Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Regulation of phosphorylation at the postsynaptic density during different activity states of Ca/calmodulin-dependent protein kinase II

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), the most abundant kinase at the postsynaptic density (PSD), is expected to be involved in activity-induced regulation of synaptic properties. CaMKII is activated when it binds calmodulin in the presence of Ca²⁺ and, once autophosphorylated on T-286/7, remains active in the absence of Ca²⁺ (autonomous form). In the present study we used a quantitative mass spectrometric strategy (iTRAQ) to identify sites on PSD components phosphorylated upon CaMKII activation. Phosphorylation in isolated PSDs was monitored under conditions where CaMKII is: (1) mostly inactive (basal state), (2) active in the presence of Ca²⁺, and (3) active in the absence of Ca²⁺. The quantification strategy was validated through confirmation of previously described autophosphorylation characteristics of CaMKII. The effectiveness of phosphorylation of major PSD components by the activated CaMKII in the presence and absence of Ca²⁺ varied. Most notably, autonomous activity in the absence of Ca²⁺ was more effective in the phosphorylation of three residues on SynGAP. Several PSD scaffold proteins were phosphorylated upon activation of CaMKII. The strategy adopted allowed the identification, for the first time, of CaMKII-regulated sites on SAPAPs and Shanks, including three conserved serine residues near the C-termini of SAPAP1, SAPAP2, and SAPAP3. Involvement of CaMKII in the phosphorylation of PSD scaffold proteins suggests a role in activity-induced structural re-organization of the PSD.     

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes

Oxidative stress remodels Ca²⁺ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca²⁺ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca²⁺ current (I_Ca,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H₂O₂ induced an increase in I_Ca,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H₂O₂-induced LTF of I_Ca,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca²⁺ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H₂O₂ via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.     

Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91^phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91^phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91^phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91^phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.     

High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.     

S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ

NO is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implied that NO can activate calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinase II (CaMKII) in neurons and the heart. Here we use our novel sensor of CaMKII activation, Camui, to monitor changes in the conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca²⁺/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the Cys-290 site. However, exposure of CaMKIIδ to GSNO prior to Ca²⁺/CaM exposure strongly suppresses kinase activation and conformational change by Ca²⁺/CaM. This NO-induced inhibition was ablated by mutation of the Cys-273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity.     

Nitric Oxide Induces Ca2+-independent Activity of the Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)

Both signaling by nitric oxide (NO) and by the Ca²⁺/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca²⁺-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca²⁺/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca²⁺. Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death.     

Catechol-O-methyltransferase: Effects of the val108met polymorphism on protein turnover in human cells

A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMT^H and COMT^L, respectively. Tissues and cells with a COMT^LL genotype have decreased COMT activity compared to COMT^HH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMT^L protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMT^LL compared to COMT^HH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMT^H was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMT^L compared to S-COMT^H protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMT^LL individuals. Subtle differences appear to be able to affect the stability of the S-COMT^L protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.     

Reactive sulfur species impair Ca2+/calmodulin-dependent protein kinase II via polysulfidation

We previously reported that Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is inhibited by S-nitrosylation of Cys⁶ in cells. Herein, we show that polysulfidation of CaMKII at Cys⁶ limits its enzyme activity following reactive sulfur species (RSS) stimulus. In vitro incubation of CaMKII with the RSS donor, Na₂S₄, induced the inhibition of the enzyme via its polysulfidation. Treatment with dithiothreitol reversed the polysulfidation and the subsequent inhibition. The inhibition of CaMKII by Na₂S₄ is competitive with ATP but not with the peptide substrate Syntide-2. In transfected cells expressing CaMKII, the enzyme activity decreased upon treatment with Na₂S₄, whereas cells expressing mutant CaMKII (C6A) were resistant to this treatment. In addition, the endogenous CaMKII was inhibited by treatment with Na₂S₄ in RAW264.7 murine macrophage cells. These results suggest a novel regulation of CaMKII by RSS via its Cys⁶ polysulfidation in cells.     

Genetically encoded fluorescent indicator for imaging NAD/NADH ratio changes in different cellular compartments

Background

The ratio of NAD⁺/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD⁺/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD⁺/NADH are fundamentally new approach for studying the NAD⁺/NADH dynamics.

Methods

We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy.

Results

The sensor, named RexYFP, reports changes in the NAD⁺/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD⁺/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD⁺/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore.

Conclusion

RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.

General significance

RexYFP has several advantages over existing NAD⁺/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.     

Regulation of CaMKII by phospho-Thr253 or phospho-Thr286 sensitive targeting alters cellular function

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated.     

Fluorescent protein recruitment assay for demonstration and analysis of in vivo protein interactions in plant cells and its application to Tobacco mosaic virus movement protein

We describe a simple fluorescent protein‐based method to investigate interactions with a viral movement protein in living cells that relies on the in vivo re‐localization of proteins in the presence of their interaction partners. We apply this method in combination with fluorescence lifetime imaging microscopy (FLIM) to demonstrate that a domain of the Tobacco mosaic virus (TMV) movement protein (MP) previously predicted to mediate protein:protein interactions is dispensable for these contacts. We suggest that this method can be generalized for analysis of other protein interactions in planta.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K⁺-dependent Na⁺/Ca²⁺-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K⁺-dependent Na⁺/Ca²⁺-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca²⁺ signal dynamics in olfactory and other neurons. Synaptic Ca²⁺ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca²⁺ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca²⁺ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca²⁺ signalling by activating a signal damping and termination pathway.     

A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD⁺ after the deacetylation was determined by converting NAD⁺ to a highly fluorescent cyclized α-adduct compound. By this assay, we found that nicotinamide, Cu²⁺, and Zn²⁺ antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.     

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.     

Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A)

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr³⁹³/Ser³⁹⁵) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.     

Characterization of CoPK02, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea

We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca²⁺/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca²⁺/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca²⁺-signaling in C. cinerea.     

Synergy between CaMKII substrates and β-adrenergic signaling in regulation of cardiac myocyte Ca(2+) handling

Cardiac excitation-contraction coupling is a highly coordinated process that is controlled by protein kinase signaling pathways, including Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA). Increased CaMKII expression and activity (as occurs during heart failure) destabilizes EC coupling and may lead to sudden cardiac death. To better understand mechanisms of cardiac CaMKII function, we integrated dynamic CaMKII-dependent regulation of key Ca²⁺ handling targets with previously validated models of cardiac EC coupling, Ca²⁺/calmodulin-dependent activation of CaMKII, and β-adrenergic activation of PKA. Model predictions are validated against CaMKII-overexpression data from rabbit ventricular myocytes. The model demonstrates how overall changes to Ca²⁺ handling during CaMKII overexpression are explained by interactions between individual CaMKII substrates. CaMKII and PKA activities during β-adrenergic stimulation may synergistically facilitate inotropic responses and contribute to a CaMKII-Ca²⁺-CaMKII feedback loop. CaMKII regulated early frequency-dependent acceleration of relaxation and EC coupling gain (which was highly sarcoplasmic reticulum Ca²⁺ load-dependent). Additionally, the model identifies CaMKII-dependent ryanodine receptor hyperphosphorylation as a proarrhythmogenic trigger. In summary, we developed a detailed computational model of CaMKII and PKA signaling in cardiac myocytes that provides unique insights into their regulation of normal and pathological Ca²⁺ handling.