Dependent Protein kinaseII Research Articles

Binary-FRET reveals transient excited-state structure associated with activity-dependent CaMKII - NR2B binding and adaptation

Synaptic functions are mediated and modulated by a coordinated choreography of protein conformational changes and interactions in response to intracellular calcium dynamics. Time-lapse Förster resonance energy transfer can be used to study the dynamics of both conformational changes and protein-protein interactions simultaneously under physiological conditions if two resonance energy transfer reactions can be multiplexed. Binary-FRET is a technique developed to independently monitor the dynamics of calcium-calmodulin dependent protein kinase-II catalytic-domain pair separation in the holoenzyme, and its role in establishing activity-dependent holoenzyme affinity for the NR2B binding fragment of the N-methyl-D-aspartate receptor. Here we show that a transient excited-state intermediate exists where paired catalytic-domains in the holoenzyme first separate prior to subsequent NR2B association. Additionally, at non-saturating free calcium concentrations, our multiplexed approach reveals that the holoenzyme exhibits a biochemical form of plasticity, calcium dependent adaptation of T-site ligand binding affinity.

Compound Chai Jin Jie Yu Tablets, Acts as An Antidepressant by Promoting Synaptic Function in the Hippocampal Neurons

ObjectiveTo investigate the effectiveness of compound Chai Jin Jie Yu Tablets (CJJYT) in ameliorating cognitive impairment associated with depression and its potential mechanism of action. MethodsIn vitro experiments, the hippocampus was isolated from the whole brain of the fetal rat and cultured into hippocampal neuron cells. 50 μM corticosterone (CORT) was added to each group 18 h before the experiment for modeling depression, with the exception of the control group. After modeling, the blank serum group was added with 10% blank serum, the CJJYT group and the venlafaxine group were added with the corresponding 10% drug-containing serum, and the control group and the model group were added with equal volumes of culture medium. The intracellular Ca2+ mean fluorescence intensity, miniature excitatory postsynaptic current (mEPSC) current amplitude, and frequency of different hippocampal neurons were evaluated as indicators of synaptic function in the hippocampal neurons. In addition, the expression of synaptic plasticity related proteins, synaptophysin-α (SYN-α), N-methyl-D-aspartate receptor 2A (NR2A), N-methyl-D- aspartate receptor 2B (NR2B), post synaptic density 95 protein (PSD-95), calcium/calmodulin dependent protein kinaseⅡ (CaMKⅡ) and synaptic associated protein (SynGAP) were detected in the hippocampal neurons by immunofluorescence staining and high content analysis (HCA) system. Then, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of SYN-α, NR2A, NR2B, PSD- 95, CaMKⅡ and SynGAP. For in vivo experiments, except for those in the blank control group, all rats were treated within a single cage for chronic unpredictable stress-induced depression modeling and subjected to corresponding drug interventions. Behavioral tests were used to detect depressive behavior and determine learning, memory and other cognitive abilities, whereas enzyme-linked immunosorbent assay (ELISA) was used to detect the CORT levels. Golgi-Cox staining was used to observe changes in the synaptic morphology of parahippocampal gyrus CA1 area (CA1) and dentategyrus (DG). ResultsIn vitro, CJJYT treatment reduced the intracellular Ca2+ mean flurorescence intensity in the hippocampal neurons (P < 0.05), causing a reduction in the frequency and current amplitude of mEPSC (P < 0.05), and thus inhibited the excessive activation of post-synaptic receptors. CJJYT treatment reduced the protein and mRNA expression of SYN-α, NR2A, NR2B and PSD-95 in the hippocampal neurons (P < 0.05), increased the mRNA and protein expression of CaMKⅡ and SynGAP (P < 0.05), and thereby improved the synaptic plasticity of the hippocampal neurons. In vivo, CJJYT intervention improved sucrose preference, voluntary activity , learning and memory ability of Morris water maze test, and suppressed appetite (P < 0.05), and increased the despair feeling of forced swimming test (P < 0.05). The CORT level was reduced (P < 0.05), leading to the repair of synaptic damage in the hippocampal neurons. ConclusionsCJJYT can improve the synaptic function of hippocampal neurons and has obvious protective effects on neurons. It can repair the structural damage in the hippocampal neurons, improving the cognitive ability of the depressed model rats. The mechanism of CJJYT improving cognition in depressed rats may be related to the transmission and function of SYN-α/NR and its downstream neurotransmitters.

