochondrial homeostasis are major contributors for the etiology of cardiac aging, although the precise underlying mechanisms remains elusive. Shank3, a heart-enriched protein, has recently been reported to regulate aging-related neurodegenerative diseases. This study aimed to examine the role of Shank3 in the pathogenesis of cardiac senescence and the possible mechanisms involved. Cardiac-specific conditional Shank3 knockout (Shank3CKO) mice were subjected to natural aging. Mitochondrial function and mitophagy activity were determined in vivo, in mouse hearts and in vitro, in cardiomyocytes. Here, we showed that cardiac Shank3 expression exhibited a gradual increase during the natural progression of the aging, accompanied by overtly decreased mitophagy activity and a decline in cardiac function. Ablation of Shank3 promoted mitophagy, reduced mitochondria-derived superoxide (H2O2 and O2•−) production and apoptosis, and protected against cardiac dysfunction in the aged heart. In an in vitro study, senescent cardiomyocytes treated with D-gal exhibited reduced mitophagy and significantly elevated Shank3 expression. Shank3 knock-down restored mitophagy, leading to increased mitochondrial membrane potential, decreased mitochondrial oxidative stress, and reduced apoptosis in senescent cardiomyocytes, whereas Shank3 overexpression mimicked D-gal-induced mitophagy inhibition and mitochondrial dysfunction in normally cultured cardiomyocytes. Mechanistically, the IP assay revealed that Shank3 directly binds to CaMKII, and this interaction was further increased in the aged heart. Enhanced Shank3/CaMKII binding impedes mitochondrial translocation of CaMKII, resulting in the inhibition of parkin-mediated mitophagy, which ultimately leads to mitochondrial dysfunction and cardiac damage in the aged heart. Our study identified Shank3 as a novel contributor to aging-related cardiac damage. Manipulating Shank3/CaMKII-induced mitophagy inhibition could thus be an optional strategy for therapeutic intervention in clinical aging-related cardiac dysfunctions.

Abstract 14868: Identification of Novel Small Molecule Inhibitors of Calcium/Calmodulin-Dependent Protein Kinase II Delta for Treatment of Beta-Blocker Resistant Arrhythmia

Introduction. CaMKIId is a validated drug target in atrial fibrillation (AF), ventricular arrhythmia, and HF. As a key mediator of Ca 2+ action, it is hyperactivated by dysregulated Ca 2+ homeostasis with pathological consequences, which can be normalized by a variety of genetic and pharmacological manipulations. Objectives. Develop an ATP competitive CaMKIId inhibitor with nanomolar potency for initial efficacy in an AF mouse model and further optimization of selectivity, PK and low CNS penetration. Methods and Results. Molecular modeling using the crystal structure of human CaMKIId with several scaffolds as a starting point, led to development of a consistent structure activity relationship model and design of AS283. The molecule is an ATP competitive inhibitor with an IC 50 of 6nM and Ki of 1nM. Unlike KN-93, a common tool inhibitor of CaMKII in preclinical studies, AS283 inhibits the autophosphorylated, Ca 2+ -independent form of the kinase. AS283 shows preclinical proof of concept in both ventricular arrhythmia and AF models, with the latter described here. Mice with a gain of function mutation in the RyR2 receptor (R 178Q/+ ) do not undergo spontaneous AF but have an increased vulnerability to AF in response to atrial pacing. Intracardial electrophysiology with recording of atrial and ventricular electrograms (Na Li and Xander Wehrens, Baylor College) were used to select mutant and wild-type mice that exhibited AF in pacing trials. AF-positive animals were randomly assigned to treatment with placebo or AS283, with KN-93 as control. AS283 completely suppressed incidence of AF in comparison to placebo and exhibited a dose response. Autophosphorylation of CaMKIId at Thr287 was used as a biomarker of target engagement. As anticipated, atrial pacing increases intracellular Ca 2+ leading to activation of CaMKII and increased P-Thr287. AS283 blocked the increase in P-Thr287. Conclusion. AS283 is a novel, potent CaMKIId inhibitor with robust preclinical efficacy, supporting its further optimization for PK and low CNS penetration. Consistent with studies using genetic and pharmacological tools, an optimized, small molecule inhibitor of CaMKII has a promise in treating arrhythmias that are sub-optimally managed with or contraindicated to beta blockers.

β-hydroxybutyrate reduces reinstatement of cocaine conditioned place preference through hippocampal CaMKII-α β-hydroxybutyrylation.

