Calmodulin-dependent Protein Kinase II Inhibitor Research Articles

CaMKII Exacerbates Doxorubicin-Induced Cardiotoxicity by Promoting Ubiquitination Through USP10 Inhibition.

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

Calmodulin kinase II inhibition suppresses atrioventricular conduction by regulating intracellular Ca2+ homeostasis

BackgroundCa2+/calmodulin-dependent protein kinase II (CaMKII) inhibition decelerates atrioventricular node (AVN) conduction, providing a potential treatment of tachycardia. However, the effectiveness of CaMKII inhibition on tachycardia and its underlying mechanism remains unclear. ObjectiveWe aimed to assess the effectiveness of CaMKII inhibition in reducing ventricular rates during atrial fibrillation and to elucidate the underlying mechanism in affecting AVN electrophysiology. MethodsCardiac CaMKII inhibition (AC3-I) mice were used. Transesophageal atrial pacing was performed to evaluate AVN conduction function and to induce atrial fibrillation. Patch-clamp techniques were employed to record action potentials and ionic currents in AVN cells. Intracellular Ca2+ transients and sarcomere length measurements were obtained with the IonOptix system. Masson trichrome stain was used to evaluate fibrosis in the AVN region. Western blotting and immunofluorescence techniques were employed to detect connexin expression and localization. ResultsCaMKII inhibition decreased the ventricular rate during atrial fibrillation and isoproterenol-induced tachycardia. Esophageal electrocardiogram results from AC3-I mice showed longer AVN conduction than in wild-type mice. AN- and N-type AVN cells from AC3-I mice exhibited slower action potential frequencies and diastolic depolarization rates than those of wild-type mice. The study revealed that CaMKII inhibition reduced AVN cell sarcoplasmic reticulum (SR) Ca2+ content, Ca2+ release rate from the SR during diastole, Ca2+ transient amplitude, and SR Ca2+ uptake rate. In addition, CaMKII inhibition prolonged the sarcomere diastole duration and enhanced the sensitivity of sarcomeres to Ca2+. ConclusionCaMKII inhibition effectively decreases the ventricular rate during atrial fibrillation and tachycardia by slowing down AVN conduction through suppressing Ca2+ overload in AVN cells.

SGLT2 inhibitors protect against diabetic cardiomyopathy and atrial fibrillation through a CaMKII independent mechanism.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as an important mediator of the increasingly evident cardioprotective benefits exerted by sodium-glucose transport protein 2 channel inhibitors (SGLT2i). However, the exact nature of the relationship between CaMKII and SGLT2i remains unclear. Here, we find that empagliflozin but not dapagliflozin attenuated susceptibility to atrial fibrillation (AF) in a type 2 diabetic (T2D) mouse model. However, both empagliflozin and dapagliflozin protected from diabetic cardiomyopathy in T2D mice. We then used real-time microscopy of neonatal rat ventricular cardiomyocytes (NRVMs) with the CaMKII biosensor - CaMKAR to demonstrate that direct inhibition of CaMKII is not essential for the effects of SGLT2i in these cells. Therefore, we conclude that the benefits of SGLT2i in heart disease likely occur through indirect modulation of CaMKII activity, or possibly through an alternative pathway altogether.

CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction

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

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

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

Abstract Or102: Macrophage-Mediated Interleukin-6 Signaling Drives Ryanodine Receptor-2 Calcium Leak in Postoperative Atrial Fibrillation

