Calmodulin-dependent Protein Kinase II Activity Research Articles

Prolonged β-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure.

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged β-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of β-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.

A Novel CaMKII Inhibitory Peptide Blocks Relapse to Morphine Seeking by Influencing Synaptic Plasticity in the Nucleus Accumbens Shell.

Drugs of abuse cause enduring functional disorders in the brain reward circuits, leading to cravings and compulsive behavior. Although people may rehabilitate by detoxification, there is a high risk of relapse. Therefore, it is crucial to illuminate the mechanisms of relapse and explore the therapeutic strategies for prevention. In this research, by using an animal model of morphine self-administration in rats and a whole-cell patch–clamp in brain slices, we found changes in synaptic plasticity in the nucleus accumbens (NAc) shell were involved in the relapse to morphine-seeking behavior. Compared to the controls, the amplitude of long-term depression (LTD) induced in the medium spiny neurons increased after morphine self-administration was established, recovered after the behavior was extinguished, and increased again during the relapse induced by morphine priming. Intravenous injection of MA, a new peptide obtained by modifying Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor “myr-AIP”, decreased CaMKII activity in the NAc shell and blocked the reinstatement of morphine-seeking behavior without influence on the locomotor activity. Moreover, LTD was absent in the NAc shell of the MA-pretreated rats, whereas it was robust in the saline controls in which morphine-seeking behavior was reinstated. These results indicate that CaMKII regulates morphine-seeking behavior through its involvement in the change of synaptic plasticity in the NAc shell during the relapse, and MA may be of great value in the clinical treatment of relapse to opioid seeking.

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca2+-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.

CaMKII binds both substrates and activators at the active site.

SUMMARYCa2+/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. When activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to the well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A long-standing model invokes two distinct S and T sites. If an interactor binds at the T-site, then it will preclude autoinhibition and allow substrates to be phosphorylated at the S site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model where persistent CaMKII activity is facilitated by high-affinity binding partners that kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.

Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by Inhibitor of Receptor Interacting Protein Kinase 3 Alleviates Necroptosis in Glycation End Products-Induced Cardiomyocytes Injury.

Necroptosisis a regulatory programmed form of necrosis. Receptor interacting protein kinase 3 (RIPK3) is a robust indicator of necroptosis. RIPK3 mediates myocardial necroptosis through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) in cardiac ischemia-reperfusion (I/R) injury and heart failure. However, the exact mechanism of RIPK3 in advanced glycation end products (AGEs)-induced cardiomyocytes necroptosis is not clear. In this study, cardiomyocytes were subjected to AGEs stimulation for 24 h. RIPK3 expression, CaMKII expression, and necroptosis were determined in cardiomyocytes after AGEs stimulation. Then, cardiomyocytes were transfected with RIPK3 siRNA to downregulate RIPK3 followed by AGEs stimulation for 24 h. CaMKIIδ alternative splicing, CaMKII activity, oxidative stress, necroptosis, and cell damage were detected again. Next, cardiomyocytes were pretreated with GSK′872, a specific RIPK3 inhibitor to assess whether it could protect cardiomyocytes against AGEs stimulation. We found that AGEs increased the expression of RIPK3, aggravated the disorder of CaMKII δ alternative splicing, promoted CaMKII activation, enhanced oxidative stress, induced necroptosis, and damaged cardiomyocytes. RIPK3 downregulation or RIPK3 inhibitor GSK′872 corrected CaMKIIδ alternative splicing disorder, inhibited CaMKII activation, reduced oxidative stress, attenuated necroptosis, and improved cell damage in cardiomyocytes.

The Regulatory Mechanism and Effect of Receptor-Interacting Protein Kinase 3 on Phenylephrine-Induced Cardiomyocyte Hypertrophy.