Studies have shown the therapeutic effects of a ketogenic diet (KD) on epilepsy, but the effect of a KD on drug reinstatement is largely unclear. This study aims to investigate whether KD consumption possesses therapeutic potential for cocaine reinstatement and the molecular mechanism. We find that a KD significantly reduces cocaine-induced reinstatement in mice, which is accompanied by a markedly elevated level of β-hydroxybutyrate (β-OHB), the most abundant ketone body, in the hippocampus. The underlying mechanism is that β-OHB posttranslationally modifies CaMKII-α with β-hydroxybutyrylation, resulting in significant inhibition of T286 autophosphorylation and downregulation of CaMKII activity. Collectively, our results reveal that β-hydroxybutyrylation is a posttranslational modification of CaMKII-α that plays a critical role in mediating the effect of KD consumption in reducing cocaine reinstatement.

A multifarious exploration of synaptic tagging and capture hypothesis in synaptic plasticity: Development of an integrated mathematical model and computational experiments

The synaptic tagging and capture (STC) hypothesis not only explain the integration and association of synaptic activities, but also the formation of learning and memory. The synaptic pathways involved in the synaptic tagging and capture phenomenon are called STC pathways. The STC hypothesis provides a potential explanation of the neuronal and synaptic processes underlying the synaptic consolidation of memories. Several mechanisms and molecules have been proposed to explain the process of memory allocation and synaptic tags, respectively. However, a clear link between the STC hypothesis and memory allocation is still missing because the encoding of memories in neural circuits is mainly associated with strongly recurrently connected groups of neurons. To explore the mechanisms of potential synaptic tagging candidates and their involvement in the process of memory allocation, we develop a mathematical model for a single dendritic spine based on five essential criteria of a synaptic tag. By developing a mathematical model, we attempt to understand the roles of the potentially critical molecular networks underlying the STC and the essential attributes of a synaptic tag. We include essential memory molecules in the STC model that have been identified in earlier studies as crucial for STC pathways. CaMKII activation is critical for the setting of the initial tag; however, coordinated activities with other kinases and the biochemical pathways are necessary for the tag to be stable. PKA modulates NMDAR-mediated Ca2+ signalling. Similarly, PKA and ERK crosstalk is essential for Ca2+ - mediated protein synthesis during l-LTP. Our theoretical model explains the quantitative contribution of Tags and protein synthesis during l-LTP in synaptic strength.

Oxidative activation of CaMKII mediates arrhythmias and contractile dysfunction in an obstructive sleep apnea mouse model

Abstract Background/Introduction Atrial arrhythmias frequently occur in patients with obstructive sleep apnea (OSA), but the underlying mechanisms remain insufficiently understood. We recently demonstrated that CaMKII-dependent pro-arrhythmic activity is increased in patients with OSA, but the mechanisms of CaMKII activation remain unknown. Interestingly, OSA can lead to increased reactive oxygen species production, which may facilitate CaMKII activation by oxidation at methionine residues 281/282. Purpose We tested if oxidation of CaMKII is involved in the development of arrhythmias and contractile dysfunction in vitro and in vivo in a mouse model of obstructive sleep apnea by tongue enlargement. Methods Experiments were performed using 41 wild-type (WT) and 25 genetically modified mice lacking oxidative CaMKII activation due to mutation of methionine 281/282 to valine (MMVV mice). Polytetrafluorethylene (PTFE, 100 μl) was injected into the tongue of 23 WT and 12 MMVV mice to induce OSA by sustained tongue enlargement as previously established. Whole body plethysmography was performed to confirm success of intervention. Echocardiography was performed at baseline and after 8 weeks to assess left-ventricular function. After 8 weeks, isolated atrial and ventricular cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 AM (5 μM, 15 min) and analyzed with epifluorescence microscopy under regular electrical field stimulation (1 Hz). Results PTFE injection resulted in an increased frequency of inspiratory flow limitations (IFLs/h) from 27.4±5.95 to 59.6±6.22 in PTFE mice (p<0.001, fig. 1A). Interestingly, in WT PTFE mice, left ventricular ejection fraction (LVEF, in %) was reduced at 8 weeks post procedure from 57.0±1.36 to 51.5±1.85 (p=0.001, fig. 1B). Importantly, mice lacking oxidative CaMKII activation were protected from such decline in contractile function, and LVEF was higher at 8 weeks vs. WT PTFE (p<0.001, fig. 1B). Congruent with deterioration of contractility in vivo, the Ca transient amplitude after 30s pause (normalized to steady-state before pause) was decreased in ventricular cardiomyocytes of WT PTFE mice indicating increased SR Ca leak (p for interaction genotype x PTFE = 0.014, fig. 1C). In contrast to WT, the post-pause ratio was increased in MMVV PTFE mice (p=0.018, fig. 1C). In addition to contractile function, cellular arrhythmic events were analyzed as non-stimulated pro-arrhythmic events (NSEs) during steady-state electrical field stimulation at 1 Hz (fig. 1D, red arrows). NSE frequency was increased in atrial cardiomyocytes from WT PTFE mice with 0.050±0.005 compared to 0.018±0.003 s–1 in WT CTRL (p<0.001, fig. 1D). Intriguingly, MMVV mice were protected from NSEs after PTFE treatment (0.020±0.004, p<0.001 vs. WT PTFE, Fig. 1D). Conclusion In a mouse model of obstructive sleep apnea, contractile dysfunction and pro-arrhythmic activity are modulated by oxidative CaMKII activation, which may have therapeutic implications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft; Medical Faculty at University of Regensburg

