Ca2+/calmodulin-dependent kinase IIδC-induced chronic heart failure does not depend on sarcoplasmic reticulum Ca2+ leak.

AIMS: Hyperactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII-dependent RyR2 phosphorylation for CaMKII-induced heart failure development in vivo. METHODS AND RESULTS: We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2-S2814A knock-in mice that are resistant to CaMKII-dependent RyR2 phosphorylation. Ca2+-spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F0 vs. 1.88 ± 0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+-leak induction in the CaMKIIδC TG, both regarding Ca2+-spark size and frequency, demonstrating that the CaMKIIδC-induced SR Ca2+ leak entirely depends on the CaMKII-specific RyR2-S2814 phosphorylation. Yet, the RyR2-S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. CONCLUSIONS: S2814 phosphoresistance of RyR2 prevents the CaMKII-dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms-independent of SR Ca2+ leak-are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure.

Mitochondrial protein prohibitin promotes learning memory recovery in mice following intracerebral hemorrhage via CAMKII/CRMP signaling pathway.

Prohibitin (PHB) is a mitochondrial inner membrane protein with neuroprotective, antioxidant, and apoptosis-reducing effects. This study aimed to explore the role of PHB in pathological symptoms, behavioral deficits, and cognitive impairment in a collagenase-IV-induced intracerebral hemorrhage (ICH) murine model. In this study, mice that received collagenase IV injection were pretreated with PHB or saline 21 days prior to modeling. The role of PHB in memory and learning ability was monitored using the Morris water maze, Y-maze, and rotarod, social, startle, and nest-building tests. The effect of PHB on depression-like symptoms was examined using the forced swimming, tail suspension, and sucrose preference tests. Subsequently, mouse samples were analyzed using immunohistochemistry, western blotting, Perls staining, Nissl staining, and gene sequencing. Results showed that collagenase IV significantly induced behavioral deficits, brain edema, cognitive impairment, and depressive symptoms. PHB overexpression effectively alleviated memory, learning, and motor deficits in mice with ICH. PHB markedly inhibited the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells and protein levels of ionized calcium-binding adapter molecule 1, glial fibrillary acidic protein, and interleukin-1ß in the perihematomal region of ICH mice. PHB overexpression also remarkably promoted production of neurologin1 (NLGL1), and upregulated levels of Ca2+-calmodulin-dependent kinase II (CaMKII) and collapsin response mediator protein-1 (CRMP1) proteins. In conclusion, PHB overexpression can effectively alleviate the neurological deficits and neurodegeneration around the hematoma region. This may play a protective role by upregulating the expression of NLGL1 and promoting expression of CaMKII and CRMP1.

Differential changes in cyclic adenosine 3'-5' monophosphate (cAMP) effectors and major Ca2+ handling proteins during diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is associated with differential and time-specific regulation of ß-adrenergic receptors and cardiac cyclic nucleotide phosphodiesterases with consequences for total cyclic adenosine 3'-5' monophosphate (cAMP) levels. We aimed to investigate whether these changes are associated with downstream impairments in cAMP and Ca2+ signalling in a type 1 diabetes (T1D)-induced DCM model. T1D was induced in adult male rats by streptozotocin (65 mg/kg) injection. DCM was assessed by cardiac structural and molecular remodelling. We delineated sequential changes affecting the exchange protein (Epac1/2), cAMP-dependent protein kinase A (PKA) and Ca2+ /Calmodulin-dependent kinase II (CaMKII) at 4, 8 and 12 weeks following diabetes, by real-time quantitative PCR and western blot. Expression of Ca2+ ATPase pump (SERCA2a), phospholamban (PLB) and Troponin I (TnI) was also examined. Early upregulation of Epac1 transcripts was noted in diabetic hearts at Week 4, followed by increases in Epac2 mRNA, but not protein levels, at Week 12. Expression of PKA subunits (RI, RIIα and Cα) remained unchanged regardless of the disease stage, whereas CaMKII increased at Week 12 in DCM. Moreover, PLB transcripts were upregulated in diabetic hearts, whereas SERCA2a and TnI gene expression was unchanged irrespective of the disease evolution. PLB phosphorylation at threonine-17 was increased in DCM, whereas phosphorylation of both PLB at serine-16 and TnI at serine-23/24 was unchanged. We show for the first time differential and time-specific regulations in cardiac cAMP effectors and Ca2+ handling proteins, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Increased Risk for Atrial Alternans in Rabbit Heart Failure: The Role of Ca2+/Calmodulin-Dependent Kinase II and Inositol-1,4,5-trisphosphate Signaling.