As a critical regulatory molecule, receptor-interacting protein kinase 3 (RIPK3) can mediate the signaling pathway of programmed necrosis. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been proved as a new substrate for RIPK3-induced necroptosis. In this study, we aimed to investigate the regulatory mechanism of RIPK3 on phenylephrine (PE)-induced cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy was induced by exposure to PE (100 μM) for 48 hours. Primary cardiomyocytes were pretreated with RIPK3 inhibitor GSK'872 (10 μM), and RIPK3 siRNA was used to deplete the intracellular expression of RIPK3. The indexes related to myocardial hypertrophy, cell injury, necroptosis, CaMKII activation, gene expression, oxidative stress, and mitochondrial membrane potential were measured. We found that after cardiomyocytes were stimulated by PE, the expressions of hypertrophy markers, atrial and brain natriuretic peptides (ANP and BNP), were increased, the release of lactate dehydrogenase was increased, the level of adenosine triphosphate (ATP) was decreased, the oxidation and phosphorylation levels of CaMKII were increased, and CaMKIIδ alternative splicing was disturbed. However, both GSK'872 and depletion of RIPK3 could reduce myocardial dysfunction, inhibit CaMKII activation and necroptosis, and finally alleviate myocardial hypertrophy. In addition, the pretreatment of RIPK3 could also lessen the accumulation of reactive oxygen species (ROS) induced by PE and stabilize the membrane potential of mitochondria. These results indicated that targeted inhibition of RIPK3 could suppress the activation of CaMKII and reduce necroptosis and oxidative stress, leading to alleviated myocardial hypertrophy. Collectively, our findings provided valuable insights into the clinical treatment of hypertrophic cardiomyopathy.

SIRT1‐mediated Lysine Crotonylation is Involved in the Regulation of CaMKII Activity in Myocardium

ObjectivesPost‐translational modifications (PTMs) can modify proteins and regulate their functions. Lysine crotonylation is a novel protein PTM that can be regulated by silent mating type information regulation 2 homolog 1 (SIRT1). Ca2+/calmodulin‐dependent protein kinase II (CaMKII) plays a significant role in mediating cellular responses to external signaling molecules. Little is known regarding the effect of SIRT1 on CaMKII crotonylation and the impact of crotonylation on the activity of this enzyme.MethodsCre‐Loxp technology was used to generate conditional cardiac‐specific SIRT1 knockout mice (SIRT1 KO). Protein crotonylation modification was studied by PTMomics. CaMKII activity was determined by ELISA. Western blot and co‐immunoprecipitation (CO‐IP) were performed to examine the expression and crotonylation of CaMKIIResultsPTMomics showed changes in protein crotonylation profiles in the myocardium of cardiac‐specific SIRT1 knockout mice. Crotonylation of CaMKII was significantly increased at lysine 301 and the enhanced CaMKII crotonylation was further confirmed by CO‐IP (2.37±0.25 in SIRT1 KO vs. 0.96±0.10 in wild‐type and 1.04±0.10 in SIRT1fl/fl mice, p<0.01). Cardiac‐specific knockout of SIRT1 did not affect the protein expression of CaMKII whereas significantly increased CaMKII activity (2.75±0.17 m units/μL in SIRT1 KO vs. 2.10±0.13 m units/μL in wild‐type and 1.98±0.17 m units/μL in SIRT1fl/fl mice, p<0.05). CO‐IP showed no physical interaction between SIRT1 and CaMKII.ConclusionsSIRT1 deficiency increases CaMKII activity in myocardium at least partially through enhancing lysine crotonylation of CaMKII, which advances our understanding of the role played by PTM in the regulation of CaMKII.