Empagliflozin inhibits increased Na influx in HFpEF cardiomyocytes and reduces arrhythmic activity in human atrial trabeculae

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is a major health problem associated with substantial morbidity and mortality. However, the underlying pathophysiological mechanisms are poorly understood, and effective treatment strategies are scarce. Importantly, SGLT2i, which have been suggested to improve cellular Na and Ca homeostasis in HFrEF, have recently been shown to also improve clinical outcomes in patients with HFpEF. Interestingly, post-hoc analyses of clinical data suggest an involvement of anti-arrhythmic effects of SGLT2i. Purpose We tested, if isolated human atrial cardiomyocytes from patients with HFpEF exhibit an increased Na influx that is responsive to treatment with the SGLT2i empagliflozin (Empa) and if Empa has anti-arrhythmic properties in human atrial trabeculae. Methods Atrial biopsies were obtained from 101 patients undergoing elective cardiac surgery. Na influx was measured as increase in [Na]i during Na/K-ATPase inhibition in isolated cardiomyocytes loaded with the Na-sensitive fluorescence dye Asante Natrium Green–2 AM (ANG-2). Western Blot and HDAC4 pulldown assay were used to investigate NaV1.5 expression/phosphorylation as well as CaMKII expression/autophosphorylation and activity. Anti-arrhythmic effects of Empa were evaluated as the reduction in premature atrial complexes (PACs), which were induced in electrically field-stimulated (1Hz) human atrial trabeculae by superfusion with isoproterenol (100 nM) and high Ca (3.5 mM). Results Compared to patients without heart failure (NF), Na influx was almost doubled in HFpEF patients (NF vs HFpEF: 0.21±0.02 vs 0.38±0.04 mmol/L/min (N=7 vs 18); p=0.005) (Fig. 1D, E). CaMKII expression, CaMKII autophosphorylation, CaMKII activity, and CaMKII-dependent NaV1.5 phosphorylation were significantly increased in atrial biopsies of HFpEF patients, whereas NaV1.5 protein abundance remained unchanged (Fig. 1A–C). Consistent with these results, the increased Na influx was significantly reduced by treatment with the specific CaMKII inhibitor autocamtide-2 related inhibitory peptide (AIP) and the late INa inhibitor tetrodotoxin (TTX) (Fig. 1D, E). Importantly, Empa also abolished the increased Na influx in HFpEF cardiomyocytes (Fig. 1D, E). Multivariate linear regression analysis, adjusting for clinical co-variates, revealed HFpEF to be an independent predictor of cardiomyocyte Na handling. In line with Empa-mediated inhibition of Na influx, the frequency of PACs in human atrial trabeculae was significantly reduced by Empa (Fig. 1F, G). Conclusion This is the first study to demonstrate increased Na influx in human cardiomyocytes from HFpEF patients potentially by an increased CaMKII-dependent NaV1.5 phosphorylation. Excitingly, treatment with Empa decreases this Na influx in HFpEF cardiomyocytes and reduces isoproterenol-induced arrhythmic activity in human atrial trabeculae, which could contribute to the cardioprotective effects of this drug in patients with HFpEF. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Else Kröner-Fresenius-Stiftung,Deutsche Forschungsgemeinschaft

Chai-Hu-San-Shen Capsule Ameliorates Ventricular Arrhythmia Through Inhibition of the CaMKII/FKBP12.6/RyR2/Ca2+ Signaling Pathway in Rats with Myocardial Ischemia.