Heart failure (HF) increases the probability of cardiac arrhythmias, including atrial fibrillation (AF), but the mechanisms linking HF to AF are poorly understood. We investigated disturbances in Ca2+ signaling and electrophysiology in rabbit atrial myocytes from normal and failing hearts and identified mechanisms that contribute to the higher risk of atrial arrhythmias in HF. Ca2+ transient (CaT) alternans-beat-to-beat alternations in CaT amplitude-served as indicator of increased arrhythmogenicity. We demonstrate that HF atrial myocytes were more prone to alternans despite no change in action potentials duration and only moderate decrease of L-type Ca2+ current. Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition suppressed CaT alternans. Activation of IP3 signaling by endothelin-1 (ET-1) and angiotensin II (Ang II) resulted in acute, but transient reduction of CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ load, and lowered the alternans risk. However, prolonged exposure to ET-1 and Ang II enhanced SR Ca2+ release and increased the degree of alternans. Inhibition of IP3 receptors prevented the transient ET-1 and Ang II effects and by itself increased the degree of CaT alternans. Our data suggest that activation of CaMKII and IP3 signaling contribute to atrial arrhythmogenesis in HF.

Lockdown, a selective small-molecule inhibitor of the integrin phosphatase PPM1F, blocks cancer cell invasion.

Phosphatase PPM1F is a regulator of cell adhesion by fine-tuning integrin activity and actin cytoskeleton structures. Elevated expression of this enzyme in human tumors is associated with high invasiveness, enhanced metastasis, and poor prognosis. Thus, PPM1F is a target for pharmacological intervention, yet inhibitors of this enzyme are lacking. Here, we use high-throughput screening to identify Lockdown, a reversible and non-competitive PPM1F inhibitor. Lockdown is selective for PPM1F, because this compound does not inhibit other protein phosphatases in vitro and does not induce additional phenotypes in PPM1F knockout cells. Importantly, Lockdown-treated glioblastoma cells fully re-capitulate the phenotype of PPM1F-deficient cells as assessed by increased phosphorylation of PPM1F substrates and corruption of integrin-dependent cellular processes. Ester modification yields LockdownPro with increased membrane permeability and prodrug-like properties. LockdownPro suppresses tissue invasion by PPM1F-overexpressing human cancer cells, validating PPM1F as a therapeutic target and providing an access point to control tumor cell dissemination.

L-type Ca2+ channel recovery from inactivation in rabbit atrial myocytes.

Adaptation of the myocardium to varying workloads critically depends on the recovery from inactivation (RFI) of L-type Ca2+ channels (LCCs) which provide the trigger for cardiac contraction. The goal of the present study was a comprehensive investigation of LCC RFI in atrial myocytes. The study was performed on voltage-clamped rabbit atrial myocytes using a double pulse protocol with variable diastolic intervals in cells held at physiological holding potentials, with intact intracellular Ca2+ release, and preserved Na+ current and Na+ /Ca2+ exchanger (NCX) activity. We demonstrate that the kinetics of RFI of LCCs are co-regulated by several factors including resting membrane potential, [Ca2+ ]i , Na+ influx, and activity of CaMKII. In addition, activation of CaMKII resulted in increased ICa amplitude at higher pacing rates. Pharmacological inhibition of NCX failed to have any significant effect on RFI, indicating that impaired removal of Ca2+ by NCX has little effect on LCC recovery. Finally, RFI of intracellular Ca2+ release was substantially slower than LCC RFI, suggesting that inactivation kinetics of LCC do not significantly contribute to the beat-to-beat refractoriness of SR Ca2+ release. The study demonstrates that CaMKII and intracellular Ca2+ dynamics play a central role in modulation of LCC activity in atrial myocytes during increased workloads that could have important consequences under pathological conditions such as atrial fibrillations, where Ca2+ cycling and CaMKII activity are altered.