Postoperative atrial fibrillation (poAF) is self-limited AF that occurs days after cardiac surgery in one-third of patients. The degree of perioperative increase in systemic inflammation, particularly involving interleukin (IL)-6, correlates with poAF risk. However, no studies to-date have identified the cell types involved nor mechanistically linked IL-6 signaling to fundamental arrhythmia mechanisms. To study poAF, we developed a mouse model consisting of atrial pericardiectomy and transient aortic cross-clamping followed by intracardiac burst pacing on postoperative day three to assess poAF inducibility. Single-cell RNA sequencing of atrial non-myocytes revealed macrophages to be the most prominently altered atrial cell type – increased 2.4-fold ( P <0.001) in mice with versus without poAF. Pseudotime trajectory analyses revealed IL-6 to be the top cytokine altered in macrophages, which we confirmed by western blot demonstrating a 1.7-fold ( P =0.012) increase in atrial IL-6 protein in mice with versus without poAF. Indeed, both macrophage depletion and conditional knockout of IL-6Ra in macrophages decreased poAF inducibility by 5.2-fold ( P =0.048) and 5.5-fold ( P =0.027), respectively. Downstream inhibition using an FDA-approved STAT3 inhibitor also rescued poAF (6.0-fold decrease, P =0.022). At the single atrial cardiomyocyte (ACM) level, IL-6 challenge led to a 6.3-fold ( P <0.001) increase in calcium (Ca 2+ ) spark frequency after normalization to sarcoplasmic reticulum Ca 2+ load, indicative of ryanodine receptor-2 (RyR2) dysfunction. Indeed, RyR2 phosphorylation at Ser2814, the target of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), was increased in the atria of mice with poAF, and CaMKII inhibition was sufficient to rescue arrhythmogenic Ca 2+ sparks in ACMs from mice with poAF. We corroborated our findings in human ACMs and atrial tissue to demonstrate translational relevance. Taken together, we use an integrated approach incorporating mouse and human biochemical, functional, and single-cell sequencing data to demonstrate that infiltrating atrial macrophages, specifically IL-6Ra from macrophages, are fundamentally required for poAF development ( Fig. 1 ). Molecularly, enhanced atrial IL-6 signaling led to arrhythmogenic RyR2 dysfunction through CaMKII. Our findings portend therapeutic utility by providing mechanistic evidence that targeting the IL-6 axis may be used for poAF prevention and treatment in a clinically amenable timeframe.

Angiotensin II-mediated oxidative stress increased the vulnerability of ventricular arrhythmia in cardiac hypertrophy rabbit model, which is suppressed by CaMKII inhibitor

Abstract Objectives Angiotensin system is a major cause of heart failure and arrhythmia. Despite a lack of direct evidence that oxidative stress causes ventricular arrhythmia, reversal of oxidative stress is considered a plausible therapy. This study evaluated the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor could suppress arrhythmia in cardiac hypertrophy rabbit model. Methods Angiotensin II (Ang II) or saline was administrated for 2 weeks via osmotic minipumps implanted subcutaneously in the midclavicular region. Hearts were perfused, mapped optically to analyze action potential durations (APD), and restitution kinetics, and tested for VF vulnerability. The intracellular calcium dynamics were measured in cardiomyocyte treated with Ang II (10 ng/ml) for 1 hours. Results In Ang II rabbit groups, 2(30%) rabbits died and had ventricular arrhythmia and hearts had enlarged left ventricle, longer APD90, slower conduction velocity (CV; P<0.01 versus control) and higher levels of transcripts for CaMKII, PLB, RyR2 (P<0.05 versus control). N-acetylcysteine treatment reversed the transcripts for APD90 and CV (P<0.01). Programmed stimulation triggered VF in Ang II (n=5/6) and Ang II + saline (n=4/6), but not in control (n=0/10, P<0.01). Cardiomyocyte induced with Ang II showed increased spontaneous Ca2+ release (Ca2+ Wave Frequency; 1.0±0.0 vs. 5.1±0.5, p=0.001, Ca2+ Amplitude; 1.0±0.0 vs. 1.3±0.1, p=0.001) with control. CaMKII inhibitor reversed the change of Ca2+. Conclusion The Ang II group had an increased incidence of arrhythmia caused by increased phosphorylation of Ca2+ handling proteins. These changes were partially reversed by CaMKII inhibition.