Elucidating the Mechanisms of CaMKII‐CaMKAP Interactions

Dynamic changes of intracellular calcium ion (Ca2+) concentrations are critical signaling mechanisms in all cells and organisms, and Ca2+ oscillations must be precisely decoded by intracellular signaling molecules, such as the ubiquitous Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Four mammalian CaMKII genes encode diverse alternative mRNA splice variants, which are thought to be targeted to different subcellular compartments to regulate numerous cell responses (exocytosis, gene expression, cell cycle). Our lab hypothesized that the localization, activity, and biological functions of CaMKII are precisely modulated by CaMKII‐associated proteins (CaMKAPs), and we have detected over 100 CaMKAPs associated with brain CaMKII holoenzymes. Some CaMKII‐CaMKAP interactions are critical for normal physiological function. CaMKIIδ binding to the L‐type Ca2+ channel (LTCC) b2a subunit is essential for cardiac function1, and binding of neuronal CaMKII to the GluN2B subunit of the NMDA‐type glutamate receptor is necessary for synaptic plasticity2. While b2a, GluN2B and most other CaMKAPs preferentially interact with activated CaMKII, the exact molecular mechanisms for these interactions, and their physiological roles, have yet to be determined. Several residues in the catalytic domain of CaMKII have been implicated in CaMKAP binding. Previous mutagenesis studies from our lab indicated that mutation of valine 102 (V102) to glutamate disrupts CaMKII binding to LTCC α1 subunits, but not to b2a, GluN2B, or densin, and also disrupts neuronal excitation‐transcription coupling3. Additionally, a naturally‐occurring CaMKIIα Glu183Val mutation linked to autism spectrum disorder and intellectual disability disrupted interactions with all CaMKAPs tested4. To further probe the molecular basis of CaMKII‐CaMKAP interactions, we are exploiting available structural information to design additional CaMKII catalytic domain mutations, and then test their effect on CaMKII‐CaMKAP interactions, as well as CaMKII activity toward multiple substrates. We are also examining the impact of additional human CaMKII mutations. Initial studies indicate that some CaMKII mutations differentially affect binding to various CaMKAPs, CaMKII autophosphorylation, and phosphorylation of a representative substrate, the GluA1 subunit of AMPA‐type glutamate receptors. Our ongoing studies are further exploring the impact of these CaMKII mutations. Expanding our knowledge of mechanisms underlying CaMKII‐CaMKAP interactions is required to better understand CaMKII biology, the role of CaMKAPs, and the molecular basis of Ca2+signaling. Koval et al., 2010. PNAS 107:4996. Halt et al., 2012. EMBO J. 31:1203. Wang et al., 2017. J Biol Chem 292:17324. Stephenson et al., 2017. J Neurosci 37:2216.

Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Hepatitis B Virus Replication from cccDNA via AMPK Activation and AKT/mTOR Suppression.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.

P21-Activated Kinase 1 Promotes Breast Tumorigenesis via Phosphorylation and Activation of the Calcium/Calmodulin-Dependent Protein Kinase II.

p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.

CaMKIIβ knockdown decreases store-operated calcium entry in hippocampal dendritic spines.

Calcium/calmodulin-dependent protein kinase II (CaMKII) and neuronal store-operated calcium entry (nSOCE) have been implicated in the development of Alzheimer's disease (AD). nSOCE is involved in regulation of dendritic spine shape, particularly in stability of mushroom spines that play role in formation of strong synapses. CaMKII is involved in regulation of induction of long-term potentiation, that is needed for shaping of memory. In the present study, we demonstrated that inhibition of kinase activity of CaMKII by KN-62 decreases nSOCE amplitude in soma of primary hippocampal neurons. We have shown that knockdown of CaMKIIβ leads to the downregulation of nSOCE in dendritic spines. In agreement with previously published data, we have also observed that CaMKIIβ knockdown causes mushroom spine loss in primary hippocampal culture. The effect of CaMKIIβ knockdown on the nSOCE may be associated with a decrease of dendritic spine head size.

The Pharmacological Inhibition of CaMKII Regulates Sodium Chloride Cotransporter Activity in mDCT15 Cells.

