kn-93 and Heart-Failure

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Endothelial Cells; Heart Failure; Myocytes, Cardiac; NADPH Oxidase 2; Neovascularization, Physiologic; Rats; STAT3 Transcription Factor; Sulfonamides; Vascular Endothelial Growth Factor Receptor-2; Ventricular Remodeling

The present study was to explore the impact of KN93 - a specific inhibitor of CaMKII - on cardiac function and cardiac reserve in HF mice.. We have generated pressure-overload HF mice using modified transverse aortic constriction (TAC) method. For acute inhibition (AI) experiment, HF mice were randomly divided into HF group, HF + KN93 AI group and HF + KN92 AI group, using sham mice as control. Mice in HF + KN93 AI group and HF + KN92 AI group were injected with CaMKII inhibitor KN93 or its inactive analogue KN92 on post-TAC day 15, while mice in HF group and Sham group were treated with saline. For chronic inhibition (CI) experiment, mice were injected daily with KN93, KN92 or saline for one week. At baseline and after isoproterenol (Iso) injection, in vivo cardiac function was assessed by echocardiography and left ventricular pressure-volume catheter.. Acute inhibition of CaMKII leads to decreased -dP/dtmin, increased EF, FS, longitudinal strain, longitudinal strain rate, ESPVR, dP/dtmax-EDV, PRSW, Tau and EDPVR, and unaltered reactivity to Iso in HF mice. Chronic inhibition results in increased EF, FS, longitudinal strain, longitudinal strain rate, ESPVR, dP/dtmax-EDV and PRSW, without alteration in -dP/dtmin, Tau and EDPVR. In addition, chronic inhibition reverses the effect of Iso on HF mice.. Although acute CaMKII inhibition can repair systolic function in HF mice, it also exacerbates the diastolic function, whereas chronic inhibition improves both systolic function and cardiac reserve to β-adrenergic stimulation without impairing diastolic function.

Topics: Animals; Benzylamines; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disease Models, Animal; Echocardiography; Electrocardiography; Heart; Heart Failure; Isoproterenol; Male; Mice; Mice, Inbred C57BL; Sulfonamides; Ventricular Function, Left

Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented.. To prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes.. Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet-induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Mice treated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenesis in vivo, despite ROS increases.. RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic cardiovascular complications and/or evolution.

Topics: Amino Acids; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Chromium; Diabetes Mellitus, Type 2; Disease Models, Animal; Fructose; Heart Failure; Male; Mice; Myocytes, Cardiac; Nicotinic Acids; Phosphorylation; Prediabetic State; Protein Kinase Inhibitors; Rats; Rats, Wistar; Reactive Oxygen Species; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sulfonamides

Calcium/calmodulin-dependent protein kinase II (CaMKII) is activated in heart failure (HF) and can contribute to arrhythmias induced by β-adrenergic receptor-mediated sarcoplasmic reticulum calcium leak.. To evaluate the effect of CaMKII inhibition on ventricular tachycardia (VT) induction in conscious HF and naive rabbits.. Nonischemic HF was induced by aortic insufficiency and constriction. Electrocardiograms were recorded in rabbits pretreated with vehicle (saline) or the CaMKII inhibitor KN-93 (300 μg/kg); VT was induced by infusion of increasing doses of norepinephrine (1.56-25 μg·kg⁻¹·min⁻¹) in naive (n = 8) and HF (n = 7) rabbits. With saline, median VT dose threshold in HF was 6.25 versus 12.5 μg·kg⁻¹·min⁻¹ norepinephrine in naive rabbits (P = 0.06). Pretreatment with KN-93 significantly increased VT threshold in HF and naive rabbits (median = 25 μg·kg⁻¹·min⁻¹, P < 0.05 vs. saline for both groups). Mean cycle length of VT initiation was shorter in HF (221 ± 20 milliseconds) than naive (296 ± 23 milliseconds, P < 0.05) rabbits with saline; this difference was not significant after treatment with KN-93.. KN-93 significantly reduced arrhythmia inducibility and slowed initiation of VT, suggesting that CaMKII inhibition may have antiarrhythmic effects in the failing human heart.