The adipose-neural axis is involved in epicardial adipose tissue-related cardiac arrhythmias

Dysfunction of the sympathetic nervous system and increased epicardial adipose tissue (EAT) have been independently associated with the occurrence of cardiac arrhythmia. However, their exact roles in triggering arrhythmia remain elusive. Here, using an invitro coculture system with sympathetic neurons, cardiomyocytes, and adipocytes, we show that adipocyte-derived leptin activates sympathetic neurons and increases the release of neuropeptide Y (NPY), which in turn triggers arrhythmia in cardiomyocytes by interacting with the Y1 receptor (Y1R) and subsequently enhancing the activity of the Na+/Ca2+ exchanger (NCX) and calcium/calmodulin-dependent protein kinase II (CaMKII). The arrhythmic phenotype can be partially blocked by a leptin neutralizing antibody or an inhibitor of Y1R, NCX, or CaMKII. Moreover, increased EAT thickness and leptin/NPY blood levels are detected in atrial fibrillation patients compared with the control group. Our study provides robust evidence that the adipose-neural axis contributes to arrhythmogenesis and represents a potential target for treating arrhythmia.

Differential sex-dependent susceptibility to diastolic dysfunction and arrhythmia in cardiomyocytes from obese diabetic HFpEF model.

Sex-differences in heart failure with preserved ejection fraction (HFpEF) are important, but key mechanisms involved are incompletely understood. While animal models can inform about sex-dependent cellular and molecular changes, many previous preclinical HFpEF models have failed to recapitulate sex-dependent characteristics of human HFpEF. We tested for sex-differences in HFpEF using a two-hit mouse model (leptin receptor-deficient db/db mice plus aldosterone infusion for 4 weeks; db/db+Aldo). We performed echocardiography, electrophysiology, intracellular Ca2+ imaging, and protein analysis. Female HFpEF mice exhibited more severe diastolic dysfunction in line with increased titin N2B isoform expression and PEVK element phosphorylation, and reduced troponin-I phosphorylation. Female HFpEF mice had lower BNP levels than males despite similar comorbidity burden (obesity, diabetes) and cardiac hypertrophy in both sexes. Male HFpEF mice were more susceptible to cardiac alternans. Male HFpEF cardiomyocytes (versus female) exhibited higher diastolic [Ca2+], slower Ca2+ transient decay, reduced L-type Ca2+ current, more pronounced enhancement of the late Na+ current, and increased short-term variability of action potential duration (APD). However, male and female HFpEF myocytes showed similar downregulation of inward rectifier and transient outward K+ currents, APD prolongation, and frequency of delayed afterdepolarizations. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed all pathological APD changes in HFpEF in both sexes, and empagliflozin pretreatment mimicked these effects of CaMKII inhibition. Vericiguat had only slight benefits, and these effects were larger in HFpEF females. We conclude that the db/db+Aldo preclinical HFpEF murine model recapitulates key sex-specific mechanisms in HFpEF and provides mechanistic insights into impaired excitation-contraction coupling and sex-dependent differential arrhythmia susceptibility in HFpEF with potential therapeutic implications. In male HFpEF myocytes, altered Ca2+ handling and electrophysiology aligned with diastolic dysfunction and arrhythmias, while worse diastolic dysfunction in females may depend more on altered myofilaments properties.

PFOS Elicits Cytotoxicity in Neuron Through Astrocyte-Derived CaMKII-DLG1 Signaling In Vitro Rat Hippocampal Model.

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

Rem2 interacts with CaMKII at synapses and restricts long-term potentiation in hippocampus.