Simple SummaryThe renal sodium chloride cotransporter (NCC) plays an important role in the total body electrolyte balance and blood pressure control. The regulation of this protein by the actin cytoskeleton has not been thoroughly studied. Here, we investigate a novel association between the actin cytoskeleton protein filamin A and the NCC using a mouse cellular model and in the native kidney. Our results show for the first time that the disruption of the actin cytoskeleton reduces NCC activity and filamin A plays an essential role in NCC protein expression in cells of the distal convoluted tubule. We further show that the pharmacological inhibition of the Ca2+/calmodulin dependent protein kinase II (CAMKII) augments NCC protein expression. These results introduce a new mechanism for the regulation of the NCC.The thiazide-sensitive sodium chloride cotransporter (NCC) in the distal convoluted tubule is responsible for reabsorbing up to one-tenth of the total filtered load of sodium in the kidney. The actin cytoskeleton is thought to regulate various transport proteins in the kidney but the regulation of the NCC by the actin cytoskeleton is largely unknown. Here, we identify a direct interaction between the NCC and the cytoskeletal protein filamin A in mouse distal convoluted tubule (mDCT15) cells and in the native kidney. We show that the disruption of the actin cytoskeleton by two different mechanisms downregulates NCC activity. As filamin A is a substrate of the Ca2+/calmodulin-dependent protein kinase II (CaMKII), we investigate the physiological significance of CaMKII inhibition on NCC luminal membrane protein expression and NCC activity in mDCT15 cells. The pharmacological inhibition of CaMKII with the compound KN93 increases the active form of the NCC (phospho-NCC) at the luminal membrane and also increases NCC activity in mDCT15 cells. These data suggest that the interaction between the NCC and filamin A is dependent on CaMKII activity, which may serve as a feedback mechanism to maintain basal levels of NCC activity in the distal nephron.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

The 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. A 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. Our 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. AKAP18δ 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.

Late Na+ Current Is [Ca2+]i-Dependent in Canine Ventricular Myocytes.

Enhancement of the late sodium current (INaL) increases arrhythmia propensity in the heart, whereas suppression of the current is antiarrhythmic. In the present study, we investigated INaL in canine ventricular cardiomyocytes under action potential voltage-clamp conditions using the selective Na+ channel inhibitors GS967 and tetrodotoxin. Both 1 µM GS967 and 10 µM tetrodotoxin dissected largely similar inward currents. The amplitude and integral of the GS967-sensitive current was significantly smaller after the reduction of intracellular Ca2+ concentration ([Ca2+]i) either by superfusion of the cells with 1 µM nisoldipine or by intracellular application of 10 mM BAPTA. Inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) by KN-93 or the autocamtide-2-related inhibitor peptide similarly reduced the amplitude and integral of INaL. Action potential duration was shortened in a reverse rate-dependent manner and the plateau potential was depressed by GS967. This GS967-induced depression of plateau was reduced by pretreatment of the cells with BAPTA-AM. We conclude that (1) INaL depends on the magnitude of [Ca2+]i in canine ventricular cells, (2) this [Ca2+]i-dependence of INaL is mediated by the Ca2+-dependent activation of CaMKII, and (3) INaL is augmented by the baseline CaMKII activity.

Mitochondrial Contributions in the Genesis of Delayed Afterdepolarizations in Ventricular Myocytes.

Mitochondria fulfill the cell’s energy demand and affect the intracellular calcium (Ca2+) dynamics via direct Ca2+ exchange, the redox effect of reactive oxygen species (ROS) on Ca2+ handling proteins, and other signaling pathways. Recent experimental evidence indicates that mitochondrial depolarization promotes arrhythmogenic delayed afterdepolarizations (DADs) in cardiac myocytes. However, the nonlinear interactions among the Ca2+ signaling pathways, ROS, and oxidized Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways make it difficult to reveal the mechanisms. Here, we use a recently developed spatiotemporal ventricular myocyte computer model, which consists of a 3-dimensional network of Ca2+ release units (CRUs) intertwined with mitochondria and integrates mitochondrial Ca2+ signaling and other complex signaling pathways, to study the mitochondrial regulation of DADs. With a systematic investigation of the synergistic or competing factors that affect the occurrence of Ca2+ waves and DADs during mitochondrial depolarization, we find that the direct redox effect of ROS on ryanodine receptors (RyRs) plays a critical role in promoting Ca2+ waves and DADs under the acute effect of mitochondrial depolarization. Furthermore, the upregulation of mitochondrial Ca2+ uniporter can promote DADs through Ca2+-dependent opening of mitochondrial permeability transition pores (mPTPs). Also, due to much slower dynamics than Ca2+ cycling and ROS, oxidized CaMKII activation and the cytosolic ATP do not appear to significantly impact the genesis of DADs during the acute phase of mitochondrial depolarization. However, under chronic conditions, ATP depletion suppresses and enhanced CaMKII activation promotes Ca2+ waves and DADs.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