Adeno-associated virus-mediated expression of an inactive CaMKIIß mutant enhances muscle mass and strength in mice.

A concurrent reduction in muscle mass and strength is frequently observed in numerous conditions, including neuromuscular disease, ageing, and muscle inactivity due to limb immobilization or prolonged bed rest. Thus, identifying the molecular mechanisms that control skeletal muscle mass and strength is fundamental for developing interventions aimed at counteracting muscle loss (muscle atrophy). It was recently reported that muscle atrophy induced by denervation of motor nerves was associated with increased expression of Ca2+/calmodulin-dependent protein serine/threonine kinase II ß (CaMKIIß) in muscle. In addition, treatment with KN-93 phosphate, which inhibits CaMKII-family kinases, partly suppressed denervation-induced muscle atrophy. Therefore, to test a possible role for CaMKIIß in muscle mass regulation, we generated and injected recombinant adeno-associated virus (AAV) vectors encoding wild-type (AAV-WT), inactive (AAV-K43 M), or constitutively active (AAV-T287D) CaMKIIß into the left hindlimb tibialis anterior muscle of mice at three months of age. Although AAV-WT infection induced expression of exogenous CaMKIIß in the hindlimb muscle, no significant changes in muscle mass and strength were observed. By contrast, AAV-K43 M or AAV-T287D infection induced exogenous expression of the corresponding mutants and significantly increased or decreased the muscle mass and strength of the infected hind limb, respectively. Together, these findings demonstrate the potential of CaMKIIß as a novel therapeutic target for enhancing muscle mass and strength.

CAMK2/CaMKII activates MLKL in short-term starvation to facilitate autophagic flux.

MLKL (mixed lineage kinase domain like pseudokinase) is a well-known core component of necrosome that executes necroptotic cell death upon phosphorylation by RIPK3 (receptor interacting serine/threonine kinase 3). Recent studies also implicate a role of MLKL in endosomal trafficking, which is not always dependent on RIPK3. Using mouse Neuro-2a and L929 as well as human HEK293 and HT29 cells, we show here that MLKL is phosphorylated in response to serum and amino acid deprivation from the culture medium, in a manner that depends on CAMK2/CaMKII (calcium/calmodulin dependent protein kinase II) but not RIPK3. The starvation-induced increase in MLKL phosphorylation was accompanied by decreases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II) and SQSTM1/p62 (sequestosome 1), markers of autophagosomes. These changes were prevented by disrupting either MLKL or CAMK2 by pharmacology and genetic manipulations. Moreover, disrupting MLKL or CAMK2 also inhibited the incorporation of LC3-II into autolysosomes, demonstrating a role of the CAMK2-MLKL pathway in facilitating autophagic flux during short-term starvation, in contrast to necroptosis which suppressed autophagic flux. Furthermore, unlike the necroptotic pathway, the starvation-evoked CAMK2-mediated MLKL phosphorylation protected cells from starvation-induced death. We propose that upon nutrient deprivation, MLKL is activated by CAMK2, which in turn facilitates membrane scission needed for autophagosome maturation, allowing the proper fusion of the autophagosome with lysosome and the subsequent substance degradation. This novel function is independent of RIPK3 and is not involved in necroptosis, implicating new roles for this pseudokinase in cell survival, signaling and metabolism.Abbreviations: CAMK2/CaMKII: calcium/calmodulin dependent protein kinase II; DIABLO/SMAC: direct inhibitor of apoptosis-binding protein with low pI/second mitochondria-derived activator of caspase; ECS: extracellular solution; ESCRT: endosomal sorting complexes required for transport; FBS: fetal bovine serum; GSK3B: glycogen synthase kinase 3 beta; HBSS: Hanks' balanced salt solution; KO: knockout; LC3-II: lipidated microtubule associated protein 1 light chain 3 beta; LDH: lactate dehydrogenase; MLKL: mixed lineage kinase domain like pseudokinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; N2a: Neuro-2a neuroblastoma; Nec-1: necrostatin-1; NSA: necrosulfonamide; PBS: phosphate-buffered saline; PI: propidium iodide; PK-hLC3: pHluorin-mKate2-human LC3; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; ROS: reactive oxygen species; RPS6KB1/S6K: ribosomal protein S6 kinase B1; shRNA: short hairpin RNA; siRNA: small interference RNA; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TSZ, treatment with TNF + DIABLO mimetics + z-VAD-FMK.

