Ca/calmodulin-dependent Protein Kinase II Research Articles

Glycolytic inhibition causes spontaneous ventricular fibrillation in aged hearts

Selective glycolytic inhibition (GI) promotes electromechanical alternans and triggered beats in isolated cardiac myocytes. We sought to determine whether GI promotes triggered activity by early afterdepolarization (EAD) or delayed afterdepolarizations in intact hearts isolated from adult and aged rats. Dual voltage and intracellular calcium ion (Ca(i)(2+)) fluorescent optical maps and single cell glass microelectrode recordings were made from the left ventricular (LV) epicardium of isolated Langendorff-perfused adult (∼4 mo) and aged (∼24 mo) rat hearts. GI was induced by replacing glucose with 10 mM pyruvate in oxygenated Tyrode's. Within 20 min, GI slowed Ca(i)(2+) transient decline rate and shortened action potential duration in both groups. These changes were associated with ventricular fibrillation (VF) in the aged hearts (64 out of 66) but not in adult hearts (0 out of 18; P < 0.001). VF was preceded by a transient period of focal ventricular tachycardia caused by EAD-mediated triggered activity leading to VF within seconds. The VF was suppressed by the ATP-sensitive K (K(ATP)) channel blocker glibenclamide (1 μM) but not (0 out of 7) by mitochondrial K(ATP) block. The Ca-calmodulin-dependent protein kinase II (CaMKII) blocker KN-93 (1 μM) prevented GI-mediated VF (P < 0.05). Block of Na-Ca exchanger (NCX) by SEA0400 (2 μM) prevented GI-mediated VF (3 out of 6), provided significant bradycardia did not occur. Aged hearts had significantly greater LV fibrosis and reduced connexin 43 than adult hearts (P < 0.05). We conclude that in aged fibrotic unlike in adult rat hearts, GI promotes EADs, triggered activity, and VF by activation of K(ATP) channels CaMKII and NCX.

Irradiation Leads to Disturbed Excitation-Contraction Coupling in Cardiac Myocytes Through ROS-Dependent CaMKII Activation

Introduction: Chest radiotherapy is part of therapeutic concepts for malignant diseases. However, radiation-induced cardiotoxic effects have become clinically relevant. The underlying pathomechanisms are poorly understood.Hypothesis: We investigated whether excitation-contraction coupling in cardiac myocytes, which depends on intact intracellular Ca cycling, may be directly affected by irradiation (IR).Methods: Isolated ventricular mouse myocytes were exposed to graded IR (0 Gy sham IR, 4 Gy and 20 Gy). Ca and Na handling properties and intracellular reactive oxygen species (ROS) levels were measured using epifluorescence microscopy (Fura-2, SBFI and CMH2-DCFDA, respectively) and confocal Ca (Fluo-4) imaging. Trypan blue staining indicated cellular injury. Western Blots revealed protein phosphorylation levels.Results: IR increased systolic sarcoplasmic reticulum (SR) Ca release leading to an acute positive-inotropic effect with Ca transients of 0.21±0.01 ratio units (r.u., F340nm/F380nm) at 20 Gy (N=89) vs. 0.15±0.01 r.u. in untreated sham control myocytes (N=98, P<0.05 using one-way ANOVA). Although SR Ca content was unaltered, an increased diastolic SR Ca leak measured as Ca sparks (752±113 Ca sparks∗pL−1∗s−1 at 20 Gy, N=32 vs. 208±34 sparks∗pL−1∗s−1 at 0 Gy, N=12, P<0.05 using one-way ANOVA) was accompanied by diastolic Ca overload and increased cellular injury. Furthermore, a rise in intracellular Na from 0.76± 0.01 r.u. (N=27) at 0 Gy to 0.79± 0.01 (N=39, P<0.05 using one-way ANOVA) at 20 Gy was observed after IR.IR-dependent elevation of ROS levels (by ∼667% at 20 Gy) contributed to Ca/calmodulin-dependent protein kinase II (CaMKII) activation. CaMKII-inhibition and ROS-scavenging prevented IR-dependent Ca overload, cellular dysfunction, and cell death.Conclusions: IR severely disturbs cardiac Ca handling and decreases myocyte viability. As underlying pathomechanism, a ROS/CaMKII signaling pathway was identified.

Spontaneous Ca waves in ventricular myocytes from failing hearts depend on Ca2+-calmodulin-dependent protein kinase II

Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca]SRT) versus control. At similar [Ca]SRT (121μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 and 1.29±0.39 SCaWs cell−1, respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca]SRT. Inhibiting CaMKII reversed this effect (14%). At similar [Ca]SRT (71µM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5±0.5; 1.6±0.7 vs. 0.36±0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis.

Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)delta(C) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10(-8) M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca](i) rose clearly on ISO in TG but not in wild-type cells (+20+/-5% versus +3+/-4% at 10(-6) M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9+/-0.5 versus 2.0+/-0.4 sparks per 100 microm(-1).s(-1), P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIdelta-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIdelta(C) mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

Ca/Calmodulin Kinase II Differentially Modulates Potassium Currents

Potassium currents contribute to action potential duration (APD) and arrhythmogenesis. In heart failure, Ca/calmodulin-dependent protein kinase II (CaMKII) is upregulated and can alter ion channel regulation and expression. We examine the influence of overexpressing cytoplasmic CaMKIIdelta(C), both acutely in rabbit ventricular myocytes (24-hour adenoviral gene transfer) and chronically in CaMKIIdelta(C)-transgenic mice, on transient outward potassium current (I(to)), and inward rectifying current (I(K1)). Acute and chronic CaMKII overexpression increases I(to,slow) amplitude and expression of the underlying channel protein K(V)1.4. Chronic but not acute CaMKII overexpression causes downregulation of I(to,fast), as well as K(V)4.2 and KChIP2, suggesting that K(V)1.4 expression responds faster and oppositely to K(V)4.2 on CaMKII activation. These amplitude changes were not reversed by CaMKII inhibition, consistent with CaMKII-dependent regulation of channel expression and/or trafficking. CaMKII (acute and chronic) greatly accelerated recovery from inactivation for both I(to) components, but these effects were acutely reversed by AIP (CaMKII inhibitor), suggesting that CaMKII activity directly accelerates I(to) recovery. Expression levels of I(K1) and Kir2.1 mRNA were downregulated by CaMKII overexpression. CaMKII acutely increased I(K1), based on inhibition by AIP (in both models). CaMKII overexpression in mouse prolonged APD (consistent with reduced I(to,fast) and I(K1)), whereas CaMKII overexpression in rabbit shortened APD (consistent with enhanced I(K1) and I(to,slow) and faster I(to) recovery). Computational models allowed discrimination of contributions of different channel effects on APD. CaMKII has both acute regulatory effects and chronic expression level effects on I(to) and I(K1) with complex consequences on APD.

Propofol and Ketamine-induced Anesthetic Depth-dependent Decrease of CaMKII Phosphorylation Levels in Rat Hippocampus and Cortex

Ca/calmodulin-dependent protein kinase II (CaMKII) activation through autophosphorylation at threonine 286 was involved in the modulation of neuronal excitability and neurotransmission. Both propofol and ketamine may affect the intracellular Ca levels through N-methyl-D-aspartate receptors or voltage-dependent Ca channels, but they have different mechanisms in general anesthesia. The purpose of this study was to investigate the effects of propofol and ketamine on CaMKII total protein and phosphorylation (p-CaMKII) levels in the brain of rats. We found that both propofol and ketamine could induce a decrease of p-CaMKII, not CaMKII total protein, in an anesthetic depth-dependent manner, whereas only ketamine caused a dose (50, 100, and 150 mg/kg)-dependent depression of p-CaMKII in hippocampus and frontal cortex of rats after intraperitoneal injections for 30 minutes. The significant depressions of p-CaMKII started at 5 minutes both in hippocampus and frontal cortex of rats after 100 mg/kg propofol treatment, whereas 100 mg/kg ketamine-induced significant depression of p-CaMKII initiated at 30 minutes in hippocampus and 5 minutes (no reduction observed at 15 min) in frontal cortex of rats. The maximum reduction of p-CaMKII with both drugs was at 60 minutes, and then restored to control level at 240 minutes. In addition, we confirmed the depression of p-CaMKII in hippocampus and frontal cortex of rats after 100 mg/kg of propofol or ketamine treatment for 60 minutes by using immunostaining. These results suggested that decreased p-CaMKII levels correlate with anesthetic depths achieved by propofol and ketamine, which may be related to the effects of propofol and ketamine on central nervous system function and their clinical effect.

Use of Fluorescence Anisotropy to Explore the Subunit Composition of Ca2+/Calmodulin Protein Kinase II Holoenzymes

