Calmodulin-dependent Kinase II Research Articles

GRK5 Controls SAP97-Dependent Cardiotoxic β1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

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

P943The effects of ovariectomy on guinea pig cardiomyocyte intracellular calcium regulation and phosphorylation

Abstract Background Differences in cardiovascular disease risk between men and women have been partly attributed to the cardioprotective effects of oestrogen. Long-term oestrogen deficiency has been shown to alter cardiomyocyte intracellular calcium handling, but little is known about the mechanisms by which these changes occur. Oestrogen is thought to induce both genomic and non-genomic effects on cardiomyocytes, the latter including phosphorylation of calcium handling proteins. Purpose This study addresses the hypothesis that long-term oestrogen deficiency increases protein kinase A (PKA) and calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation in cardiomyocytes, resulting in altered intracellular calcium regulation. Methods Female guinea pigs underwent sham (n = 7) or ovariectomy (OVx) (n = 8) operations and 150 days later, left ventricular myocytes were enzymatically isolated and loaded with fluo-4AM to monitor intracellular calcium. Calcium transients (CaT) were recorded using confocal microscopy. PKA and CaMKII phosphorylation were inhibited by superfusing cells with specific inhibitors, PKI and AIP, respectively. Experiments were carried out both in the presence and absence of β-agonist, isoprenaline (ISO), and relative changes to CaT parameters compared between OVx and sham cells. Results CaT amplitude was greater (p < 0.05) in the OVx group (ΔF/Fo= 2.51 ± 0.57) compared with sham (ΔF/Fo = 2.16 ± 0.57). Inhibition of CaMKII phosphorylation increased CaT amplitude in the sham but not OVx group, both in the presence (by 22%, p < 0.01) and absence of ISO (by 19%, p < 0.01). Time to peak of the CaT increased to a greater extent following inhibition of PKA and CaMKII phosphorylation in the OVx group compared with sham, both in the presence (by 69%, p < 0.0001) and absence (by 162%, p < 0.0001) of ISO respectively. CaT decay time significantly increased (by 21%, p < 0.01) in the sham group following inhibition of PKA and CaMKII together, whilst decay times in the OVx group remained unchanged in the presence and absence of ISO. At higher pacing rates, time to peak of the CaT decreased significantly (by 48%, p < 0.01) in the OVx group but not sham with inhibition of phosphorylation. Conclusion Our findings suggest ovariectomy alters intracellular calcium regulation and some of these effects appear to be mediated by alterations in phosphorylation of calcium handling proteins and/or changes to sites of phosphorylation.

CaMKII inhibitor 1 (CaMK2N1) mRNA is upregulated following LTP induction in hippocampal slices.

CaMK2N1 and CaMK2N2 (also known as CaMKIINα and β) are endogenous inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), an enzyme critical for memory and long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning. CaMK2N1/2 mRNAs are rapidly and differentially upregulated in the hippocampus and amygdala after acquisition or retrieval of fear memory. Moreover, CaMK2N2 protein levels increase after contextual fear conditioning. Therefore, it was proposed that CaMK2N1/2 genes (Camk2n1/2) could be immediate-early genes transcribed promptly (30-60min) after training. As a first approach to explore a role in synaptic plasticity, we assessed a possible regulation of Camk2n1/2 during the expression phase of LTP in hippocampal CA3-CA1 connections in rat brain slices. Quantitative PCR revealed that Camk2n1, but not Camk2n2, is upregulated 60min after LTP induction by Schaffer collaterals high-frequency stimulation. We observed a graded, significant positive correlation between the magnitude of LTP and Camk2n1 change in individual slices, suggesting a coordinated regulation of these properties. If mRNA increment actually resulted in the protein upregulation in plasticity-relevant subcellular locations, CaMK2N1 may be involved in CaMKII fine-tuning during LTP maintenance or in the regulation of subsequent plasticity events (metaplasticity).