Antidepressant-like effects of the Guanxin Danshen formula via mediation of the CaMK II-CREB-BDNF signalling pathway in chronic unpredictable mild stress-induced depressive rats.

Depression is a chronic and recurrent syndrome of mood disorder causing immense social and economic burden; thus, treatment should be improved. Guanxin Danshen formula (GXDSF), a natural botanical drug composition prescription, has significant cardiovascular protective effects and is widely used in the clinical treatment of myocardial ischaemic diseases. However, it is still unclear and seldom studied whether GXDSF has neuroprotective effects against depressive disorders. This study explored whether GXDSF has antidepressant-like effects in rats exposed to chronic unpredictable mild stress (CUMS) and analysed the possible underlying neurotrophic expression and psychotropic mechanisms. The present study was designed to investigate the antidepressant effects of GXDSF treatment in a CUMS-induced rat model. Based on the clinical doses, the drug-treated group was intragastrically administered GXDSF for 30 days, and rats were simultaneously exposed to CUMS stimulation for 30 days. After induction and drug administration, the depression-like behaviours were determined via the sucrose preference test (SPT), the open field test (OFT), the tail suspension test (TST), and the forced swim test (FST). ELISA kits were used to examine the monoaminergic neurotransmitters, monoamine oxidase (MAO) and Ca2+ levels in the hippocampus. Moreover, we measured and analysed the brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels and the upstream regulation and signal pathways of BDNF and NGF to explore their related mechanisms in this animal model of depression, including calcium-calmodulin dependent protein kinase-II (CaMKII) and cAMP response element-binding (CREB). The results revealed that GXDSF may possess significant antidepressant-like effects via improving body weight, raising the sucrose preference in the SPT, increasing the total distance, the number of upright stands, and the residence time of the central zone in the open field test (OPF) and reducing the immobility time in the TST and FST. In addition, GXDSF significantly upregulated the relative levels of neurotransmitters, including dopamine (DA), norepinephrine (NE), and serotonin (5-HT), in a dose-dependent manner and inhibited MAO activities in the hippocampus. Moreover, GXDSF reversed the decline in intracellular CREB and p-CREB expression induced by CUMS, downregulated the phosphorylation levels of intracellular CaMKII and its two subunits CaMKIIα and CaMKIIβ in the hippocampus, and thus, clearly upregulated the downstream effector protein expression levels of BDNF, NGF, and synitaxine-1 in the hippocampus. These data suggest that the antidepressant effects of GXDSF have a potential relationship with regulating changes in the CaMKII-CREB-BDNF pathway. Despite several limitations of this study, the results have suggested that GXDSF administration possesses antidepressant-like effects in CUMS-treated rats and provide the first in vivo demonstration of a possible mechanism of GXDSF via regulating changes in the CaMKII-CREB-BDNF signalling pathway. These findings provide a novel potential substrate by which herbal antidepressants may exert therapeutic effects in the treatment of depression.

Effect of Electroacupuncture on Intracellular Calcium Ion Concentration and Expression of Calcium/Calmodulin Dependent Protein Kinase Ⅱ in Lumbar Spinal Cord in Rats with Neuropathic Pain

To observe the changes of intracellular calcium ([Ca2＋]i) concentration and expression of calcium/calmodulin dependent protein kinaseⅡ (CaMKⅡ) in spinal dorsal horn neurons of spared nerve injury (SNI) rats, so as to explore its mechanisms underlying improvement of neuropathic pain. One hundred and ten SD rats were randomly divided into 5 groups: sham control, model, EA, AP-5 and L-NAME groups. The sham group underwent only a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right "Weizhong" (BL 40) and "Huantiao" (GB 30) for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg·kg－1·d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase [NOS], 60 mg·kg－1·d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, and the calcium fluorescence intensity (shown by Fluo-3/AM calcium fluorescence indicator) of the superficial layer of the lumbar spinal cord (L 4－L 6) was measured by immunohistochemical staining and the expression of spinal cord (L 4－L 6) CaMK Ⅱ protein was detected by Western blot (WB). After modeling, the mechanical pain threshold was significantly decreased on day 10 and 16 after operation in comparison with the sham operation group and baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA, AP-5 and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05), while the effect of EA was significantly superior to that of AP-5 and L-NAME groups (P<0.05), suggesting a reduction of EA analgesia after administration of AP-5 and L-NAME. The concentration of intracellular [Ca2＋]i was significantly higher in the model group than in the sham group, and considerably lower in the EA, AP-5 and L-NAME groups than in the model group (P<0.01, P<0.05). Moreover, the expression level of CaMKⅡ shown by WB and immunohistochemical staining was significantly higher in the model group than in the sham group (P<0.05) and obviously lower in the EA group (not the AP-5 and L-NAME groups) than in the model group on day 16 after the intervention (P<0.05). It suggests an involvement of glutamate NMDA receptor and NMDAR-NOS/NO signaling in the analgesic effect and CaMKⅡ expression down-regulation of EA. EA can ease pain in rats with neuropathic pain, which is closely related to its effect in reducing the calcium concentration and the expression of CaMKⅡ in the lumbar spinal cord, possibly mediated by glutamate NMDA receptor and NMDAR-NOS/NO signaling.

Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions

Between 8 to 14 calcium-calmodulin (Ca2+/CaM) dependent protein kinase-II (CaMKII) subunits form a complex that modulates synaptic activity. In living cells, the autoinhibited holoenzyme is organized as catalytic-domain pairs distributed around a central oligomerization-domain core. The functional significance of catalytic-domain pairing is not known. In a provocative model, catalytic-domain pairing was hypothesized to prevent ATP access to catalytic sites. If correct, kinase-activity would require catalytic-domain pair separation. Simultaneous homo-FRET and fluorescence correlation spectroscopy was used to detect structural changes correlated with kinase activation under physiological conditions. Saturating Ca2+/CaM triggered Threonine-286 autophosphorylation and a large increase in CaMKII holoenzyme hydrodynamic volume without any appreciable change in catalytic-domain pair proximity or subunit stoichiometry. An alternative hypothesis is that two appropriately positioned Threonine-286 interaction-sites (T-sites), each located on the catalytic-domain of a pair, are required for holoenzyme interactions with target proteins. Addition of a T-site ligand, in the presence of Ca2+/CaM, elicited a large decrease in catalytic-domain homo-FRET, which was blocked by mutating the T-site (I205K). Apparently catalytic-domain pairing is altered to allow T-site interactions.

Characterization of Calcium-Calmodulin Kinase II Inhibitor Protein (CaMKIIN) by Fluorescence Polarization and Fluctuation Analysis

Calcium-calmodulin (Ca/CaM) dependent protein Kinase-II (CaMKII) is a major mediator of various cellular signaling processes and has been implicated in schizophrenia, heart failure, arthritis, and certain types of cancers. CaMKII subunits assemble to form a large dodecameric holoenzyme with which numerous proteins are known to interact. Among these regulators are CaMKIIN-alpha and -beta, small 8 kDa proteins that are expressed almost exclusively in brain and testis. CaMKIIN proteins are highly specific inhibitors of CaMKII activity, but the physiological role and oligomeric state of these proteins are not known. It is also not known if interactions with the holoenzyme are stable or transient, or if these interactions are stoichiometric with kinase subunits. To address these questions we used Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid method that simultaneously measures Homo-FRET and fluorescence correlation spectroscopy (FCS). FPFA studies reveal that C-terminal Venus-tagged CaMKIIN is a monomeric protein that can bind stably to Ca/CaM-activated CaMKII. Up to six CaMKIIN monomers can bind to the holoenzyme, suggesting that one CaMKIIN inhibitory protein interacts with two CaMKII subunits in the holoenzyme. Furthermore, Homo-FRET analysis of Venus-tagged CaMKII holoenzyme co-incubated with untagged CaMKIIN revealed the separation of catalytic domain pairs, but only after activation with Ca/CaM. This is consistent with our previous observation of catalytic domain unpairing in hippocampal neurons upon calcium influx through NMDA receptors. The C-terminus of CaMKIIN is thought to play a key role in inhibition of CaMKII because a reduction on inhibitory potency was observed when its C-terminal was tagged with Venus. Our results suggest that in addition to inhibiting the kinase, CaMKIIN regulates catalytic domain pairing.