Synaptic plasticity, the process whereby neuronal connections are either strengthened or weakened in response to stereotyped forms of stimulation, is widely believed to represent the molecular mechanism that underlies learning and memory. The holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) plays a well-established and critical role in the induction of a variety of forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) and depotentiation. Previously, we identified the GTPase Rem2 as a potent, endogenous inhibitor of CaMKII. Here, we report that knock out of Rem2 enhances LTP at the Schaffer collateral to CA1 synapse in hippocampus, consistent with an inhibitory action of Rem2 on CaMKII in vivo. Further, re-expression of WT Rem2 rescues the enhanced LTP observed in slices obtained from Rem2 conditional knock out (cKO) mice, while expression of a mutant Rem2 construct that is unable to inhibit CaMKII in vitro fails to rescue increased LTP. In addition, we demonstrate that CaMKII and Rem2 interact in dendritic spines using a 2pFLIM-FRET approach. Taken together, our data lead us to propose that Rem2 serves as a brake on synaptic potentiation via inhibition of CaMKII activity. Further, the enhanced LTP phenotype we observe in Rem2 cKO slices reveals a previously unknown role for Rem2 in the negative regulation of CaMKII function.

Ursolic acid inhibits the synaptic release of glutamate and prevents glutamate excitotoxicity in rats

The present study evaluated the effect of ursolic acid, a natural pentacyclic triterpenoid, on glutamate release in rat cortical nerve terminals (synaptosomes) and its neuroprotection in a kainic acid-induced excitotoxicity rat model. In cortical synaptosomes, ursolic acid produced a concentration-dependent inhibition of evoked glutamate release with a half-maximum inhibition of release value of 9.5 μM, and calcium-free medium and the P/Q -type Ca2+ channel blocker, ω-agatoxin IVA, but not ω-conotoxin GVIA, an N-type Ca2+ channel blocker, prevented the ursoloic acid effect. The molecular docking study indicated that ursolic acid interacted with P/Q-type Ca2+ channels. Ursolic acid also significantly decreased the depolarization-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the subsequent phosphorylation of synapsin I, and the ursolic acid effect on evoked glutamate release was inhibited by the CaMKII inhibitor KN 62 in synaptosomes. In addition, in rats that were intraperitoneally injected with ursolic acid 30 min before kainic acid intraperitoneal injection, cortical neuronal degeneration was attenuated. This effect of ursolic acid in the improvement of kainic acid-induced neuronal damage was associated with the reduction of kainic acid-induced glutamate increase in the cortex of rats; this was characterized by the reduction of glutamate and glutaminase levels and elevation of glutamate dehydrogenase, glutamate transporter 1, glutamate-aspartate transporter, and glutamine synthetase protein levels. These results suggest that ursolic acid inhibits glutamate release from cortical synaptosomes by decreasing P/Q-type Ca2+ channel activity and subsequently suppressing CaMKII and exerts a preventive effect against glutamate neurotoxicity by controlling glutamate levels.

Hippocampal CaMKII inhibition induces reactivation-dependent amnesia for extinction memory and causes fear relapse

Hippocampal GluN2B subunit-containing NMDAR (GluN2B-NMDAR) activation during recall destabilizes fear extinction memory, which must undergo brain-derived neurotrophic factor (BDNF)-dependent reconsolidation to persist. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr protein kinase essential for hippocampus-dependent memory processing that acts downstream GluN2B-NMDAR and controls BDNF expression, but its participation in fear extinction memory reconsolidation has not yet been studied. Using a combination of pharmacological and behavioral tools, we found that in adult male Wistar rats, intra dorsal-CA1 administration of the CaMKII inhibitors autocamtide-2-related inhibitory peptide (AIP) and KN-93, but not of their inactive analogs scrambled AIP and KN-92, after fear extinction memory recall impaired extinction and caused GluN2B-NMDAR-dependent recovery of fear. Our results indicate that hippocampal CaMKII is necessary for fear extinction reconsolidation, and suggest that modulation of its activity around the time of recall controls the inhibition that extinction exerts on learned fear.