Cyclic AMP represses pathological MEF2 activation by myocyte-specific hypo-phosphorylation of HDAC5

Class IIa histone deacetylases (HDACs) critically regulate cardiac function through the repression of the activity of myocyte enhancer factor 2 (MEF2)-dependent gene programs. Protein kinase D (PKD) and Ca2+/Calmodulin-dependent kinase II (CaMKII) activate MEF2 by phosphorylating distinct HDAC isoforms and thereby creating 14–3-3 binding sites for nucleo-cytoplasmic shuttling. Recently, it has been shown that this process is counteracted by cyclic AMP (cAMP)-dependent signaling. Here, we investigated the specific mechanisms of how cAMP-dependent signaling regulates distinct HDAC isoforms and determined their relative contributions to the protection from pathological MEF2 activation. We found that cAMP is sufficient to induce nuclear retention and to blunt phosphorylation of the 14–3-3 binding sites of HDAC5 (Ser259/498) and HDAC9 (Ser218/448) but not HDAC4 (Ser246/467/632). These regulatory events could be observed only in cardiomyocytes and myocyte-like cells but not in non-myocytes, pointing to an indirect myocyte-specific mode of action. Consistent with one previous report, we found that blunted phosphorylation of HDAC5 and HDAC9 was mediated by protein kinase A (PKA)-dependent inhibition of PKD. However, we show by the use of neonatal cardiomyocytes derived from genetic HDAC mouse models that endogenous HDAC5 but not HDAC9 contributes specifically to the repression of endogenous MEF2 activity. HDAC4 contributed significantly to the repression of MEF2 activity but based on the mechanistic findings of this study combined with previous results we attribute this to PKA-dependent proteolysis of HDAC4. Consistently, cAMP-induced repression of agonist-driven cellular hypertrophy was blunted in cardiomyocytes deficient for both HDAC5 and HDAC4. In conclusion, cAMP inhibits MEF2 through both nuclear accumulation of hypo-phosphorylated HDAC5 and through a distinct HDAC4-dependent mechanism.

CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts

Previous studies indicated that Ca2+/calmodulin-dependent kinase II (CaMKII), a kinase involved in the modulation of ryanodine receptor activity, activates Ca2+-regulated protease μ-calpain to promote myocardial ischemia/reperfusion injury. This study was performed to explore the underlying mechanisms in CaMKII-induced calpain activation to better understand heart injury. To examine the Ca2+ paradox and ischemia/reperfusion injury, isolated rat hearts were subjected to a Ca2+-free solution for 3 min, or left coronary artery occlusion for 40 min, prior to restoration of normal perfusion. Blockade of trans-sarcoplasmic reticulum Ca2+ flux using ryanodine and thapsigargin failed to prevent Ca2+ paradox-induced heart injury. In contrast, the Ca2+ paradox increased CaMKII auto-phosphorylation at Thr287, while the CaMKII inhibitor KN-62 and the Na+/Ca2+ exchanger inhibitor KB-R7943 alleviated heart injury and calpain activity. Intriguingly, the binding of μ-calpain large subunit calpain-1 (CAPN1) to phospho-CaMKII was blunted by both inhibitors. Thus, a Ca2+ leak via the ryanodine receptor is not an essential element in CaMKII-elicited calpain activation. In hearts receiving vector injection, ischemia/reperfusion caused elevated calpain activity and α-fodrin degradation, along with membrane integrity damage, similar to the effects noted in control hearts. Importantly, all these alterations were diminished with delivery of adeno-associated virus expressing mutant CaMKIIδC T287A. Ischemia/reperfusion increased CaMKII auto-phosphorylation and binding of CAPN1 to phospho-CaMKII, and facilitated the translocation of phospho-CaMKII and CAPN1 to the plasma membrane, all of which were reversed by injecting CaMKII mutant. Furthermore, the relocation capacity and the interaction of CaMKII with CAPN1 appeared to be dependent upon CaMKII autophosphorylation, as its mutant delivery increased the level of CaMKII, but did not increase membrane content of CaMKII and CAPN1, or their interactions. Together, CaMKII/calpain interaction represents a new avenue for mediating myocardial ischemia/reperfusion injury, and CaMKII likely serves as both a kinase and a carrier, thereby promoting calpain membrane translocation and activation.