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Disease Models, Animal; Electrocardiography; Enzyme Activation; Female; Heart Failure; Heart Rate; Male; Norepinephrine; Protein Kinase Inhibitors; Rabbits; Sulfonamides; Tachycardia, Ventricular

Activation of Wnt signaling results in maladaptive cardiac remodeling and cardiomyopathy. Recently, calcium/calmodulin-dependent protein kinase II (CaMKII) was reported to be a pivotal participant in myocardial remodeling. Because CaMKII was suggested as a downstream target of noncanonical Wnt signaling, we aimed to elucidate the role of CaMKII in dishevelled-1-induced cardiomyopathy and the mechanisms underlying its function. Dishevelled-1-induced cardiomyopathy was reversed by deletion of neither CaMKIIδ nor CaMKIIγ. Therefore, dishevelled-1-transgenic mice were crossed with CaMKIIδγ double-knockout mice. These mice displayed a normal cardiac phenotype without cardiac hypertrophy, fibrosis, apoptosis, or left ventricular dysfunction. Further mechanistic analyses unveiled that CaMKIIδγ couples noncanonical Wnt signaling to histone deacetylase 4 and myosin enhancer factor 2. Therefore, our findings indicate that the axis, consisting of dishevelled-1, CaMKII, histone deacetylase 4, and myosin enhancer factor 2, is an attractive therapeutic target for prevention of cardiac remodeling and its progression to left ventricular dysfunction.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Benzylamines; beta Catenin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dishevelled Proteins; Fibrosis; Heart Failure; Histone Deacetylases; Hypertrophy, Left Ventricular; MAP Kinase Signaling System; MEF2 Transcription Factors; Mice; Mice, Knockout; Myocardium; Phenotype; Phosphoproteins; Protein Kinase C; Sulfonamides; Ultrasonography; Ventricular Dysfunction, Left; Ventricular Remodeling; Wnt Proteins; Wnt Signaling Pathway

Sustained activation of Gαq transgenic (Gq) signaling during pressure overload causes cardiac hypertrophy that ultimately progresses to dilated cardiomyopathy. The molecular events that drive hypertrophy decompensation are incompletely understood. Ca(2+)/calmodulin-dependent protein kinase II δ (CaMKIIδ) is activated downstream of Gq, and overexpression of Gq and CaMKIIδ recapitulates hypertrophy decompensation.. To determine whether CaMKIIδ contributes to hypertrophy decompensation provoked by Gq.. Compared with Gq mice, compound Gq/CaMKIIδ knockout mice developed a similar degree of cardiac hypertrophy but exhibited significantly improved left ventricular function, less cardiac fibrosis and cardiomyocyte apoptosis, and fewer ventricular arrhythmias. Markers of oxidative stress were elevated in mitochondria from Gq versus wild-type mice and respiratory rates were lower; these changes in mitochondrial function were restored by CaMKIIδ deletion. Gq-mediated increases in mitochondrial oxidative stress, compromised membrane potential, and cell death were recapitulated in neonatal rat ventricular myocytes infected with constitutively active Gq and attenuated by CaMKII inhibition. Deep RNA sequencing revealed altered expression of 41 mitochondrial genes in Gq hearts, with normalization of ≈40% of these genes by CaMKIIδ deletion. Uncoupling protein 3 was markedly downregulated in Gq or by Gq expression in neonatal rat ventricular myocytes and reversed by CaMKIIδ deletion or inhibition, as was peroxisome proliferator-activated receptor α. The protective effects of CaMKIIδ inhibition on reactive oxygen species generation and cell death were abrogated by knock down of uncoupling protein 3. Conversely, restoration of uncoupling protein 3 expression attenuated reactive oxygen species generation and cell death induced by CaMKIIδ. Our in vivo studies further demonstrated that pressure overload induced decreases in peroxisome proliferator-activated receptor α and uncoupling protein 3, increases in mitochondrial protein oxidation, and hypertrophy decompensation, which were attenuated by CaMKIIδ deletion.. Mitochondrial gene reprogramming induced by CaMKIIδ emerges as an important mechanism contributing to mitotoxicity in decompensating hypertrophy.