Fluorescence Polarization and Fluctuation Analysis Reveals Covert Changes in CaMKII Holoenzyme Organization Triggered by Calmodulin and Camkiintide

The calcium-calmodulin dependent protein kinase-II (CaMKII) is a multimeric kinase involved in memory and synaptic modulation. In the auto-inhibited state, a CaMKII holoenzyme is composed of catalytic domain pairs that are distributed around a central core of association domains. Upon binding calcium/calmodulin (Ca2+/CaM) the kinase is activated. Ca2+/CaM binding is also thought to trigger a conformational change that exposes the T interaction site on the catalytic domain, The T-site allows the kinase to interact with several synaptic proteins, including NMDA receptors. In the context of the intact holoenzyme, little is known about the conformational changes initiated by Ca2+/CaM binding to the regulatory domain or by subsequent T-site interactions. We have used Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid microscopy technique to assess these changes. FPFA simultaneously measures Forster resonance energy transfer (FRET, a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and diffusion time (an attribute sensitive to the mass and shape of a protein complex). In homogenates, activation with Ca2+/CaM triggered catalytic-domain pair extension (as indicated by diffusion time) without catalytic-domain pair separation (as assayed by homo-FRET). CaMKIINtide inhibitor peptide, a T-site ligand, triggered catalytic-domain pair separation, but only when co-incubated with Ca2+/CaM. Furthermore, a T-site mutant, CaMKII (I205K), which cannot bind to NMDA receptors or translocate to spines, exhibited an increase in FRET (catalytic-domain dimerization) upon activated by Ca2+/CaM, and also prevented CaMKIINtide triggered catalytic-domain pair separation. These observations suggest that catalytic-domains remain paired in the activated holoenzyme by virtue of T-site regulated interactions. Catalytic-domain pairs only separate if exogenous T-site ligands can effectively compete for the sites holding catalytic domains together.

Expression of calcium/calmodulin dependent protein kinaseIIin spinal cord of the rats followed lidocaine hydrochloride intrathecal injection

Objective To observe the expression of calcium/calmodulin dependent protein kinaseⅡ(CaMKⅡ) in the spinal cord of the rats followed lidocaine hydrochloride intrathecal injection. Methods 48 male SD rats weight(230±20)g, after intrathecal indwelling catheter, were randomly divided into four groups (n=12, 8 rats for behavioral detection and 4 rats for western blotting): normal group (C group), sham group (S group), DMSO group (D group), 10% lidocaine group (L group). Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL) were detected before and after 2 h, 4 h, 8 h, 12 h, 1 d, 2 d, 3 d , 4 d and 5 d with drug treatment. Intumescentia lumbalis of the spinal cord were collected to measure the expression of CaMKⅡwith western blotting after drug treatment for 12 h. Results The based MWT of the rats in C, S and D group were (11.2±3.1)g, (11.8±2.2)g and (11.4±2.4)g respectively. There were no differences among the every time points (n=8, P>0.05). The MWT of the rats in L group significantly increased at 2 h, 4 h, 8 h, 12 h, 1 d , 2 d, 3 d and 4 d after treatment with lidocaine hydrochloride, and the data were (22.0±6.6)g, (22.2±5.3)g, (20.5±5.8)g, (18.5±4.3)g, (16.7±3.2)g, (15.2±3.1)g, (15.5±3.5)g , (13.7±2.4)g respectively(n=8, P 0.05). The TWL of the rats in L group significantly increased at 2 h, 4 h, 8 h, 12 h, 1 d, 2 d and 3 d after treatment with lidocaine hydrochloride(n=8, P<0.01). The expression of CaMKⅡ of the rats in C group, S group, D group and L group were 0.17±0.03, 0.16±0.03, 0.19±0.05, 0.42±0.11, and significantly upregulated in L goup (n=4, P<0.01). Conclusion Lidocaine hydrochloride intrathecal injection can increase the expression of the CaMKⅡin the spinal cord of the rats.Those indicate that CaMKⅡ may be involved with the nerve damage induced by lidocaine hydrochloride. Key words: Lidocaine hydrochloride; Spinal cord; Nerve damage; Calcium/calmodulin dependent protein kinaseⅡ