Activation of nicotinic acetylcholine receptors induces potentiation and synchronization within in vitro hippocampal networks

Nicotinic acetylcholine receptors (nAChRs) are known to play a role in cognitive functions of the hippocampus, such as memory consolidation. Given that they conduct Ca2+ and are capable of regulating the release of glutamate and γ‐aminobutyric acid (GABA) within the hippocampus, thereby shifting the excitatory‐inhibitory ratio, we hypothesized that the activation of nAChRs will result in the potentiation of hippocampal networks and alter synchronization. We used nicotine as a tool to investigate the impact of activation of nAChRs on neuronal network dynamics in primary embryonic rat hippocampal cultures prepared from timed‐pregnant Sprague‐Dawley rats. We perturbed cultured hippocampal networks with increasing concentrations of bath‐applied nicotine and performed network extracellular recordings of action potentials using a microelectrode array (MEA). We found that nicotine modulated network dynamics in a concentration‐dependent manner; it enhanced firing of action potentials as well as facilitated bursting activity. In addition, we used pharmacological agents to determine the contributions of discrete nAChR subtypes to the observed network dynamics. We found that β4‐containing nAChRs are necessary for the observed increases in spiking, bursting and synchrony, while the activation of α7 nAChRs augments nicotine‐mediated network potentiation but is not necessary for its manifestation. We also observed that antagonists of N‐methyl‐D‐aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs) partially blocked the effects of nicotine. Furthermore, nicotine exposure promoted autophosphorylation of Ca2+/calmodulin‐dependent kinase II (CaMKII) and serine 831 phosphorylation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) subunit GluA1. These results suggest that nicotinic receptors induce potentiation and synchronization of hippocampal networks and glutamatergic synaptic transmission. Findings from this work highlight the impact of cholinergic signaling in generating network‐wide potentiation in the form of enhanced spiking and bursting dynamics that coincide with molecular correlates of memory such as increased phosphorylation of CaMKII and GluA1.Support or Funding InformationNational Science Foundation (PHY‐1205919 and IOS‐ 1755033), National Institutes of Health (RF1 AG056603‐01) and the Georgetown‐MedStar CERSI Scholars program.

Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.

An interaction between CaMKII and calpain mediates myocardial ischemia/reperfusion injury

We have previously demonstrated that Ca2+/calmodulin-dependent kinase II (CaMKII), a kinase involved in modulating intracellular Ca2+ homeostasis, activates the Ca2 + −regulated protease m-calpain and promotes myocardial ischemia/reperfusion injury. This study was to investigate the underneath regulatory mechanisms.

The Role of Calmodulin in Tumor Cell Migration, Invasiveness, and Metastasis.

Calmodulin (CaM) is the principal Ca2+ sensor protein in all eukaryotic cells, that upon binding to target proteins transduces signals encoded by global or subcellular-specific changes of Ca2+ concentration within the cell. The Ca2+/CaM complex as well as Ca2+-free CaM modulate the activity of a vast number of enzymes, channels, signaling, adaptor and structural proteins, and hence the functionality of implicated signaling pathways, which control multiple cellular functions. A basic and important cellular function controlled by CaM in various ways is cell motility. Here we discuss the role of CaM-dependent systems involved in cell migration, tumor cell invasiveness, and metastasis development. Emphasis is given to phosphorylation/dephosphorylation events catalyzed by myosin light-chain kinase, CaM-dependent kinase-II, as well as other CaM-dependent kinases, and the CaM-dependent phosphatase calcineurin. In addition, the role of the CaM-regulated small GTPases Rac1 and Cdc42 (cell division cycle protein 42) as well as CaM-binding adaptor/scaffold proteins such as Grb7 (growth factor receptor bound protein 7), IQGAP (IQ motif containing GTPase activating protein) and AKAP12 (A kinase anchoring protein 12) will be reviewed. CaM-regulated mechanisms in cancer cells responsible for their greater migratory capacity compared to non-malignant cells, invasion of adjacent normal tissues and their systemic dissemination will be discussed, including closely linked processes such as the epithelial–mesenchymal transition and the activation of metalloproteases. This review covers as well the role of CaM in establishing metastatic foci in distant organs. Finally, the use of CaM antagonists and other blocking techniques to downregulate CaM-dependent systems aimed at preventing cancer cell invasiveness and metastasis development will be outlined.

Benzenesulfonamide Derivatives as Calcium/Calmodulin-Dependent Protein Kinase Inhibitors and Antiviral Agents against Dengue and Zika Virus Infections.