Ca/calmodulin dependent protein kinase II (CaMKII) is a serine/threonine kinase which is highly enriched in the CNS and thought to be important in the development of long term potentiation. Several variants of CaMKII exist but alpha and beta subunits predominate in brain tissue. Though it is commonly believed that alpha and beta subunits combine to form heteromeric holoenzymes comprised of 12 subunits, this has not been directly demonstrated in vivo. Therefore, we used FRET imaging to directly examined the proximity of alpha and beta isoforms when they assemble to form heterologous holoenzymes. Specifically we used both time-resolved and steady-state fluorescence anisotropy measurements. Energy migration FRET (emFRET; FRET between like fluorophores) is observed in holoenzymes in which the C termini have been tagged with Venus. Fluorescence anisotropy is one of the few techniques which can detect emFRET and provide information of the number of fluorophores undergoing energy transfer. Venus tagged alpha isoforms were coexpressed with Amber (a point mutation in Venus that destroys the fluorophore) tagged beta isoforms and energy migration was seen to decrease indicating that holoenzymes are composed of both alpha and beta subunits in close proximity. Fluorescence anisotropy signatures of Venus tagged holoenzymes were invariant across a broad range of expression levels suggesting that inter-holoenzyme emFRET was not occurring. We used anisotropy “standards” consisting of concatamers of Venus and Amber to facilitate the interpretation of anisotropy curves in terms of the number of fluorescent proteins participating in emFRET. Together, these experiments indicate that alpha and beta subunits can coexist in the same holoenzyme. The availability of emFRET “standards” to the general scientific community should aid in the interpretation of anisotropy decay data.

Simulation of Ca-Calmodulin-Dependent Protein Kinase II on Rabbit Ventricular Myocyte Ion Currents and Action Potentials

Ca-calmodulin-dependent protein kinase II (CaMKII) was recently shown to alter Na + channel gating and recapitulate a human Na + channel genetic mutation that causes an unusual combined arrhythmogenic phenotype in patients: simultaneous long QT syndrome and Brugada syndrome. CaMKII is upregulated in heart failure where arrhythmias are common, and CaMKII inhibition can reduce arrhythmias. Thus, CaMKII-dependent channel modulation may contribute to acquired arrhythmic disease. We developed a Markovian Na + channel model including CaMKII-dependent changes, and incorporated it into a comprehensive myocyte action potential (AP) model with Na + and Ca 2+ transport. CaMKII shifts Na + current ( I Na) availability to more negative voltage, enhances intermediate inactivation, and slows recovery from inactivation (all loss-of-function effects), but also enhances late noninactivating I Na (gain of function). At slow heart rates, with long diastolic time for I Na recovery, late I Na is the predominant effect, leading to AP prolongation (long QT syndrome). At fast heart rates, where recovery time is limited and APs are shorter, there is little effect on AP duration, but reduced availability decreases I Na, AP upstroke velocity, and conduction (Brugada syndrome). CaMKII also increases cardiac Ca 2+ and K + currents ( I Ca and I to), complicating CaMKII-dependent AP changes. Incorporating I Ca and I to effects individually prolongs and shortens AP duration. Combining I Na, I Ca, and I to effects results in shortening of AP duration with CaMKII. With transmural heterogeneity of I to and I to downregulation in heart failure, CaMKII may accentuate dispersion of repolarization. This provides a useful initial framework to consider pathways by which CaMKII may contribute to arrhythmogenesis.

Effect of Endothelin-1 on L-type Ca Current in Rat Ventricular Myocytes

Purpose: Production of endothelin (ET-1) increases under pathophysiological conditions such as heart failure. ETA receptor is one of the Gq-coupled receptors and we have previously reported that stimulation of alpha1-adrenoceptor, another Gq-coupled receptor, increases L-type Ca current (ICa) via activation of protein kinase C (PKC) and Ca/calmodulin dependent protein kinase II (CaMKII) using a perforated patch clamp technique (O-Uchi et al, PNAS 2005;102:9400-5). Thus, ETA receptor stimulation was expected to show a similar response of ICa to that in the case of alpa1-adrenoceptor stimulation.

β-Adrenergic Enhancement of Sarcoplasmic Reticulum Calcium Leak in Cardiac Myocytes Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase

Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether beta-adrenergic receptor (beta-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca](i)) and SR Ca load ([Ca](SRT)) were assessed using fluo-4. beta-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca](i) decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2-related inhibitory peptide II or beta-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the beta-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca](i) decline rate. The results suggest that beta-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca](i).