Cellular Mechanisms Underlying the Low Cardiotoxicity of Istaroxime.

Background Istaroxime is an inhibitor of Na+/K+ ATPase with proven efficacy to increase cardiac contractility and to accelerate relaxation attributable to a relief in phospholamban-dependent inhibition of the sarcoplasmic reticulum Ca2+ ATPase. We have previously shown that pharmacologic Na+/K+ ATPase inhibition promotes calcium/calmodulin-dependent kinase II activation, which mediates both cardiomyocyte death and arrhythmias. Here, we aim to compare the cardiotoxic effects promoted by classic pharmacologic Na+/K+ ATPase inhibition versus istaroxime. Methods and Results Ventricular cardiomyocytes were treated with ouabain or istaroxime at previously tested equi-inotropic concentrations to compare their impact on cell viability, apoptosis, and calcium/calmodulin-dependent kinase II activation. In contrast to ouabain, istaroxime neither promoted calcium/calmodulin-dependent kinase II activation nor cardiomyocyte death. In addition, we explored the differential behavior promoted by ouabain and istaroxime on spontaneous diastolic Ca2+ release. In rat cardiomyocytes, istaroxime did not significantly increase Ca2+ spark and wave frequency but increased the proportion of aborted Ca2+ waves. Further insight was provided by studying cardiomyocytes from mice that do not express phospholamban. In this model, the lower Ca2+ wave incidence observed with istaroxime remains present, suggesting that istaroxime-dependent relief on phospholamban-dependent sarcoplasmic reticulum Ca2+ ATPase 2A inhibition is not the unique mechanism underlying the low arrhythmogenic profile of this drug. Conclusions Our results indicate that, different from ouabain, istaroxime can reach a significant inotropic effect without leading to calcium/calmodulin-dependent kinase II-dependent cardiomyocyte death. Additionally, we provide novel insights regarding the low arrhythmogenic impact of istaroxime on cardiac Ca2+ handling.

CaMKII exacerbates heart failure progression by activating class I HDACs.

BACKGROUND: Persistent cardiac Ca2+/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling. Class I HDACs, including HDAC1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. While it is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, the role of CaMKII in regulating class I HDACs during heart failure progression is unclear. METHODS AND RESULTS: CaMKII increases the deacetylase activity of recombinant HDAC1, HDAC2 and HDAC3 via in vitro phosphorylation assays. Phosphorylation sites on HDAC1 and HDAC3 are identified with mass spectrometry. HDAC1 activity is also increased in cardiac-specific CaMKIIδC transgenic mice (CaMKIIδC-tg). Beyond post-translational modifications, CaMKII induces HDAC1 and HDAC3 expression. HDAC1 and HDAC3 expression are significantly increased in CaMKIIδC-tg mice. Inhibition of CaMKII by overexpression of the inhibitory peptide AC3-I in the heart attenuates the upregulation of HDAC1 after myocardial infarction surgery. Importantly, a potent HDAC1 inhibitor Quisinostat improves downregulated autophagy genes and cardiac dysfunction in CaMKIIδC-tg mice. In addition to Quisinostat, selective class I HDACs inhibitors, Apicidin and Entinostat, HDAC3 specific inhibitor RGFP966, as well as HDAC1 and HDAC3 siRNA prevent CaMKII overexpression induced cardiac myocyte hypertrophy. CONCLUSION: CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression. Selective class I HDACs inhibition may be a novel therapeutic avenue to alleviate CaMKII hyperactivity induced cardiac dysfunction.