Emerging and resurging mosquito-borne flaviviruses are an important public health challenge. The increased prevalence of dengue virus (DENV) infection has had a significant socioeconomic impact on epidemic countries. The recent outbreak of Zika virus (ZIKV) has created an international public health emergency because ZIKV infection has been linked to congenital defects and Guillain-Barré syndrome. To develop potentially prophylactic antiviral drugs for combating these acute infectious diseases, we have targeted the host calcium/calmodulin-dependent kinase II (CaMKII) for inhibition. By using CaMKII structure-guided inhibitor design, we generated four families of benzenesulfonamide (BSA) derivatives for SAR analysis. Among these substances, N-(4-cycloheptyl-4-oxobutyl)-4-methoxy-N-phenylbenzenesulfonamide (9) showed superior properties as a lead CaMKII inhibitor and antiviral agent. BSA 9 inhibited CaMKII activity with an IC50 value of 0.79 μM and displayed EC50 values of 1.52 μM and 1.91 μM against DENV and ZIKV infections of human neuronal BE(2)C cells, respectively. Notably, 9 significantly reduced the viremia level and increased animal survival time in mouse-challenge models.

Noncanonical retinoic acid signaling.

All-trans retinoic acid (atRA) is the principle active metabolite of Vitamin A. atRA is well known to act through nuclear RA receptors (RARs) to regulate gene expression involved in a wide spectrum of biological processes such as growth, differentiation, and function. Recently, novel activities of atRA, independent of the action of RARs, have been increasingly reported and referred to as noncanonical activities. We have determined cellular retinoic acid binding protein 1 (CRABP1) as the primary mediator of the noncanonical activities of atRA. At the molecular level, atRA binds CRABP1, which then immediately acts as an adaptor in the formation of specific signaling scaffolds to rapidly modulate downstream signaling pathways in a cell context-dependent manner. The first established CRABP1-atRA activity is to rapidly dampen the activation of the mitogen-activated protein kinase (MAPK) cascade in response to growth factor stimulation, thereby suppressing cell cycle progression of stem cells. The second established activity is to rapidly reduce Ca2+/calmodulin dependent kinase II (CaMKII) activity in differentiated cells such as cardiomyocyte in response to β-adrenergic stimulation. This chapter describes in vivo and in vitro experimental systems and methodologies appropriate for determining the noncanonical activities of atRA that are mediated by CRABP1 and cell context dependent.

Activation of nicotinic acetylcholine receptors induces potentiation and synchronization within in vitro hippocampal networks.

Nicotinic acetylcholine receptors (nAChRs) are known to play a role in cognitive functions of the hippocampus, such as memory consolidation. Given that they conduct Ca2+ and are capable of regulating the release of glutamate and γ-aminobutyric acid (GABA) within the hippocampus, thereby shifting the excitatory-inhibitory ratio, we hypothesized that the activation of nAChRs will result in the potentiation of hippocampal networks and alter synchronization. We used nicotine as a tool to investigate the impact of activation of nAChRs on neuronal network dynamics in primary embryonic rat hippocampal cultures prepared from timed-pregnant Sprague-Dawley rats. We perturbed cultured hippocampal networks with increasing concentrations of bath-applied nicotine and performed network extracellular recordings of action potentials using a microelectrode array. We found that nicotine modulated network dynamics in a concentration-dependent manner; it enhanced firing of action potentials as well as facilitated bursting activity. In addition, we used pharmacological agents to determine the contributions of discrete nAChR subtypes to the observed network dynamics. We found that β4-containing nAChRs are necessary for the observed increases in spiking, bursting, and synchrony, while the activation of α7 nAChRs augments nicotine-mediated network potentiation but is not necessary for its manifestation. We also observed that antagonists of N-methyl-D-aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs) partially blocked the effects of nicotine. Furthermore, nicotine exposure promoted autophosphorylation of Ca2+ /calmodulin-dependent kinase II (CaMKII) and serine 831 phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluA1. These results suggest that nicotinic receptors induce potentiation and synchronization of hippocampal networks and glutamatergic synaptic transmission. Findings from this work highlight the impact of cholinergic signaling in generating network-wide potentiation in the form of enhanced spiking and bursting dynamics that coincide with molecular correlates of memory such as increased phosphorylation of CaMKII and GluA1. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Transient Chemogenetic Inhibition of D1-MSNs in the Dorsal Striatum Enhances Methamphetamine Self-Administration.