Ventricular arrhythmia is one of the main causes of sudden cardiac death, especially after myocardial ischemia. Previous studies have shown that Chai-Hu-San-Shen capsule (CHSSC) can reduce the incidence of ventricular arrhythmias following myocardial ischemia, however, the mechanisms of it are unclear. In present study, we explored the mechanism of CHSSC ameliorates ventricular arrhythmia following myocardial ischemia via inhibiting the CaMKII/FKBP12.6/RyR2/Ca2+ signaling pathway. In vivo, a myocardial ischemia rat model was established and treated with CHSSC to evaluate the therapeutic effect of CHSSC. In vitro, we established an ischemia model in H9C2 cells and treated with CHSSC, KN-93, or H-89. Then, intracellular Ca2+ content, the expression of RyR2, and the interaction between FKBP12.6 and RyR2 were detected. The results showed that CHSSC could delay the occurrence of ventricular arrhythmias and shorten the duration of ventricular arrhythmias. After myocardial ischemia, the intracellular Ca2+ content was increased, and CHSSC treatment mitigated this increase, down-regulated the levels of p-CaMKII, CaMKII, p-RyR2, and RyR2, and up-regulated the levels of p-RyR2 (Ser2808) and p-RyR2 (Ser2814). Co-immunoprecipitation showed an interaction between FKBP12.6 and RyR2, and CHSSC up-regulated the content of the FKBP12.6-RyR2 complex in ischemic cells. In conclusion, our study showed that CaMKII activation led to hyperphosphorylation of RyR2 (Ser2814) and RyR2 (Ser2808) during cardiomyocyte ischemia, which resulted in dissociation of the FKBP12.6-RyR2 complex, and increased intracellular Ca2+ content, which may contribute to the development of ventricular arrhythmias. CHSSC may reduce the incidence of ventricular arrhythmias following myocardial ischemia through inhibition of the CaMKII/RyR2/FKBP12.6/Ca2+ signaling pathway.

RGS11-CaMKII complex mediated redox control attenuates chemotherapy-induced cardiac fibrosis

Dose limiting cardiotoxicity remains a major limiting factor in the clinical use of several cancer chemotherapeutics including anthracyclines and the antimetabolite 5-fluorouracil (5-FU). Prior work has demonstrated that chemotherapeutics increase expression of R7 family regulator of G protein signaling (RGS) protein-binding partner Gβ5, which drives myocyte cytotoxicity. However, though several R7 family members are expressed in heart, the exact role of each protein in chemotherapy driven heart damage remains unclear. Here, we demonstrate that RGS11, downregulated in the human heart following chemotherapy exposure, possesses potent anti-apoptotic actions, in direct opposition to the actions of fellow R7 family member RGS6. RGS11 forms a direct complex with the apoptotic kinase CaMKII and stress responsive transcription factor ATF3 and acts to counterbalance the ability of CaMKII and ATF3 to trigger oxidative stress, mitochondrial dysfunction, cell death, and release of the cardiokine neuregulin-1 (NRG1), which mediates pathological intercommunication between myocytes and endothelial cells. Doxorubicin triggers RGS11 depletion in the murine myocardium, and cardiac-specific OE of RGS11 decreases doxorubicin-induced fibrosis, myocyte hypertrophy, apoptosis, oxidative stress, and cell loss and aids in the maintenance of left ventricular function. Conversely, RGS11 knockdown in heart promotes cardiac fibrosis associated with CaMKII activation and ATF3/NRG1 induction. Indeed, inhibition of CaMKII largely prevents the fibrotic remodeling resulting from cardiac RGS11 depletion underscoring the functional importance of the RGS11-CaMKII interaction in the pathogenesis of cardiac fibrosis. These data describe an entirely new role for RGS11 in heart and identify RGS11 as a potential new target for amelioration of chemotherapy-induced cardiotoxicity.

N-Methyl D-aspartate receptor subtype 2B/Ca2+/calmodulin-dependent protein kinase II signaling in the lateral habenula regulates orofacial allodynia and anxiety-like behaviors in a mouse model of trigeminal neuralgia.

Trigeminal neuralgia (TN) is a peripheral nerve disorder often accompanied by abnormalities in mood. The lateral habenula (LHb) plays important roles in the modulation of pain and emotion. In the present study, we investigated the involvement of the LHb in the mechanisms underlying allodynia and anxiety induced by partial transection of the infraorbital nerve (pT-ION) in mice. Our results indicated that pT-ION induced persistent orofacial allodynia and anxiety-like behaviors, which were correlated with increased phosphorylation of N-Methyl D-aspartate receptor (NMDAR) subtype 2B (p-NR2B) and Ca2+/calmodulin-dependent protein kinase II (p-CaMKII) in LHb neurons. Bilateral inhibition of NMDARs and CaMKII in the LHb attenuated the allodynia and anxiety-like behavior induced by pT-ION. Furthermore, bilateral activation of NMDARs in the LHb increased the expression of p-NR2B and p-CaMKII and induced orofacial allodynia and anxiety-like behaviors in naive mice. Adeno-associated virus (AAV)-mediated expression of hM3D(Gq) in CaMKII+ neurons of the bilateral LHb, followed by clozapine-N-oxide (CNO) administration, also triggered orofacial allodynia and anxiety-like behaviors in naïve mice with successful virus infection in LHb neurons (verified based on immunofluorescence). In conclusion, these findings suggest that activation of NMDA/CaMKII signaling in the LHb contributes to the occurrence and development of TN and related anxiety-like behaviors. Therefore, suppressing the activity of CaMKII+ neurons in the bilateral LHb by targeting NMDA/CaMKII may represent a novel strategy for treating pain and anxiety associated with TN.