CAIII expression in skeletal muscle is regulated by Ca2+-CaMKII-MEF2C signaling.

Carbonic anhydrase III (CAIII) is selectively expressed in slow-twitch myofibers in skeletal muscle. The fast-twitch to slow-twitch transformation of myofibers following denervation is accompanied by increased CAIII expression, suggesting that the effects of nerve impulses on skeletal-muscle remodeling influence CAIII expression. Here, we determined the molecular mechanisms underlying the effects of nerve conduction on CAIII expression. The results indicated that changes in skeletal-muscle [Ca2+]i altered CAIII expression. Moreover, results from the RNA-interference and over-expression experiments identified myocyte enhancer factor 2C (MEF2C) as the key transcription factor regulating [Ca2+]i-mediated changes in CAIII transcription. Additionally, chromatin immunoprecipitation experiments and luciferase assays confirmed MEF2C interaction and direct binding of the CAIII promoter between -416 and -200 base pair. Investigations of upstream cytoplasmic signaling pathways responsible for MEF2C activation revealed Ca2+/calmodulin-dependent protein kinase II (CaMKII) as the key factor involved in MEF2C-mediated regulation of CAIII expression. This study demonstrates that the Ca2+-CaMKII-MEF2C signaling pathway is the key factor involved in regulating CAIII expression in skeletal muscle. These results provide a theoretical basis supporting further investigations of changes in CAIII levels under different pathophysiological conditions and will facilitate a broader understanding of the biological functions of CAIII.

ß-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII.

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. ß-adrenergic receptor (ßAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate INaL. However, it remains unclear how each of these pathways regulates INaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on INaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that INaL had a basal level current independent of PKA, but partially dependent on CaMKII. ßAR activation (10 nM isoproterenol, ISO) further enhanced INaL via both PKA and CaMKII pathways. However, PKA predominantly increased INaL early during the AP plateau, whereas CaMKII mainly increased INaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced INaL similar to the ßAR-induced CaMKII effect, while NOS inhibition prevented the ßAR-induced CaMKII-dependent INaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced INaL activation early in the AP. Taken together, our data reveal differential modulations of INaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in INaL during AP deepens our understanding of the important role of INaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

Repetitive Transient Ischemia-Induced Cardiac Angiogenesis is Mediated by Camkii Activation.

BACKGROUND/AIMS: Coronary angiogenesis is an important protective mechanism in response to myocardial ischemia in coronary artery disease. However, the underlying mechanisms remain largely unclear. Here, we investigated the role of CaMKII activation in ischemia-induced cardiac angiogenesis. METHODS: Repetitive transient ischemia model was established in C57/BL6 mice by daily multiple episodes (3 times/day) of short time (5 min) occlusion of the left anterior descending coronary artery for 7 days. Coronary angiogenesis was detected by immunofluorescent staining. RT-qPCR and Western blot analyses were used to detect the mRNA and protein levels of CaMKII, p-CaMKII and VEGF. Primary cardiac microvascular endothelial cells (CMECs) were isolated to investigate the effects of KN93 on cell proliferation and migration in hypoxic condition. RESULTS: We found that angiogenesis was induced in the ischemic myocardium and suppressed by chronic intraperitoneal injection of CaMKII inhibitor KN93. RT-qPCR and Western blot analyses showed that myocardial ischemia induced an increased expression and autophosphorylation of CaMKII. VEGF expression was increased in the ischemia model but blunted by KN93. Moreover, KN93 suppressed the proliferation and migration of cardiac endothelial cells in hypoxic condition in which the protein expression of CaMKII, p-CaMKII and VEGF was increased. CONCLUSION: CaMKII is an important mediator for the ischemia-induced coronary angiogenesis, in which CaMKII-triggered VEGF expression plays a key role.

CaMKII-mediated phosphorylation of RyR2 plays a crucial role in aberrant Ca2+ release as an arrhythmogenic substrate in cardiac troponin T-related familial hypertrophic cardiomyopathy.