Abstract 13500: An Improved Reporter Identifies Ruxolitinib as a Potent and Cardioprotective CaMKII Inhibitor

Background: Ca 2+ /Calmodulin-dependent protein kinase II (CaMKII) hyperactivity is an established driver of cardiac arrhythmias. Despite proven benefits of CaMKII inhibition in numerous preclinical arrhythmia models, translation of CaMKII antagonists into humans has remained unsuccessful, and today, there are no clinically approved CaMKII inhibitors. Our ability to identify potent CaMKII inhibitors has been hamstrung by a lack of a CaMKII biosensor with sufficient sensitivity for high throughput drug discovery. Research question: We hypothesized that development of a CaMKII biosensor with high throughput capabilities would enable identification of CaMKII inhibitors capable of preventing arrhythmias. Methods: To address this problem, we engineered and validated a new genetically encoded CaMKII biosensor: CaMKAR (CaMKII Activity Reporter). CaMKAR enjoys a dynamic range of 227 ± 11.1 %, seconds-scale kinetics, robust specificity, and dual in cellulo and in vitro functionality. This makes CaMKAR the fastest and most sensitive CaMKII biosensor to date and the first that is amenable for in cellulo drug screening. Results: Using CaMKAR, we carried out a drug repurposing screen (4,475 compounds in clinical use) in human cells. This yielded five previously unrecognized CaMKII inhibitors: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib (false discovery-adjusted p<3 x 10 -5 ). Stand out among these, ruxolitinib, an FDA-approved Janus kinase inhibitor, blocked CaMKII kinase activity with an inhibitory constant of 23.5 ± 2.2 nM. In patient-derived cardiomyocytes, ruxolitinib ameliorated arrhythmogenic Ca 2+ release events (p<0.0001), and in mice, ruxolitinib prevented catecholaminergic polymorphic ventricular tachycardia (p<0.01) and rescued pre-existing atrial fibrillation (p<0.0001). Conclusion: Our work presents three impactful Conclusions: 1) Our biosensor, CaMKAR, provides the highest CaMKII activity reporting performance to date. 2) Long sought-after CaMKII inhibitors already exist within the human pharmacopeia. 3) Our results demonstrate that ruxolitinib—a drug already in human use—is a cardioprotective CaMKII inhibitor and a suitable candidate for cardiovascular repurposing.

Abstract 12726: Novel, Potent, and Highly Selective Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Inhibitors Reduce Substrate Phosphorylation in Rat Hearts and Prolong Survival in a Mouse Model of Severe Heart Failure

Introduction: CaMKII is a target of longstanding interest in drug development and has been validated in multiple preclinical models of heart failure, as well as in arrhythmia diseases, including catecholaminergic polymorphic ventricular tachycardia and atrial fibrillation. We describe here the pharmacological profiles of a novel oral CaMKII inhibitor, CRD-2015. We demonstrate the ability of CRD-2015 to reduce substrate phosphorylation in rat hearts, and to strongly prolong survival in the calsequestrin transgenic (CSQ-Tg) mouse model of severe heart failure. Results: CRD-2015 is a potent (IC 50 0.62nM), highly selective CaMKII inhibitor. Oral administration of CRD-2015 dose-dependently decreased phospholamban (P-PLN) and ryanodine receptor 2 (P-RyR2) phosphorylation at their CaMKII-specific sites (Thr17 and Ser2814, respectively) in normal rat hearts, with strong correlation between the plasma concentrations (PK) and the reduction in phosphorylation (PD) (Figure, A). In CSQ-Tg mice, CRD-2015 also decreased cardiac P-PLN and P-RyR2 in a dose-dependent manner. Chronic administration of CRD-2015 prolonged survival in a dose-dependent manner (Figure, B). At the maximum dose level, all CSQ-Tg mice survived for 27 days (n=15/15) with the treatment of CRD-2015, while no mice survived past 24 days in the vehicle-treated group (n=0/15). Similar dose and concentration-dependent reductions in P-PLN and P-RyR2 and prolongation of survival in CSQ-Tg mice were also detected with a second potent (IC 50 0.40nM) and selective CaMKII inhibitor, CRD-2959. Conclusions: Our novel CaMKII inhibitor, CRD-2015 shows a clear PK/PD relationship in normal rats and prolongs survival in a mouse model of severe heart failure. These results demonstrate the successful creation of specific CaMKII inhibitors with efficacy in preclinical models and support their clinical development for the treatment of heart failure and other cardiovascular diseases.