Topics: Acetylcysteine; Animals; Apoptosis; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomegaly; Cardiomyopathy, Dilated; Cells, Cultured; Disease Progression; Gene Expression Profiling; GTP-Binding Protein alpha Subunits, Gq-G11; Heart Failure; Ion Channels; Male; Mice; Mice, Knockout; Mice, Transgenic; Mitochondria, Heart; Mitochondrial Proteins; Myocytes, Cardiac; Oxidative Stress; Point Mutation; PPAR alpha; Pressure; Rats; Reactive Oxygen Species; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sequence Analysis, RNA; Sulfonamides; Transfection; Uncoupling Protein 3

Aberrant calcium signaling is considered one of the key mechanisms contributing to arrhythmias, especially in the context of heart failure. In human heart failure, there is significant down-regulation of the sarcoplasmic reticulum (SR) protein junctin, and junctin deficiency in mice is associated with stress-induced arrhythmias.. The purpose of this study was to determine whether the increased SR Ca(2+) leak and arrhythmias associated with junctin ablation may be associated with increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phosphorylation of the cardiac ryanodine receptor (RyR2) and whether pharmacologic inhibition of CaMKII activity may prevent these arrhythmias.. Using a combination of biochemical, cellular, and in vivo approaches, we tested the ability of KN-93 to reverse aberrant CaMKII phosphorylation of RyR2. Specifically, we performed protein phosphorylation analysis, in vitro cardiomyocyte contractility and Ca(2+) kinetics, and in vivo ECG analysis in junctin-deficient mice.. In the absence of junctin, RyR2 channels displayed CaMKII-dependent hyperphosphorylation. Notably, CaMKII inhibition by KN-93 reduced the in vivo incidence of stress-induced ventricular tachycardia by 65% in junctin null mice. At the cardiomyocyte level, KN-93 reduced the percentage of junctin null cells exhibiting spontaneous Ca(2+) aftertransients and aftercontractions under stress conditions by 35% and 37%, respectively. At the molecular level, KN-93 blunted the CaMKII-mediated hyperphosphorylation of RyR2 and phospholamban under stress conditions.. Our data suggest that CaMKII inhibition is effective in preventing arrhythmogenesis in the setting of junctin ablation through modulation of both SR Ca(2+) release and uptake. Thus, it merits further investigation as promising molecular therapy.

Topics: Ablation Techniques; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium Signaling; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disease Models, Animal; Heart Failure; Mice; Models, Cardiovascular; Myocytes, Cardiac; Phosphorylation; Protein Kinase Inhibitors; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sulfonamides

Dilated cardiomyopathy (DCM), characterized by dilatation and dysfunction of the left ventricle, is an important cause of heart failure. Many mutations in various genes, including cytoskeletal protein genes and contractile protein genes, have been identified in DCM patients, but the mechanisms of how such mutations lead to DCM remain unknown.. We established the mouse model of DCM by expressing a mutated cardiac alpha-actin gene, which has been reported in patients with DCM, in the heart (mActin-Tg). mActin-Tg mice showed gradual dilatation and dysfunction of the left ventricle, resulting in death by heart failure. The number of apoptotic cardiomyocytes and protein levels of p53 were increased in the hearts of mActin-Tg mice. Overexpression of Bcl-2 or downregulation of p53 decreased the number of apoptotic cardiomyocytes and improved cardiac function. This mouse model showed a decrease in myofilament calcium sensitivity and activation of calcium/calmodulin-dependent kinase IIdelta (CaMKIIdelta). The inhibition of CaMKIIdelta prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function.. CaMKIIdelta plays a critical role in the development of heart failure in part by accumulation of p53 and induction of cardiomyocyte apoptosis in the DCM mouse model.