The dorsal striatum is important for the development of drug addiction; however, the role of dopamine D1 receptor (D1R) expressing medium-sized spiny striatonigral (direct pathway) neurons (D1-MSNs) in regulating excessive methamphetamine intake remains elusive. Here we seek to determine if modulating D1-MSNs in the dorsal striatum alters methamphetamine self-administration in animals that have demonstrated escalation of self-administration. A viral vector-mediated approach was used to induce expression of the inhibitory (Gi coupled-hM4D) or stimulatory (Gs coupled-rM3D) designer receptors exclusively activated by designer drugs (DREADDs) engineered to specifically respond to the exogenous ligand clozapine-N-oxide (CNO) selectively in D1-MSNs in the dorsal striatum. CNO in animals expressing hM4D increased responding for methamphetamine compared to vehicle in a within subject treatment paradigm. CNO in animals that did not express DREADDs (DREADD naïve-CNO) or expressed rM3D did not alter responding for methamphetamine, demonstrating specificity for hM4D-CNO interaction in increasing self-administration. Postmortem tissue analysis reveals that hM4D-CNO animals had reduced Fos immunoreactivity in the dorsal striatum compared to rM3D-CNO animals and DREADD naïve-CNO animals. Cellular mechanisms in the dorsal striatum in hM4D-CNO animals reveal enhanced expression of D1R and Ca2+/calmodulin-dependent kinase II (CaMKII). Conversely, rM3D-CNO animals had enhanced activity of extracellular signal-regulated kinase (Erk1/2) and Akt in the dorsal striatum, supporting rM3D-CNO interaction in these animals compared with drug naïve controls, DREADD naïve-CNO and hM4D-CNO animals. Our studies indicate that transient inhibition of D1-MSNs-mediated strengthening of methamphetamine addiction-like behavior is associated with cellular adaptations that support dysfunctional dopamine signaling in the dorsal striatum.

IK1 channel agonist zacopride suppresses ventricular arrhythmias in conscious rats with healing myocardial infarction

AimsArrhythmogenesis of chronic myocardial infarction (MI) is associated with the prolongation of action potential, reduction of inward rectifier potassium (IK1, Kir) channels and hyper-activity of Calcium/calmodulin-dependent kinase II (CaMKII) in cardiomyocytes. Zacopride, a selective IK1 agonist, was applied to clarify the cardioprotection of IK1 agonism via a CaMKII signaling on arrhythmias post-MI. MethodsMale SD rats were implanted wireless transmitter in the abdominal cavity and subjected to left main coronary artery ligation or sham operation. The telemetric ECGs were monitored per day throughout 4 weeks. At the endpoint, isoproterenol (1.28 mg/kg, i.v.) was administered for provocation test. The expressions of Kir2.1 (dominant subunit of IK1 in ventricle) and CaMKII were detected by Western-blotting. Key findingsIn the telemetric rats post-MI, zacopride significantly reduced the episodes of atrioventricular conduction block (AVB), premature ventricular contraction (PVC), ventricular tachycardia (VT) and ventricular fibrillation (VF), without significant effect on superventricular premature contraction (SPVC). In provocation test, zacopride suppressed the onset of ventricular arrhythmias in conscious PMI or sham rats. The expression of Kir2.1 was significantly downregulated and p-CaMKII was upregulated post-MI, whereas both were restored by zacopride treatment. SignificanceIK1/Kir2.1 might be an attractive target for pharmacological controlling of lethal arrhythmias post MI.

Activation of CaMKII via ER-stress mediates coxsackievirus B3-induced cardiomyocyte apoptosis.