Merazin hydrate produces rapid antidepressant effects by activating CaMKII to promote neuronal activities and proliferation in hippocampus

In our previous studies, we demonstrated that merazin hydrate (MH) had rapid antidepressant effects, but the deep mechanism needed to be further investigated. In this study, we used depressive-like model, behavioral tests, molecular biology and pharmacological interventions to reveal the underlying mechanisms of MH’s rapid antidepressants. We found that a single administration of MH was able to produce rapid antidepressant effects in chronic unpredictable mild stress (CUMS) exposed mice at 1 day later, similar to ketamine. Moreover, MH could not only significantly up-regulated the expressions of cFOS, but also obviously increased the number of Ki67 positive cells in hippocampal dentate gyrus (DG). Furthermore, we also found that the phosphorylated expression of calcium/calmodulin-dependent protein kinase II (CaMKII) was significantly reduced by CUMS in hippocampus, which was also reversed by MH. In addition, pharmacological inhibition of CaMKII by using KN-93 (a CaMKII antagonist) blocked the MH’s up-regulation of cFOS and Ki67 in hippocampal DG. To sum up, this study demonstrated that MH produced rapid antidepressant effects by activating CaMKII to promote neuronal activities and proliferation in hippocampus.

Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signalling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response a Ca2+ stimulus, the increase in CAMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CAMKII and regulates cardiomyocyte Ca2+ signalling which could have important implications for cardiac function.

Abstract P3195: Mitochondrial CaMKII Drives Cardiometabolic Dysfunction In Heart Failure

Background: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is an established negative regulator of cardiac injury. Both the expression and activity levels of CaMKII delta, the primary cardiac isoform of this kinase, are elevated in models of heart failure (HF) such as ischemia-reperfusion (I/R) injury and following myocardial infarction (MI). This upregulation is due in part to CaMKII’s role in the regulation of excitation-contraction coupling, apoptosis, activation of hypertrophic programming, arrhythmias and pro-inflammatory signaling. Recently, a pool of mitochondrial CaMKII (mtCaMKII) has been identified; we have demonstrated that there is an observed increase in mitochondrial CaMKII activation with left ventricular dilation acutely following injury in a mouse model of MI. This deleterious post-MI phenotype is rescued with genetic mitochondrial CaMKII inhibition; conversely, mice with myocardial mitochondrial CaMKII overexpression present with a severe dilated cardiomyopathy phenotype, alterations in electron transport chain (ETC) activity, and decreased ATP production. Objective: To date, the molecular mechanisms by which CaMKII regulates mitochondrial energetics are unknown and present a novel therapeutic target for HF which we are currently studying. Methods: As we have identified changes in the activity of enzymes, particularly complexes I and II, in the mitochondrial ETC as well as TCA cycle in response to increased CaMKII levels or activity, we are in the process of assessing changes in the mitochondrial CaMKII interactome between uninjured and failing hearts. Utilizing liquid chromatography-mass spectrometry (LCMS) and proteomics analysis of both scaffolding interactions alongside a novel ATP analogue labeling technique, we have mapped novel mitochondrial proteins which interact with mtCaMKII. Significance: The identification of previously unknown mitochondrial pathways involving CaMKII may uncover promising pharmacological targets for cardiovascular therapeutics, as this kinase is a critical player in the progression of HF.