CaMKII as a therapeutic target in catecholaminergic polymorphic ventricular tachycardia type 1

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority Background In catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) adrenergic activation of the cardiac ryanodine receptor (RyR2) causes spontaneous calcium release and triggers arrhythmias. Calcium/calmodulin-dependent protein kinase II (CaMKII) can contribute to such arrhythmogenic calcium release and has been proposed as a therapeutic target in CPVT1. To predict the efficacy and safety of this strategy, it is necessary to know whether the mechanism for CaMKII activation is important for its arrhythmogenic effects, and if inhibition has proarrhythmic effects. We tested (1) if oxidation of CaMKII contributes to spontaneous calcium release in CPVT1 and (2) if inhibition of CaMKII in this condition can induce calcium alternans. Methods Mice with the CPVT1-causative mutation RyR2-R2474S (RyR2-RS) were crossed with mice with the CaMKII M281/282V (MMVV) mutation that prevents CaMKII M281/282 oxidation, to create double mutants (RyR2-RSxMMVV). Telemetric ECG surveillance was used to study in vivo arrhythmias following an adrenergic challenge by i.p. administration of the beta-adrenoceptor agonist isoprenaline. Confocal line-scan imaging and whole-cell calcium imaging were used to study arrhythmogenic calcium release in isolated left ventricular cardiomyocytes during stimulation with isoprenaline. Results As expected, RyR2-RS mice exhibited more arrhythmic events and spontaneous calcium release (i.e. calcium sparks and calcium waves) compared to wild-type mice. Treatment of RyR2-RS cardiomyocytes with either the CaMKII inhibitor KN-93 or the antioxidant n-acetyl-cysteine reduced spontaneous calcium release (i.e. calcium sparks and calcium waves, for KN-93 and n-acetyl-cysteine, respectively). Interestingly, CaMKII inhibition by KN-93 also increased both incidence and degree of arrhythmogenic calcium alternans in RyR2-RS cardiomyocytes. This adverse effect was a result of prolonged refractoriness of calcium release. Furthermore, to test whether the protective effect of antioxidant treatment in RyR2-RS was mediated via CaMKII oxidation, we compared arrhythmias and spontaneous calcium release (i.e. calcium waves) in RyR2-RSxMMVV with RyR2-RS. However, these two genotypes did not differ in either incidence or severity of arrhythmias, and showed similar degree of spontaneous calcium release. Conclusions Inhibition of CaMKII protects against spontaneous calcium release in CPVT1, and is a promising therapeutic strategy. However, the fact that such inhibition also induces calcium alternans needs further exploration. Antioxidative agents also attenuate arrhythmogenic calcium release in CPVT1 cardiomyocytes, but this effect does not seem to involve the M281/282 CaMKII oxidation site. Future studies should explore other oxidation sites.