Cardiomyocyte apoptosis contributes to the development of coxsackievirus B3 (CVB3)-induced myocarditis, but the mechanism for the apoptosis by CVB3 infection remains unclear. Here, we showed that CVB3-induced endoplasmic reticulum (ER) stress response and apoptosis in cultured H9c2 cardiomyocytes. We found that Ca2+ -calmodulin-dependent kinase II (CaMKII) was activated by ER stress-dependent intracellular Ca2+ overload in the CVB3-infected H9c2 cardiomyocytes. Treatment with an inhibitor of ER stress, 4-phenylbutyric acid (4-PBA), attenuated intracellular Ca2+ accumulation indirectly and reduced CaMKII activity. Inhibition of CaMKII with pharmacological inhibitor (KN-93) or short hairpin RNA reduced CVB3-induced H9c2 apoptosis and repressed cytochrome c release from mitochondria to cytoplasm; whereas overexpression of the activated mutant of CaMKII (CaMKII-T287D) enhanced CVB3-induced H9c2 apoptosis and mitochondrial cytochrome c release, which could be alleviated by blocking of mitochondrial Ca2+ uniporter or mitochondrial permeability transition pore. Further in vivo investigation revealed that blocking of CaMKII with KN-93 prevented cardiomyocytes apoptosis and improved cardiac contractile function in CVB3-infected mouse heart. Collectively, these findings provide a novel evidence that CaMKII plays a vital role in the promotion of CVB3-induced cardiomyocyte apoptosis, which links ER stress and mitochondrial Ca2+ uptake.

The Neuroprotective Effect of Forskolin and Its Influence of Mitochondrial Dysfunction in Neurons in Primary Rat Cerebellum Cultures

Activation of cyclic adenosine monophosphate (cAMP) synthesis is known to lead to triggering of neuroprotective signal cascades. It is therefore interesting to assess the effects of forskolin as an activator of adenylate cyclase and cAMP production on the neurotoxic action of glutamate in rat cerebellar neurons and to identify the participants in this signal cascade. This approach was used in experiments with a protein kinase A (PKA) inhibitor, the protein kinase C (PKC) blocker chelerythrine, and the calmodulin-dependent kinase II (CaMKII) blocker KN93, which showed that the neuroprotective effect of forskolin (1 μM) on long-term (24 h) exposure to glutamate (100 μM) involves PKA and CaMKII. Additional analysis of the dynamics of the development of mitochondrial dysfunction on exposure to glutamate showed that forskolin can prevent the drop in mitochondrial membrane potential in rat cerebellar neurons typical of excitotoxic stress.

Calmodulin kinase II regulates atrial myocyte late sodium current, calcium handling, and atrial arrhythmia

Atrial fibrillation (AF) is the most common type of arrhythmia. Abnormal atrial myocyte Ca2+ handling promotes aberrant membrane excitability and remodeling that are important for atrial arrhythmogenesis. The sequence of molecular events leading to loss of normal atrial myocyte Ca2+ homeostasis is not established. Late Na+ current (INa,L) is increased in atrial myocytes from AF patients together with an increase in activity of Ca2+/calmodulin-dependent kinase II (CaMKII). The purpose of this study was to determine whether CaMKII-dependent phosphorylation at Ser571 on NaV1.5 increases atrial INa,L, leading to aberrant atrial Ca2+ cycling, altered electrophysiology, and increased AF risk. Atrial myocyte electrophysiology, Ca2+ handling, and arrhythmia susceptibility were studied in wild-type and Scn5a knock-in mice expressing phosphomimetic (S571E) or phosphoresistant (S571A) NaV1.5 at Ser571. Atrial myocytes from S571E but not S571A mice displayed an increase in INa,L and action potential duration, and with adrenergic stress have increased delayed afterdepolarizations. Frequency of Ca2+ sparks and waves was increased in S571E atrial myocytes compared to wild type. S571E mice showed an increase in atrial events induced by adrenergic stress and AF inducibility invivo. Isolated S571E atria were more susceptible to spontaneous atrial events, which were abrogated by inhibiting sarcoplasmic reticulum Ca2+ release, CaMKII, or the Na+/Ca2+ exchanger. Expression of phospho-NaV1.5 at Ser571 and autophosphorylated CaMKII were increased in atrial samples from human AF patients. This study identified CaMKII-dependent regulation of NaV1.5 as an important upstream event in Ca2+ handling defects and abnormal impulse generation in the setting of AF.