NMDA receptor inhibitor MK801 alleviated pro-inflammatory polarization of BV-2 microglia cells

Microglia have both protective and pathogenic properties, while polarization plays a decisive role in their functional diversity. Apart from being an energetic organelle, mitochondria possess biological capabilities of signaling and immunity involving mitochondrial dynamics. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotransmission, excitatory neurotoxicity and pro-inflammatory properties in a membrane location- and cell context-dependent manner. In this study, we have provided experimental evidence showing that by acting on mitochondrial dynamics, NMDA receptors displayed pro-inflammatory properties, while its non-competitive inhibitor MK801 exhibited anti-inflammatory potential in Lipopolysaccharide (LPS)-challenged BV-2 microglia cells. LPS stimulation increased the protein phosphorylation of cells regarding their NMDA receptor component subunits and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), along with mobilizing intracellular calcium. Additionally, parallel changes occurred in the activation of Transforming Growth Factor-β (TGF-β)-Activated Kinase 1 (TAK1), NF-κB p65 and NF-κB DNA binding activity, acquisition of pro-inflammatory M1 polarization and expression of pro-inflammatory cytokines. LPS-treated cells further displayed signs of mitochondrial dysfunction with higher expressions of the active form of Dynamin-Related Protein 1 (Drp1), NADPH Oxidase-2 (NOX2) expression and the generation of DCFDA-/MitoSOX-sensitive Reactive Oxygen Species (ROS). NMDA receptor blockade by MK801, along with CaMKII inhibitor KN93, Drp1 inhibitor Mdivi-1 and antioxidant apocynin alleviated LPS-induced pro-inflammatory changes. Other than the reported CaMKII/TAK1/NF-κB axis, our in vitro study revealed the CaMKII/Drp1/ROS/NF-κB axis being an alternative cascade for shaping pro-inflammatory phenotypes of microglia upon LPS stimulation, and MK801 having the potential for inhibiting microglia activation and any associated inflammatory damages.

Increased CaMKII activation and contrast changes of cardiac β1-and β3-Adrenergic signaling pathways in a humanized angiotensinogen model of hypertension

AimsUpregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) contributes to the pathogenesis of cardiovascular disease, including hypertension. Transgenic rats expressing the human angiotensinogen gene [TGR (hAGT)L1623] are a new novel humanized model of hypertension that associates with declines in cardiac contractile function and β-adrenergic receptor (AR) reserve. The molecular mechanisms are unclear. We tested the hypothesis that in TGR (hAGT)L1623 rats, left ventricular (LV) myocyte CaMKIIδ and β3-AR are upregulated, but β1-AR is down-regulated, which are important causes of cardiac dysfunction and β-AR desensitization. Main methodsWe compared LV myocyte CaMKIIδ, CaMKIIδ phosphorylation (at Thr287) (pCaMKIIδ), and β1-and β3-AR expressions and determined myocyte functional and [Ca2+]I transient ([Ca2+]iT) responses to β-AR stimulation with and without pretreatment of myocytes using an inhibitor of CaMKII, KN-93 (10−6 M, 30 min) in male Sprague Dawley (SD; N = 10) control and TGR (hAGT)L1623 (N = 10) adult rats. Key findingsHypertension in TGR (hAGT)L1623 rats was accompanied by significantly increased LV myocyte β3-AR protein levels and reduced β1-AR protein levels. CaMKIIδ phosphorylation (at Thr287), pCaMKIIδ was significantly increased by 35%. These changes were followed by significantly reduced basal cell contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. Isoproterenol (10−8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax, and [Ca2+]iT. Moreover, only in TGR (hAGT)L1623 rats, pretreatment of LV myocytes with KN-93 (10−6 M, 30 min) fully restored normal basal and isoproterenol-stimulated myocyte contraction, relaxation, and [Ca2+]iT. SignificanceLV myocyte CaMKIIδ overactivation with associated contrast changes in β3-AR and β1-AR may be the key molecular mechanism for the abnormal contractile phenotype and β-AR desensitization in this humanized model of hypertension.