Serine-phosphorylated STAT1 is a prosurvival factor in Wilms' tumor pathogenesis

Wilms' tumor (WT), one of the most common pediatric solid cancers, arises in the developing kidney as a result of genetic and epigenetic changes that lead to the abnormal proliferation and differentiation of the metanephric blastema. As activation of signal transducers and activators of transcription (STATs) plays an important role in the maintenance/growth and differentiation of the metanephric blastema, and constitutively activated STATs facilitate neoplastic behaviors of a variety of cancers, we hypothesized that dysregulation of STAT signaling may also contribute to WT pathogenesis. Accordingly, we evaluated STAT phosphorylation patterns in tumors and found that STAT1 was constitutively phosphorylated on serine 727 (S727) in 19 of 21 primary WT samples and two WT cell lines. An inactivating mutation of S727 to alanine reduced colony formation of WT cells in soft agar by more than 80% and induced apoptosis under conditions of growth stress. S727-phosphorylated STAT1 provided apoptotic resistance for WT cells via upregulation of expression of the heat-shock protein (HSP)27 and antiapoptotic protein myeloid cell leukemia (MCL)-1. The kinase responsible for STAT1 S727 phosphorylation in WT cells was identified based upon the use of selective inhibitors as protein kinase CK2, not p38, MAP-kinase kinase (MEK)1/2, phosphatidylinositol 3'-kinase, protein kinase C or Ca/calmodulin-dependent protein kinase II (CaMKII). The inhibition of CK2 blocked the anchorage-independent growth of WT cells and induced apoptosis under conditions of growth stress. Our findings suggest that serine-phosphorylated STAT1, as a downstream target of protein kinase CK2, plays a critical role in the pathogenesis of WT and possibly other neoplasms with similar STAT1 phosphorylation patterns.

Long-Term Potentiation Is Mediated by Multiple Kinase Cascades Involving CaMKII or Either PKA or p42/44 MAPK in the Adult Rat Dentate Gyrus In Vitro

The induction of NMDA-receptor-dependent long-term potentiation (LTP) in adult CA1 is contingent on activation of Ca/calmodulin-dependent protein kinase II (CaMKII). However, little is known about kinase mediation of LTP in the dentate gyrus. In the present study, the involvement of the kinases CaMKII, PKA, and MAPK in the induction of LTP was studied in the dentate gyrus of adult rats. Individual application of selective inhibitors of CaMKII, MEK, or PKA did not inhibit induction of LTP. In contrast, coapplication of a CaMKII inhibitor with either a PKA or MEK inhibitor resulted in a strong block of LTP. Induction of LTP was blocked by the coapplication of the inhibitors CaMKII and PKA or MEK, both when they were applied 1 h before the induction stimulus and also when they were applied after the induction stimulus. Thus LTP is mediated by either of two parallel cascades, one involving CaMKII and the other PKA or MAPK. Moreover, these cascades are active for a certain period after the induction stimulus.

A-Cell: A Computer Software for a Virtual Neuron

Virtual neuron is a model neuron, which is expressed by a computer-executable format. Virtual neuron comprises all biochemical, biophysical, electrical and mechanical mechanisms lying behind the phenomena of a real neuron. Here, we show A-Cell software, which was developed for modeling and simulation of virtual neuron. A model for Ca/calmodulin-dependent protein kinase II (CaMKII) activation in a spine was constructed. The simulation of the A-Cell model showed the localized distribution of activated CaMKII near PSD (postsynaptic density). This localization could be a basis for the translocation of activated CaMKII to PSD, which was observed in experiments.

Ca 2+ /Calmodulin–Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca 2+ Leak in Heart Failure

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.

Frequency-dependent Acceleration of Relaxation in the Heart Depends on CaMKII, but not Phospholamban

J. Desantiago, L. S. Maier and D. M. Bers. Frequency-dependent Acceleration of Relaxation in the Heart Depends on CaMKII, but not Phospholamban. Journal of Molecular and Cellular Cardiology (2002) 34, 975–984. Frequency-dependent acceleration of relaxation (FDAR) is an intrinsic physiological mechanism, which allows more rapid ventricular diastolic filling at higher heart rates. FDAR is also observed in isolated myocardial trabeculae and cardiac myocytes, but its mechanism is still poorly understood. We tested the hypothesis that FDAR results mainly from Ca/calmodulin-dependent protein kinase II (CaMKII) dependent stimulation of sarcoplasmic reticulum (SR) Ca transport, but does not require phospholamban. Experiments were performed at 23 or 35°C in isolated ventricular muscle and single myocytes from wild-type (WT) and phospholamban knockout (PLB-KO) mice and rat ventricular myocytes. Isometric twitch force of muscles and unloaded shortening and Ca transients in myocytes were measured ([Ca]o=1mM) in the absence and presence of CaMKII inhibitors (1μM KN-93 or 20μM autocamtide-2 related inhibitory peptide, AIP). Stimulation frequency was altered over a wide range (0.2–8Hz) and post-rest vs steady state twitches were also compared. In both WT and PLB-KO mouse muscles FDAR of twitch force was prominent, but was largely suppressed by KN-93. FDAR of twitch contractions was associated with FDAR of Ca transients in PLB-KO myocytes, and both were inhibited by KN-93. Similarly, a different CaMKII inhibitor (AIP) inhibited FDAR of contraction and Ca transients in rat ventricular myocytes. We conclude that FDAR results mainly from CaMKII-dependent stimulation of SR Ca transport, but does not require phospholamban.