Topics: Actin Cytoskeleton; Actins; Animals; Apoptosis; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomyopathy, Dilated; Disease Models, Animal; Enzyme Activation; Heart Failure; Humans; Mice; Mice, Transgenic; Myocytes, Cardiac; Protein Kinase Inhibitors; Sulfonamides; Tumor Suppressor Protein p53

QT prolongation may increase the risk of torsades de pointes (TdP). Early afterdepolarizations (EADs) and transmural dispersion of repolarization have been known to serve as physiological substrates and predictors for TdP. Abnormal Ca(2+) cycling is the proximate cause of EADs, and Ca(2+) cycling is abnormal in heart failure (HF). However, the mechanisms for drug-induced TdP in HF are poorly understood. The purpose of this study was to search for torsadogenic-modifying effects of verapamil, ryanodine, KB-R7943, W-7, KN-93, and H-8 on ventricular premature depolarizations (VPD) and TdP in rabbits with HF. Rabbits with HF were pretreated with propranolol followed by test articles before continuous infusion of dofetilide to induce TdP. In the control hearts, VPD and TdP were induced in all rabbits and the onsets of VPD and TdP were 3.6 +/- 1.3 minutes and 10.3 +/- 1.4 minutes, respectively. Dofetilide lengthened RR, QT and QTc. Verapamil, ryanodine and H-8 significantly delayed onset of VPD (P < 0.05) and suppressed TdP (P < 0.01). KB-R7943, W-7, and KN-93 accelerated onset of TdP. Blockades of L-type Ca(2+) channel, ryanodine channel, and protein kinase A prevent dofetilide-induced TdP, suggesting roles for intracellular Ca(2+) overload and Ca(2+) signaling pathways in drug-induced TdP.

Topics: Animals; Anti-Arrhythmia Agents; Benzylamines; Disease Models, Animal; Heart Failure; Isoquinolines; Male; Protein Kinase Inhibitors; Rabbits; Ryanodine; Sulfonamides; Thiourea; Torsades de Pointes; Ventricular Premature Complexes; Verapamil

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.

Topics: Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium Channels, L-Type; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disease Models, Animal; Heart Failure; Isoproterenol; Membrane Potentials; Mice; Mice, Knockout; Mice, Transgenic; Myocytes, Cardiac; Protein Kinase Inhibitors; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sulfonamides; Time Factors

Augmented and slowed late Na(+) current (I(NaL)) is implicated in action potential duration variability, early afterdepolarizations, and abnormal Ca(2+) handling in human and canine failing myocardium. Our objective was to study I(NaL) modulation by cytosolic Ca(2+) concentration ([Ca(2+)](i)) in normal and failing ventricular myocytes. Chronic heart failure was produced in 10 dogs by multiple sequential coronary artery microembolizations; 6 normal dogs served as a control. I(NaL) fine structure was measured by whole cell patch clamp in ventricular myocytes and approximated by a sum of fast and slow exponentials produced by burst and late scattered modes of Na(+) channel gating, respectively. I(NaL) greatly enhanced as [Ca(2+)](i) increased from "Ca(2+) free" to 1 microM: its maximum density increased, decay of both exponentials slowed, and the steady-state inactivation (SSI) curve shifted toward more positive potentials. Testing the inhibition of CaMKII and CaM revealed similarities and differences of I(NaL) modulation in failing vs. normal myocytes. Similarities include the following: 1) CaMKII slows I(NaL) decay and decreases the amplitude of fast exponentials, and 2) Ca(2+) shifts SSI rightward. Differences include the following: 1) slowing of I(NaL) by CaMKII is greater, 2) CaM shifts SSI leftward, and 3) Ca(2+) increases the amplitude of slow exponentials. We conclude that Ca(2+)/CaM/CaMKII signaling increases I(NaL) and Na(+) influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na(+) influx provides a novel Ca(2+) positive feedback mechanism (via Na(+)/Ca(2+) exchanger), enhancing contractions at higher beating rates but worsening cardiomyocyte contractile and electrical performance in conditions of poor Ca(2+) handling in heart failure.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Chronic Disease; Cytosol; Disease Models, Animal; Dogs; Heart Failure; Heart Ventricles; Ion Channel Gating; Kinetics; Models, Cardiovascular; Myocytes, Cardiac; Patch-Clamp Techniques; Peptide Fragments; Protein Kinase Inhibitors; Research Design; Signal Transduction; Sodium; Sodium Channels; Sulfonamides

Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKIIdelta and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5-green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (F(nuc)/F(cyto) 3.3+/-0.3 vs 7.2+/-0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKIIdelta(C) expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP F(nuc)/F(cyto) toward control, and simultaneous inhibition restored F(nuc)/F(cyto) to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKIIdelta(B), or CaMKIIdelta(C) reduced baseline HDAC5 F(nuc)/F(cyto) in control myocytes (3.4+/-0.5, 3.8+/-0.5, and 5.2+/-0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.

Topics: Active Transport, Cell Nucleus; Adult; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Carbazoles; Cell Nucleus; Cells, Cultured; Cytosol; Disease Models, Animal; Endothelin-1; Enzyme Activation; Female; Heart Failure; Heart Ventricles; Histone Deacetylases; Humans; Indoles; Male; Middle Aged; Myocardium; Myocytes, Cardiac; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Rabbits; Recombinant Fusion Proteins; Sulfonamides; Time Factors; Transcription, Genetic; Transduction, Genetic; Up-Regulation; Ventricular Function, Left

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).

Topics: Adrenergic beta-Agonists; Animals; Benzylamines; Calcium; Calcium Channels, L-Type; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cells, Cultured; Colforsin; Cyclic AMP-Dependent Protein Kinases; Diastole; Heart Failure; Isoproterenol; Isoquinolines; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Peptides; Phosphorylation; Protein Processing, Post-Translational; Rabbits; Receptors, Adrenergic, beta; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Sulfonamides; Tetracaine

We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.

Topics: Adrenergic beta-Agonists; Animals; Arrhythmias, Cardiac; Benzylamines; Blotting, Western; Calcium Signaling; Carrier Proteins; Cell Line; Cyclic AMP-Dependent Protein Kinases; Heart Failure; Humans; Ion Channel Gating; Ion Transport; Isoproterenol; Kidney; Marine Toxins; Mice; Myocytes, Cardiac; Oxazoles; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Rabbits; Rats; Recombinant Fusion Proteins; Ryanodine Receptor Calcium Release Channel; Sodium-Calcium Exchanger; Staurosporine; Structure-Activity Relationship; Sulfonamides; Transfection

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.

Topics: Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium Channels; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calcium-Transporting ATPases; Cyclic AMP-Dependent Protein Kinases; Echocardiography; Heart Failure; Inositol 1,4,5-Trisphosphate Receptors; Myocytes, Cardiac; Phosphorylation; Rabbits; Receptors, Cytoplasmic and Nuclear; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Tacrolimus Binding Protein 1A; Tacrolimus Binding Proteins

The cardiac ryanodine receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for muscle excitation-contraction coupling. Using site-directed mutagenesis, we identified the specific Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation site on recombinant RyR2, distinct from the site for protein kinase A (PKA) that mediates the "fight-or-flight" stress response. CaMKII phosphorylation increased RyR2 Ca2+ sensitivity and open probability. CaMKII was activated at increased heart rates, which may contribute to enhanced Ca2+-induced Ca2+ release. Moreover, rate-dependent CaMKII phosphorylation of RyR2 was defective in heart failure. CaMKII-mediated phosphorylation of RyR2 may contribute to the enhanced contractility observed at higher heart rates. The full text of this article is available online at http://circres.ahajournals.org.

Topics: Adrenergic beta-Agonists; Amino Acid Sequence; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cardiac Pacing, Artificial; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Heart Failure; Heart Rate; Humans; Isoproterenol; Molecular Sequence Data; Mutagenesis, Site-Directed; Myocardial Infarction; Myocardium; Phosphorylation; Phosphoserine; Protein Processing, Post-Translational; Rabbits; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Ryanodine Receptor Calcium Release Channel; Sequence Alignment; Sequence Homology, Amino Acid; Sulfonamides; Tacrolimus Binding Proteins; Ultrasonography