AIMS: Cardiac Troponin T (TnT) mutation-linked familial hypertrophic cardiomyopathy (FHC) is known to cause sudden cardiac death at a young age. Here, we investigated the role of the Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR), ryanodine receptor (RyR2), in the pathogenic mechanism of lethal arrhythmia in FHC-related TnT-mutated transgenic mice (TG; TnT-delta160E). METHODS AND RESULTS: In TG cardiomyocytes, the Ca2+ spark frequency (SpF) was much higher than that in non-TG cardiomyocytes. These differences were more pronounced in the presence of isoproterenol (ISO; 10 nM). This increase in SpF was largely reversed by a CaMKII inhibitor (KN-93), but not by a protein kinase A inhibitor (H89). CaMKII phosphorylation at Ser2814 in RyR2 was increased significantly in TG. Spontaneous Ca2+ transients (sCaTs) after cessation of a 1-5 Hz pacing, frequently observed in ISO-treated TG cardiomyocytes, were also attenuated by KN-93, but not by H89. The RyR2 stabilizer dantrolene attenuated Ca2+ sparks and sCaTs in ISO-treated TG cardiomyocytes, indicating that the mutation-linked aberrant Ca2+ release is mediated by destabilized RyR2. CONCLUSIONS: In FHC-linked TnT-mutated hearts, RyR2 is susceptible to CaMKII-mediated phosphorylation, presumably because of a mutation-linked increase in diastolic [Ca2+]i, causing aberrant Ca2+ release leading to lethal arrhythmia.

Up-regulation of Ca2+/CaMKII/CREB signaling in salicylate-induced tinnitus in rats.

The purpose of the study was to investigate the changes of Ca2+/calmodulin-dependent protein kinases II (CaMKII)/cAMP response element-binding protein (CREB) signaling pathway in a rat tinnitus model. Eighteen Wistar rats were randomly divided into three groups: normal control (NC), normal saline (NS), and tinnitus model (TM) groups. Tinnitus model was induced by intraperitoneal injection of salicylate. The concentration of intracellular calcium level in auditory cortex cells was determined using Fura-2 acetoxymethyl ester (Fura-2 AM) method with fluorospectrophotometer. Expressions of calmodulin (CaM), N-methyl-D-aspartate receptor 2B subunit (NR2B), calcium-calmodulin kinase II (CaMKII), and cAMP response element-binding protein (CREB) were detected with Western blot. Tinnitus model was successfully established by the intraperitoneal administration of salicylate in rats. Compared with rats in NC and NS groups, salicylate administration significantly elevated CaM, NR2B, phospho-CaMKII and phospho-CREB expression in auditory cortex from tinnitus model group (p < 0.05), and the free intracellular Ca2+ concentrations (p < 0.05). Our data reveal that salicylate administration causes tinnitus symptoms and elevates Ca2+/CaMKII/CREB signaling pathway in auditory cortex cells. Our study likely provides a new understanding of the development of tinnitus.

Competitive Tuning Among Ca2+/Calmodulin-Dependent Proteins: Analysis of in silico Model Robustness and Parameter Variability.