CAMKIIα Catalytic Domain Dimerization is Regulated through T-Site Interactions

The calcium-calmodulin (CaM) dependent protein Kinase-II (CaMKII) regulates various cellular process and has been implicated in illness including schizophrenia, heart failure, arthritis, and even certain types of cancers. CaMKII subunits assemble to form a large dodecameric holoenzyme. The structure of a kinase subunit is organized into 3 domains, a catalytic domain, a regulatory domain, and an oligomerization domain. Each N-terminal catalytic domain harbors the kinase catalytic site, its ATP binding site, as well as the so-called ‘T-site’, thought to interact with the T286 autophosphorylation site in the regulatory domain. The T-site is responsible for mediating the interaction of the holoenzyme with other proteins, such as NMDA receptors, in response to activation. The catalytic domain is attached to an autoinhibitory regulatory domain that contains the T286 autophosphorylation site, and a CaM-binding site. In turn: the regulatory domain is itself tethered to a C-terminal oligomerization domain that organizes the enzyme into ring-shaped oligomers. We have previously shown in living cells using FRET analysis that catalytic domains form pairs in the intact auto-inhibited holoenzyme. In Hippocampal neurons, these pairs separate in response to calcium influx through NMDA-receptors, but it is not known if this is a direct response to CaM binding, or a down stream affect of kinase activation. Here using fluorescence polarization and fluctuation analysis (FPFA), a hybrid method that simultaneously measures homo-FRET and fluorescence correlation spectroscopy (FCS), we show that CaMKIINtide, a T-site ligand, disrupts catalytic domain pairing in the intact holoenzyme. This reaction is dose dependent but was only observed upon co-incubation with CaM to activate the enzyme. The holoenzyme can be activated without catalytic domain pair separation. We also show that T-site specific mutations at I205, stabilize catalytic domain pairing and counteract CaMKIINtide's ability to disrupt catalytic domain pairing.

Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.

The Calcium/Calmodulin/Kinase System and Arrhythmogenic Afterdepolarizations in Bradycardia-Related Acquired Long-QT Syndrome

Sustained bradycardia is associated with long-QT syndrome in human beings and causes spontaneous torsades de pointes in rabbits with chronic atrioventricular block (CAVB), at least partly by downregulating delayed-rectifier K(+)-current to cause action potential (AP) prolongation. We addressed the importance of altered Ca(2+) handling, studying underlying mechanisms and consequences. We measured ventricular cardiomyocyte [Ca(2+)](i) (Indo1-AM), L-type Ca(2+)-current (I(CaL)) and APs (whole-cell perforated-patch), and Ca(2+)-handling protein expression (immunoblot). CAVB increased AP duration, cell shortening, systolic [Ca(2+)](i) transients, and caffeine-induced [Ca(2+)](i) release, and CAVB cells showed spontaneous early afterdepolarizations (EADs). I(CaL) density was unaffected by CAVB, but inactivation was shifted to more positive voltages, increasing the activation-inactivation overlap zone for I(CaL) window current. Ca(2+)-calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation was enhanced in CAVB, indicating CaMKII activation. CAVB also enhanced CaMKII-dependent phospholamban-phosphorylation and accelerated [Ca(2+)](i)-transient decay, consistent with phosphorylation-induced reductions in phospholamban inhibition of sarcoplasmic reticulum (SR) Ca(2+)-ATPase as a contributor to enhanced SR Ca(2+) loading. The CaMKII-inhibitor KN93 reversed CAVB-induced changes in caffeine-releasable [Ca(2+)](i) and I(CaL) inactivation voltage and suppressed CAVB-induced EADs. Similarly, the calmodulin inhibitor W7 suppressed CAVB-induced I(CaL) inactivation voltage shifts and EADs, and a specific CaMKII inhibitory peptide prevented I(CaL) inactivation voltage shifts. The SR Ca(2+)-uptake inhibitor thapsigargin and the SR Ca(2+) release inhibitor ryanodine also suppressed CAVB-induced EADs, consistent with an important role for SR Ca(2+) loading and release in arrhythmogenesis. AP-duration changes reached a maximum after 1 week of bradypacing, but peak alterations in CaMKII and [Ca(2+)](i) required 2 weeks, paralleling the EAD time course. CAVB-induced remodeling enhances [Ca(2+)](i) load and activates the Ca(2+)-calmodulin-CaMKII system, producing [Ca(2+)](i)-handling abnormalities that contribute importantly to CAVB-induced arrhythmogenic afterdepolarizations.

CaMKII Structure—An Elegant Design

Crystal structures of protein kinases continue to reveal new mechanisms for the regulation of catalytic activity of these enzymes. In this issue of Cell, Rosenberg et al. (2005) report the structure of the catalytic and regulatory domains of CaMKII, a protein kinase important in the cellular response to changes in intracellular calcium ion concentration. This study provides new mechanistic insights into the workings of this finely tuned molecular machine.

Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes.

Downregulation of sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is a critical marker of pathological myocardial hypertrophy. The effects of calcium-dependent signaling and of contractile activity on the regulation of myocardial SERCA2a expression remain unclear. The present study dissociates effects of calcium-dependent signaling through calcineurin (CN) and calmodulin dependent protein kinase-II (CAMK-II), from effects of contractile activity in spontaneously contracting rat neonatal ventricular cardiomyocytes (NVCM) using 2,3-butanedione monoxime (BDM), which arrests contractions but maintains calcium fluxes. SERCA2a mRNA expression was analysed using Northern hybridisation in spontaneously contracting NVCM (control) and in NVCM treated with either BDM, L-type Ca2+-channel blocker (verapamil), CN-blocker (cyclosporin A; CsA), CAMK-II blocker (KN-93), or combinations thereof. Transient transfection of the CN-dependent transcription factor nuclear factor of activated T-lymphocytes (NFATc), coupled to GFP, was used to detect NFAT nuclear translocation. The effects of CN/CAMK-II-dependent signaling were further dissected into effects of the transcription factors NFATc4 and myocyte enhancer factor 2c (MEF2c) on the activity of various SERCA2a promoter fragments using transient transfection assays. Treatment with BDM induced a 2.5-fold rise in SERCA2a mRNA, which was abolished by addition of verapamil and was reduced by addition of CsA (-40%) and KN-93 (-20%). NFAT nuclear translocation was similar in control and BDM-treated NVCM. SERCA2a promoter activity was stimulated by NFATc4 and MEF2c, but only when both factors were co-transfected. Following contractile arrest with BDM, upregulation of SERCA2a mRNA expression by CN/CAMK-II signaling becomes evident. This upregulation is likely the result of synergistic stimulation of SERCA2a promoter activity by NFATc4 and MEF2c. Contractile activity opposes this upregulation through distinct and independent pathways.

Reduced Ca2+-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure.

Studies on the status of multifunctional Ca(2+)-calmodulin (CaM)-dependent protein kinase-II (CaMKII) in failing hearts are limited and controversial. The study was performed in the left ventricular (LV) myocardium of six dogs with heart failure (HF) (LV ejection fraction, 23 +/- 2%) and six normal (NL) dogs. In the LV homogenate, CaMKII activity and its protein level were determined by using the CaMKII peptide and antibody, respectively. Furthermore, the protein level of CaM and phosphorylated phospholamban (PLB) at threonine-17 (PLB-Thr(17)) and serine-16 (PLB-Ser(16)) were also determined in the LV homogenate using a specific antibody. In addition, the level of zinc, which inhibits protein kinase A activity, was determined in the LV tissue by inductively coupled plasma mass spectrometry. CaMKII activity and phosphorylated PLB-Thr(17) and PLB-Ser(16) levels, but not CaM and Zn levels, were significantly reduced in the LV homogenate of dogs with HF compared with NL dogs. These results suggest that CaMKII activity is reduced in the failing LV myocardium, and this abnormality is associated with reduced protein expression level of the enzyme but not due to changes in CaM and zinc levels. In conclusion, reduced CaMKII activity and phosphorylated PLB level may be partly responsible for impaired sarcoplasmic reticulum function in HF.

Effect of diabetes on calcium/calmodulin dependent protein kinase-II from rat brain

Changes in the protein levels and activity of Ca 2+/Calmodulin dependent protein kinase II (CaM kinase II) level were studied in cytosolic and particulate fractions from cerebral hemisphere, cerebellum, brain stem, thalamus and hypothalamus regions of rat brain after 4 and 12 weeks of induction of diabetes. Streptozotocin induced diabetes, resulted in pronounced increase of CaM kinase II activity as determined by the kinase activity assay. The total amount of enzyme protein ( α-subunit specific) also showed increase as revealed by western blotting. Parallel studies were also made in age matched control rats and insulin treated diabetic rats. The increase in CaM kinase II activity was more pronounced in the 12 weeks diabetic group. Insulin treatment of diabetic rats, resulted in recovery of enzyme activity near to control values from majority of the brain regions studied. The expression of α-subunit specific CaM kinase II correlates with the enzyme activity in the diabetic rat brain.