Tiotropium bromide, a long acting muscarinic receptor antagonist triggers intracellular calcium signalling in the heart

Background and purposeTiotropium bromide (TB) is a long acting muscarinic receptor antagonist used to manage chronic obstructive pulmonary disease (COPD). Recent meta-analyses suggest an increased risk of cardiovascular events with TB. Ca2+/calmodulin dependent kinase II (CaMKII) and L-type Ca2+ channels regulate Ca2+ concentrations allowing management of Ca2+ across membranes. Pathological increases in Ca2+ are initially slow and progressive, however once the cytosolic concentration rises >1–3 μM from ~100 nM, calcium overload occurs and can lead to cell death. Ipratropium bromide, a short acting muscarinic receptor antagonist has previously been found to induce Ca2+ mediated eryptosis. The aim of this study was to investigate the role of Ca2+ in Tiotropium bromide mediated cardiotoxicity. Experimental approachIsolated Sprague-Dawley rat hearts were perfused with TB (10–0.1 nM) ± KN-93 (400 nM) or nifedipine (1 nM). Hearts were stained to determine infarct size (%) using triphenyltetrazolium chloride (TTC), or snap frozen to determine p-CaMKII (Thr286) expression. Cardiomyocytes were isolated using a modified Langendorff perfusion and enzymatic dissociation before preparation for Fluo 3-AM staining and flow cytometric analysis. Key resultsTB increased infarct size compared to controls by 6.91–8.41%, with no effect on haemodynamic function. KN-93/nifedipine with TB showed a 5.90/7.38% decrease in infarct size compared to TB alone, the combined use of KN-93 with TB also showed a significant increase in left ventricular developed pressure whilst nifedipine with TB showed a significant decrease in coronary flow. TB showed a 42.73% increase in p-CaMKII (Thr286) versus control, and increased Ca2+ fluorescence by 30.63% in cardiomyocytes. Conclusions and implicationsTo our knowledge, this is the first pre-clinical study to show that Tiotropium bromide induces Ca2+ signalling via CaMKII and L-type Ca2+ channels to result in cell damage. This has significant clinical impact due to long term use of TB in COPD patients, and warrants assessment of cardiac drug safety.

4966Targeting INaL by a neuronal sodium channel isoform improves survival in a CaMKII-transgenic heart failure mouse model

Abstract Background Cardiac pathologies like hypertrophy and heart failure are known to be associated with proarrhythmogenic triggers like early- (EADs) and delayed afterdepolarizations (DADs) that can be partly attributed to an augmentation of late sodium current (INaL). Enhanced INaL is closely connected with increased activity of Ca2+/calmodulin dependent-kinase II (CaMKII) in pathology as it is enhanced by CaMKII on the one hand but can also indirectly increase CaMKII-activity on the other. We recently found neuronal sodium channel NaV1.8 to be involved in INaL-augmentation in heart failure and cardiac hypertrophy. Here, we studied possible antiarrhythmic effects of NaV1.8-inhibition in a transgenic mouse model with enhanced CaMKII-expression by selectively knocking out NaV1.8. Methods/Results To investigate antiarrhythmic effects of NaV1.8-depletion in-vivo and in-vitro we crossbred CaMKII-transgenic mice (CaMKII+/T) with NaV1.8-knock-out mice (SCN10A−/−). Surprisingly, CaMKII+/T-mice lacking NaV1.8 (CaMKII+/T & SCN10A−/−) showed a significantly improved survival compared to CaMKII+/T alone (97.5 vs 72.0 days, p<0.05). Heart weight to tibia length ratio was significantly increased in CaMKII+/T-mice compared to wild-type, without any differences between CaMKII+/T and CaMKII+/T & SCN10A−/−. To investigate the underlying mechanisms out of this observation we isolated single cardiomyocytes and performed patch-clamp experiments as well as confocal microscopy to measure Ca2+-transients and diastolic Ca2+-waves. INaL-integral was significantly smaller in cardiomyocytes from CaMKII+/T & SCN10A−/−-mice compared to CaMKII+/T alone. During action potential recordings, significantly less afterdepolarizations occurred in CaMKII+/T & SCN10A−/− compared to cardiomyocytes from CaMKII+/T -mice (16.7/min vs 34.9/min, p<0.05). There was a trend of less cells exhibiting diastolic Ca2+-waves in Ca2+-measurements from CaMKII+/T & SCN10A−/− compared to CaMKII+/T (15% vs 25%, p=0.09). As some cells showed more than one event, we calculated the frequency of Ca2+-waves and found a significant reduction of Ca2+-waves in CaMKII+/T & SCN10A−/− vs. CaMKII+/T (22.8/min vs 43.0/min, p<0.05). Moreover, the time to the first event was significantly longer in CaMKII+/T & SCN10A−/−. Ca2+-transient amplitude (F/F0) was significantly lower in CaMKII+/T compared to CaMKII+/T & SCN10A−/− (4.6 vs. 5.3, p=0.05). Further, Ca2+-extrusion from the cytosol was significantly faster in CaMKII+/T & SCN10A−/−. Conclusion Our data demonstrates, that inhibition of INaL by targeting NaV1.8 has a potent antiarrhythmic potential as we found a reduction of EADs, DADs and diastolic Ca2+-waves in CaMKII+/T & SCN10A−/−-cardiomyocytes. This antiarrhythmic potential appears to be potent enough to improve survival and to rescue the proarrhythmogenic phenotype of CaMKII-overexpression. However, further in-vivo experiments are necessary to investigate NaV1.8-inhibition for a possible therapeutic approach.