INTRODUCTION: Calcium/Calmodulin-dependent (Ca2+/CaM-dependent) regulation of protein signaling has long been recognized for its importance in a number of physiological contexts. Found in almost all eukaryotic cells, Ca2+/CaM-dependent signaling participates in muscle development, immune responses, cardiac myocyte function and regulation of neuronal connectivity. In excitatory neurons, dynamic changes in the strength of synaptic connections, known as synaptic plasticity, occur when calcium ions (Ca2+) flux through NMDA receptors and bind the Ca2+-sensor calmodulin (CaM). Ca2+/CaM, in turn, regulates downstream protein signaling in actin polymerization, receptor trafficking, and transcription factor activation.The activation of downstream Ca2+/CaM-dependent binding proteins (CBPs) is a function of the frequency of Ca2+ flux, such that each CBP is preferentially "tuned" to different Ca2+ input signals. We have recently reported that competition among CBPs for CaM binding is alone sufficient to recreate in silico the observed in vivo frequency-dependence of several CBPs. However, CBP activation may strongly depend on the identity and concentration of proteins that constitute the competitive pool; with important implications in the regulation of CBPs in both normal and disease states. METHODS: Here, we extend our previous deterministic model of competition among CBPs to include phosphodiesterases, AMPAR receptors that are important in synaptic plasticity, and enzymatic function of CBPs: cAMP regulation, kinase activity, and phosphatase activity. After rigorous parameterization and validation by global sensitivity analysis using Latin Hypercube Sampling (LHS) and Partial Rank Correlation Coefficients (PRCC), we explore how perturbing the competitive pool of CBPs influences downstream signaling events. In particular, we hypothesize that although perturbations may decrease activation of one CBP, increased activation of a separate, but enzymatically-related CBP could compensate for this loss, providing a homeostatic effect. RESULTS AND CONCLUSIONS: First we compare dynamic model output of two models: a two-state model of Ca2+/CaM binding and a four-state model of Ca2+/CaM binding. We find that a four-state model of Ca2+/CaM binding best captures the dynamic nature of the rapid response of CaM and CBPs to Ca2+ flux in the system. Using global sensitivity analysis, we find that model output is robust to parameter variability. Indeed, although variations in the expression of the CaM buffer neurogranin (Ng) may cause a decrease in Ca2+/CaM-dependent kinase II (CaMKII) activation, overall AMPA receptor phosphorylation is preserved; ostensibly by a concomitant increase in adenylyl cyclase 8 (AC8)-mediated activation of protein kinase A (PKA). Indeed phosphorylation of AMPAR receptors by CaMKII and PKA is robust across a wide range of Ng concentrations, though increases in AMPAR phosphorylation is seen at low Ng levels approaching zero. Our results may explain recent counter-intuitive results in neurogranin knockout mice and provide further evidence that competitive tuning is an important mechanism in synaptic plasticity. These results may be readily translated to other Ca2+/CaM-dependent signaling systems in other cell types and can be used to suggest targeted experimental investigation to explain counter-intuitive or unexpected downstream signaling outcomes.Tamara Kinzer-Ursem is an Assistant Professor in the Weldon School of Biomedical Engineering. She received her B.S. in Bioengineering from the University of Toledo and her M.S. and Ph.D. degrees in Chemical Engineering from the University of Michigan, and her post-doctoral training in Molecular Neuroscience at the California Institute of Technology. Prior to joining Purdue she was the Head of R&D in Biochemistry at Maven Biotechnologies and Visiting Associate in Chemical Engineering at the California Institute of Technology.Research in the Kinzer-Ursem lab focuses on developing tools to advance quantitative descriptions of cellular processes and disease within three areas of expertise: 1) Using particle diffusivity measurements to quantify biomolecular processes. Particle diffusometry is being used as a sensitive biosensor to detect the presence of pathogens in environmental and patient samples. 2) Development of novel protein tagging technologies that are used to label proteins in vivo to enable quantitative description of protein function and elucidate disease mechanisms. 3) Computational modeling of signal transduction mechanisms to understand cellular processes. Using computational techniques, we have recently described "competitive tuning" as a mechanism that might be used to regulate information transfer through protein networks, with implications in cell behavior and drug target analysis.

Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation.

Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits the differentiation of human neural progenitor cells and influences the expression of proteins associated with several cellular functions. We aimed to determine whether such chronic irradiation influences the expression of proteins associated with PC12 cells. Chronic irradiation at 0.027 mGy/min resulted in inhibition of NGF-induced neurite extension. Furthermore, irradiation enhanced the nerve growth factor (NGF)-induced increase in the phosphorylation of extracellular signal-regulated kinase (ERK), but did not affect the phosphorylation of NGF receptors, suggesting that irradiation influences pathways unassociated with the activation of ERK. We then examined whether irradiation influenced the Akt-Rac1 pathway, which is unaffected by ERK activation. Chronic irradiation also enhanced the NGF-induced increase in Akt phosphorylation, but markedly inhibited the NGF-induced increase in Rac1 activity that is associated with neurite extension. These results suggest that the inhibitory effect of irradiation on neurite extension influences pathways unassociated with Akt activation. As Ca2+/calmodulin-dependent kinase II (CaMKII) is known to inhibit the NGF-induced neurite extension in PC12 cells, independent of ERK and Akt activation, we next examined the effects of irradiation on CaMKII activation. Chronic irradiation induced CaMKII activation, while application of KN-62 (a specific inhibitor of CaMKII), attenuated increases in CaMKII activation and recovered neurite extension and NGF-induced increases in Rac1 activity that was inhibited by irradiation. Our results suggest that chronic irradiation with low-dose-rate γ-rays inhibits Rac1 activity via CaMKII activation, thereby inhibiting NGF-induced neurite extension.