Calcium/calmodulin-dependent protein kinase II stimulates the inward rectifier potassium current in beta-adrenergic adaptation of ventricular cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): NEW NATIONAL EXCELLENCE PROGRAM OF THE MINISTRY FOR INNOVATION AND TECHNOLOGY FROM THE SOURCE OF THE NATIONAL RESEARCH, DEVELOPMENT AND INNOVATION FUND. Introduction and purpose Acute β-adrenergic receptor (β-AR) stimulation shortens the ventricular action potential (AP). This effect is mainly regulated by the β- adrenergic stimulation of the cardiac potassium currents. Our aim was to investigate the extent of calcium/calmodulin-dependent protein kinase II (CaMKII) involvement in mediating the effect of β-AR activation on the inward rectifier potassium current – I K1 . Methods We carried out our experiments on isolated cardiomyocytes originating from canine left ventricles. The inward rectifier potassium current – I K1 was measured under a "canonical" AP under action potential voltage clamp conditions. Data were collected in four study groups [1] Control conditions (CTRL) [2] Inhibition of CaMKII with 1 µM KN-93 (KN-93) [3] Inhibition of PKA with 3 µM H-89 (H-89) [4] Acute β-adrenergic stimulation with 10 nM isoproterenol (ISO) [5] β-adrenergic stimulation with CaMKII inhibition (KN-93 + ISO) [6] β-adrenergic stimulation with PKA inhibition (H-89 + ISO) [7] β-adrenergic stimulation with inhibited PKA and CaMKII (KN-93 + H-89 + ISO) Results I K1 current amplitude did not differ among the studied groups, the total carried charge however was significantly, about 30 % larger in the ISO group compared to CTRL, and about 20 % larger compared to KN-93 + ISO. Under beta- adrenergic stimulation, I K1 starts to activate earlier during the AP plateau. I K1 density was about 3 times greater both at +20 mV and at 0 mV membrane potential under the command "canonical" AP in ISO compared to CTRL. Similarly, I K1 density was about 60 % and 90 % larger at +20 mV and at 0 mV, respectively, in KN-93 + ISO compared to KN-93. Similar results have been obtained by conventional voltage-clamp technique. Conclusion Based on the results of our researches the CaMKII activation plays an important role in β -adrenergic stimulation the I K1 potassium current.

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

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Sunitinib and Imatinib Display Differential Cardiotoxicity in Adult Rat Cardiac Fibroblasts That Involves a Role for Calcium/Calmodulin Dependent Protein Kinase II.

Background: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodeling of the heart following insult or injury, yet their involvement in anti-cancer drug induced cardiotoxicity has been largely overlooked. Here, we examine the direct effects of sunitinib malate and imatinib mesylate on adult rat CF viability, Ca2+ handling and mitochondrial function that may contribute to TKI-induced cardiotoxicity. In particular, we investigate whether Ca2+/calmodulin dependent protein kinase II (CaMKII), may be a mediator of TKI-induced effects.Methods: CF viability in response to chronic treatment with both drugs was assessed using MTT assays and flow cytometry analysis. Calcium mobilization was assessed in CFs loaded with Fluo4-AM and CaMKII activation via oxidation was measured via quantitative immunoblotting. Effects of both drugs on mitochondrial function was determined by live mitochondrial imaging using MitoSOX red.Results: Treatment of CFs with sunitinib (0.1–10 μM) resulted in concentration-dependent alterations in CF phenotype, with progressively significant cell loss at higher concentrations. Flow cytometry analysis and MTT assays revealed increased cell apoptosis and necrosis with increasing concentrations of sunitinib. In contrast, equivalent concentrations of imatinib resulted in no significant change in cell viability. Both sunitinib and imatinib pre-treatment increased Angiotensin II-induced intracellular Ca2+ mobilization, with only sunitinib resulting in a significant effect and also causing increased CaMKII activation via oxidation. Live cell mitochondrial imaging using MitoSOX red revealed that both sunitinib and imatinib increased mitochondrial superoxide production in a concentration-dependent manner. This effect in response to both drugs was suppressed in the presence of the CaMKII inhibitor KN-93.Conclusions: Sunitinib and imatinib showed differential effects on CFs, with sunitinib causing marked changes in cell viability at concentrations where imatinib had no effect. Sunitinib caused a significant increase in Angiotensin II-induced intracellular Ca2+ mobilization and both TKIs caused increased mitochondrial superoxide production. Targeted CaMKII inhibition reversed the TKI-induced mitochondrial damage. These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in CFs, consistent with the differential selectivity of sunitinib and imatinib.