P3832Dantrolene reduces CaMKII-mediated arrhythmogenesis

Abstract Rationale In atrial and ventricular rhythm disorders, an increased diastolic sarcoplasmatic reticulum (SR) calcium leak can induce a depolarizing transient inward current, serving as a trigger for cellular arrhythmias. Dantrolene has been shown to also stabilize the cardiac ryanodine receptor. However, the detailed mechanism of the mode of action remains unknown. This study aims to investigate the effects of dantrolene on calcium/calmodulin-dependent kinase II (CaMKII) mediated arrhythmogenesis. Methods and results Right atrial cardiomyocytes (CM) were isolated from patients with atrial fibrillation. To investigate SR Ca2+ leak, measurements of diastolic SR Ca2+ sparks were performed by confocal microscopy using Fluo-4 AM. Dantrolene (10 μmol/l) potently reduced Ca2+-spark-frequency (CaSpF) by 90±26% (p<0.05, n=21 cells dantrolene vs. 19 cells control) leading to a reduction of the calculated diastolic SR-Ca2+-leak by 91±31% (p<0.05, n=21 vs. 19). Interestingly, CaMKII-inhibition using Autocamtide-2-Related Inhibitory Peptide (AIP) did not further reduced SR Ca2+ leak compared to dantrolene alone in human cardiomyocytes. This observation may suggest (secondary) inhibitory effects of dantrolene on CaMKII. To elucidate the role of CaMKII in dantrolene-mediated antiarrhythmic effects, we investigated atrial CM from mice overexpressing CaMKII (TG) and respective wildtype controls (WT). CaSpF and SR Ca2+ leak were reduced by dantrolene in both TG and WT mice (p<0.005, TG: dantrolene vs. vehicle n=132 vs 127 cells (9 mice); WT: dantrolene vs. vehicle n=61 vs 61 cells (5 mice)). However, proarrhythmogenic Ca2+ waves were only significantly reduced by dantrolene in TG mice (p<0.05, TG: dantrolene vs. vehicle 10.8% vs. 26.2%, n=154 vs 164 cells). Correspondingly, the incidence of delayed afterdepolarizations (DADs) in TG cells was significantly diminished by dantrolene (p<0.05, TG: dantrolene vs. vehicle 1/14 vs. 9/15 cells, n=5 mice). In contrast, DADs were not reduced by dantrolene in WT cells without increased CaMKII activity (p=n.s., WT: dantrolene vs vehicle 3/16 vs 2/13 cells, n=5 mice). In preliminary in vivo experiments, intraperitoneal injection of 40 mg/kg body weight dantrolene reduced the inducibility of arrhythmias by ventricular burst stimulation in CaMKII TG mice compared to vehicle (dantrolene 0/2 mice vs. vehicle 2/2 mice, p<0.05 Chi-Square). Conclusion Dantrolene beneficially altered Ca2+ homeostasis in human AF CM and murine CM. Dantrolene seems to exert its antiarrhythmic potential in a CaMKII-dependent manner. Thus, dantrolene as an already clinically approved compound might be a potential antiarrhythmic drug that merits clinical investigation. Acknowledgement/Funding Deutsche Forschungsgemeinschaft (MA 1981/5-1 and 7-1 to LSM). Marga und Walter Boll-Stiftung (SS).