Low-molecular fraction of wheat protein hydrolysate stimulates glucagon-like peptide-1 secretion in an enteroendocrine L cell line and improves glucose tolerance in rats.

The incretin hormone glucagon-like peptide-1 (GLP-1) is secreted by enteroendocrine L cells. Stimulating endogenous GLP-1 secretion by dietary factors is a promising strategy to increase GLP-1 action. Several studies have examined the specific physiological function of wheat protein hydrolysate. Some reports suggested that intake of wheat protein ameliorates hyperglycemia. We hypothesized that wheat protein hydrolysate reduces blood glucose concentration via stimulation of GLP-1 secretion. In this study, we investigated whether wheat protein hydrolysate stimulates GLP-1 secretion and its molecular mechanism in an enteroendocrine L cell line (GLUTag cells), and we examined the effect on glucose tolerance via stimulation of GLP-1 secretion followed by induction of insulin secretion in rats. The low-molecular fraction of wheat protein hydrolysate (LWP) significantly increased GLP-1 secretion, whereas the high-molecular fraction did not. This increase was found to involve activation of the Ca2+/calmodulin-dependent kinase II pathway mediated by G protein-coupled receptor family C group 6 subtype A. Moreover, preadministration of LWP ameliorated hyperglycemia via the stimulation of GLP-1 secretion followed by induction of insulin secretion in rats. Furthermore, this LWP-induced glucose-lowering effect was significantly attenuated by the administration of a GLP-1 receptor antagonist. These results demonstrate that LWP significantly increased GLP-1 secretion through activation of the Ca2+/calmodulin-dependent kinase II pathway mediated by G protein-coupled receptor family C group 6 subtype A in GLUTag cells. Moreover, preadministration of LWP ameliorated hyperglycemia via the stimulation of GLP-1 secretion followed by induction of insulin secretion in rats.

Dysregulation of neuronal calcium homeostasis in Alzheimer's disease - A therapeutic opportunity?

Alzheimer's disease (AD) is the disease of lost memories. Synaptic loss is a major reason for memory defects in AD. Signaling pathways involved in memory loss in AD are under intense investigation. The role of deranged neuronal calcium (Ca2+) signaling in synaptic loss in AD is described in this review. Familial AD (FAD) mutations in presenilins are linked directly with synaptic Ca2+ signaling abnormalities, most likely by affecting endoplasmic reticulum (ER) Ca2+ leak function of presenilins. Excessive ER Ca2+ release via type 2 ryanodine receptors (RyanR2) is observed in AD spines due to increase in expression and function of RyanR2. Store-operated Ca2+ entry (nSOC) pathway is disrupted in AD spines due to downregulation of STIM2 protein. Because of these Ca2+ signaling abnormalities, a balance in activities of Ca2+-calmodulin-dependent kinase II (CaMKII) and Ca2+-dependent phosphatase calcineurin (CaN) is shifted at the synapse, tilting a balance between long-term potentiation (LTP) and long-term depression (LTD) synaptic mechanisms. As a result, synapses are weakened and eliminated in AD brains by LTD mechanism, causing memory loss. Targeting synaptic calcium signaling pathways offers opportunity for development of AD therapeutic agents.

CaMKII and at least two unidentified kinases phosphorylate regulatory light chain in non-contracting cardiomyocytes.

In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca(2+)-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% ± 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca(2+) (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca(2+)/calmodulin-dependent kinase and a Ca(2+)/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities.