Calmodulin-dependent Protein Kinase II Pathway Research Articles

Rac1 promotes the lipopolysaccharide-induced inflammatory response and contraction-associated proteins (CAPs) expression in mouse uterine smooth muscle cells

Activation of the maternal immune system leads to a downstream cascade of proinflammatory events that culminate in the activation of spontaneous uterine contractions, which is associated with preterm birth. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a crucial protein related to cell contraction and inflammation. The main purpose of this study was to explore the role and function of Rac1′s regulation of inflammation through in- vivo and in-vitro experiments. Rac1 inhibitor was used in animal model of preterm birth and cells isolated from the uterine tissues of pregnant mice on gestational day 16 were transfected with adenovirus to knockdown or overexpress Rac1 and treated with the Calcium–calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. The expression of Rac1, uterine contraction-associated proteins (CAPs) (COX-2 and Connexin43), and inflammatory cytokines, were assessed by Western blotting and RTPCR.LPS upregulated Rac1, COX-2 and Connexin43 expression in uterine smooth muscle cells (USMCs). The expression of inflammatory cytokines, COX-2, and Connexin43 was significantly decreased in shRac1-transfected cells compared with cells stimulated with LPS only. Rac1 overexpression led to an increase in the expression of inflammatory cytokines, COX-2, and Connexin43. Furthermore, after Rac1 overexpression, KN93 reduced the expression of uterine contraction-associated proteins and inflammatory cytokines. It is thought that the effect of Rac1 on inflammatory cytokine and contraction-associated protein expression in USMCs is mediated by CaMKII. Rac1 can modulate the expression of contraction-associated proteins and inflammatory cytokines through the CaMKII pathway. Rac1 could be an effective therapeutic target for improving the outcome of preterm birth.

Proline hydroxylase domain-containing enzymes regulate calcium levels in cardiomyocytes by TRPA1 ion channel

The proline hydroxylase domain-containing enzymes (PHDs) acts as cellular oxygen sensors, inducing a series of responses to hypoxia, especially during the regulation of metabolism and energy homeostasis. The increase of Ca2+ in cardiomyocytes, induced by the opening of PHD signaling pathway, is the key initiation signal necessary for the PHD-mediated regulation of the energy metabolism pathway, but the underlying molecular mechanism remains incompletely understood. This study used PHD inhibitors (PHIs) and PHD2-specific RNA interference (PHD2shRNA) to inhibit PHD signals in cardiomyocytes to explore whether transient receptor potential ankyrin 1 (TRPA1) is involved in the regulation of calcium ion influx in the PHD activation pathway associated with to AMP-activated protein kinase (AMPK). The Fluo-3AM probe was used to measure changes in free intracellular calcium ion concentrations, and Western blot analysis was used to detect the levels of phosphorylated (P)-AMPK, TRPA1, and P–Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) levels. The PHI-mediated inhibition of PHD resulted in an increase in free Ca2+ fluorescence in cardiomyocytes, which activated AMPK, TRPA1, and CaMKⅡ. The TRPA1 inhibitor HC030031, the CaMKII inhibitor KN93, and a ryanodine inhibitor (Ryanodine) were all able to inhibit the PHI-induced increase in intracellular Ca2+ and AMPK activation. Both PHIs and PHD2shRNA were able to effectively activate CaMKII and TRPA1. However, an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor and the protein kinase A (PKA) inhibitor H89 did not significantly inhibit the PHI-induced increase in intracellular Ca2+ and AMPK activation. These results indicated that PHD might activate the CaMKⅡ pathway through the TRPA1 ion channel, inducing the release of calcium from the sarcoplasmic reticulum through ryanodine receptor 2 (RyR2), activating AMPK to initiate the protective effects of hypoxia in cardiomyocytes.

Dysregulation of cardiac CaMKII pathway is increased in aging and chronic inflammation

Chronic elevation of inflammatory mediators is common in frail older adults and predicts a host of adverse cardiovascular outcomes, including heart failure and sudden death. To date, few specific molecular mechanisms have been identified that connect chronic inflammation to age-related cardiomyopathies. Dysregulation in Ca2+and calmodulin-dependent protein kinase II (CaMKII) signaling has been implicated in promoting inflammatory responses in cardiomyopathies but is understudied in the context of aging. We compared myocardial CaMKII signaling in young (32 weeks) and old (104 weeks) wild type (WT) and interleukin 10 null mice (IL-10tm/tm), a model of chronic inflammation and frailty. Aged cohorts were treated with 0.6 mg of losartan. Hearts from mouse cohorts were assayed for total and autophosphorylated CaMKII (P-CaMKII) and the phosphorylation status of phospholamban (PLN) (downstream signaling pathway) using western blot techniques. We observed significant increases in total and activated, autophosphorylated, CaMKII (P-CaMKII) (2.4 ± 0.38 vs 1.2 ± 0.12 p< 0.02; 9.0 ± 1.9 vs 0.03 ± 0.01 p< 0.002), but decreases in the phosphorylation of PLN (0.6 ±0.3 vs 1.7 ± 0.3 p<0.03) at a validated CaMKII target site (T17) in old compared to young WT hearts. Similarly, we found increased P-CaMKII (6.9 ± 1.5 vs 0.7 ± 0.4 p< 0.004) and diminished PLN T17 phosphorylation (0.8 ± 0.2 vs 1.5 ± 0.2 p<0.02) in old compared to young IL-10tm/tm mice. Old IL-10tm/tm mouse hearts had less total CaMKII compared to old WT mouse hearts (1.1 ± 0.3 vs 2.9 ±0.2 p< 0.002). Four weeks treatment of old WT mice with Losartan was associated with reversal of observed age-related changes in the CaMKII signaling pathway. We observed decreases in total CaMKII, P-CaMKII (2.9 ± 0.2 vs.1.5 ± 0.2 p<0.001; 2.2 ± 0.9 vs 0.47 ± 0.1 p <0.03), and an increase in PLN T17 phosphorylation (0.55±0.3 vs 2.23 ± 0.4 p< 0.007). The effects of Losartan on the animals aging with chronic inflammation (old IL-10tm/tm) was less clear. We observed a losartan associated increase in total CaMKII (1.41 ± 0.02 vs 1.3 ± 0.02 p<0.02) but no impact on P-CaMKII or PLN T17 phosphorylation. We interpret our findings to suggest that changes in myocardial CaMKII signaling are a feature of aging that is affected by chronic inflammation.

Pka and CaMKII Signaling Synergistically Promotes Atrial Arrhythmia in Populations of Human Atrial Tissues

The β-adrenergic receptor (βAR)/cAMP/PKA and multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) signaling pathways are key mediators of cardiac excitation-contraction coupling (ECC) and share multiple downstream targets. Hyperactivation of both signaling pathways contributes to the initiation and maintenance of atrial fibrillation (AF), the world's most common arrhythmia. Using novel populations of computational models coupling atrial electrophysiology and Ca2+ handling with detailed descriptions of βAR-PKA and CaMKII pathways, we have previously shown that PKA and CaMKII effects on atrial ECC proteins synergistically promote a vicious cycle of Ca2+and membrane potential instabilities. Since both kinases are known to affect tissue-level modifiers of atrial electrophysiology, here we sought to test whether PKA and CaMKII signaling also synergize to mediate arrhythmias in tissue, and to determine the mechanisms underlying the increased vulnerability. Populations of 1D strand models revealed increased vulnerability to unidirectional block with hyperactive CaMKII, mediated by Na+ channel loss of function and cell-to-cell uncoupling, which was exacerbated by activation of βARs. Simulations of 2D heterogeneous tissues demonstrated synergistic enhancement of arrhythmia vulnerability by βAR stimulation and CaMKII hyperactivation through dysregulating conduction and modifying source-sink mismatch, as well as promoting tissue repolarization dispersion and invoking spontaneous Ca2+-overload-mediated action potentials. CaMKII inhibition substantially reduced the vulnerability. Collectively, our simulations reveal synergy in PKA and CaMKII effects on tissue-level outcomes, and depict a novel paradigm for Ca2+-CaMKII-dependent involvement in both enhanced triggered activity and conduction disturbances in AF. These findings suggest that interrupting the vicious cycle of ever-increasing CaMKII and Ca2+ (e.g., via CaMKII inhibition) may be a valuable pharmacotherapy approach to counteract both AF triggers and functional substrate.

Novel Synthetic Coumarin-Chalcone Derivative (E)-3-(3-(4-(Dimethylamino)Phenyl)Acryloyl)-4-Hydroxy-2H-Chromen-2-One Activates CREB-Mediated Neuroprotection in Aβ and Tau Cell Models of Alzheimer's Disease.

Abnormal accumulations of misfolded Aβ and tau proteins are major components of the hallmark plaques and neurofibrillary tangles in the brains of Alzheimer's disease (AD) patients. These abnormal protein deposits cause neurodegeneration through a number of proposed mechanisms, including downregulation of the cAMP-response-element (CRE) binding protein 1 (CREB) signaling pathway. Using CRE-GFP reporter cells, we investigated the effects of three coumarin-chalcone derivatives synthesized in our lab on CREB-mediated gene expression. Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells were used to evaluate these agents for possible antiaggregative, antioxidative, and neuroprotective effects. Blood-brain barrier (BBB) penetration was assessed by pharmacokinetic studies in mice. Of the three tested compounds, (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-4-hydroxy-2H-chromen-2-one (LM-021) was observed to increase CREB-mediated gene expression through protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) in CRE-GFP reporter cells. LM-021 exhibited antiaggregative, antioxidative, and neuroprotective effects mediated by the upregulation of CREB phosphorylation and its downstream brain-derived neurotrophic factor and BCL2 apoptosis regulator genes in Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells. Blockage of the PKA, CaMKII, or ERK pathway counteracted the beneficial effects of LM-021. LM-021 also exhibited good BBB penetration ability, with brain to plasma ratio of 5.3%, in in vivo pharmacokinetic assessment. Our results indicate that LM-021 works as a CREB enhancer to reduce Aβ and tau aggregation and provide neuroprotection. These findings suggest the therapeutic potential of LM-021 in treating AD.

Atherogenic L5 LDL induces cardiomyocyte apoptosis and inhibits KATP channels through CaMKII activation

BackgroundCardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation plays a critical role in cardiomyocyte (CM) apoptosis and arrhythmia. Functional ATP-sensitive potassium (KATP) channels are essential for cardiac protection during ischemia. In cultured CMs, L5 low-density lipoprotein (LDL) induces apoptosis and QTc prolongation. L5 is a highly electronegative and atherogenic aberrant form of LDL, and its levels are significantly higher in patients with cardiovascular-related diseases. Here, the role of L5 in cardiac injury was studied by evaluating the effects of L5 on CaMKII activity and KATP channel physiology in CMs.MethodsCultured neonatal rat CMs (NRCMs) were treated with a moderate concentration (ie, 7.5 μg/mL) of L5 or L1 (the least electronegative LDL subfraction). NRCMs were examined for apoptosis and viability, CaMKII activity, and the expression of phosphorylated CaMKIIδ and NOX2/gp91phox. The function of KATP and action potentials (APs) was analyzed by using the patch-clamp technique.ResultsIn NRCMs, L5 but not L1 significantly induced cell apoptosis and reduced cell viability. Furthermore, L5 decreased Kir6.2 expression by more than 50%. Patch-clamp analysis showed that L5 reduced the KATP current (IKATP) density induced by pinacidil, a KATP opener. The partial recovery of the inward potassium current during pinacidil washout was susceptible to subsequent inhibition by the IKATP blocker glibenclamide. Suppression of IKATP by L5 significantly prolonged the AP duration. L5 also significantly increased the activity of CaMKII, the phosphorylation of CaMKIIδ, and the expression of NOX2/gp91phox. L5-induced apoptosis was prevented by the addition of the CaMKII inhibitor KN93 and the reactive oxygen species scavenger Mn (III)TBAP.ConclusionsL5 but not L1 induces CM damage through the activation of the CaMKII pathway and increases arrhythmogenicity in CMs by modulating the AP duration. These results help to explain the harmful effects of L5 in cardiovascular-related disease.

Interplay Between β-Adrenergic Stimulation and CaMKII Signaling Favors Human Atrial Arrhythmogenesis: Insights from Populations of Models

Excessive β-adrenergic receptor (βAR) stimulation and hyperactivation of the multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) play important roles in the initiation and maintenance of atrial fibrillation (AF), the world's most common cardiac arrhythmia. It has been postulated that the interplay of these two pathways with intracellular Ca2+ signaling drives a vicious cycle that promotes arrhythmogenesis. Here, we coupled our well-established computational model of human atrial cellular electrophysiology (extended to incorporate two recently-characterized atrial-predominant K+ currents) and Ca2+ handling (Grandi et al., 2011) with detailed descriptions of βAR/cAMP/PKA and CaMKII pathways (Soltis and Saucerman 2010; Morotti et al., 2014) to investigate their respective roles and potential synergy in promoting AF.

β-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. β-adrenergic receptor (βAR) 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. βAR 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 βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-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.

Do DPP-4 Inhibitors Cause Heart Failure Events by Promoting Adrenergically Mediated Cardiotoxicity? Clues From Laboratory Models and Clinical Trials.

DPP-4 (dipeptidyl peptidase-4) inhibitors have increased the risk of heart failure events in both randomized clinical trials and observational studies, but the mechanisms that underlie their deleterious effect remain to be elucidated. Previous work has implicated a role of these drugs to promote cardiac fibrosis. This article postulates that DPP-4 inhibitors increase the risk of heart failure events by activating the sympathetic nervous system to stimulate cardiomyocyte cell death, and it crystallizes the findings from both experimental studies and clinical trials that support the hypothesis. Inhibition of DPP-4 not only potentiates the actions of GLP-1 (glucagon-like peptide-1; which can increase myocardial cAMP) but also potentiates the actions of SDF-1 (stromal cell-derived factor 1), NPY (neuropeptide Y), and substance P to activate the sympathetic nervous system and stimulate β-adrenergic receptors to cause cardiomyocyte apoptosis, presumably through a CaMKII (Ca++/calmodulin-dependent protein kinase II) pathway. An action of SDF-1 to interfere with cAMP and protein kinase A signaling may account for the absence of a clinically overt positive chronotropic effect. This conceptual framework is supported by the apparent ability of β-blocking drugs to attenuate the increased risk of DPP-4 inhibitors in a large-scale clinical trial. Sympathetic activation may explain the increased risk of heart failure produced by DPP-4 inhibitors. The proposed mechanism has major implications for clinical care because in the treatment of patients with type 2 diabetes mellitus, DPP-4 inhibitors are widely prescribed, but β-blockers are underutilized because of fears that they might mask hypoglycemia.

Secreted AGR2 promotes invasion of colorectal cancer cells via Wnt11-mediated non-canonical Wnt signaling

Human anterior gradient-2 (AGR2), a member of protein disulfide isomerase (PDI) family, is present in both intracellular and extracellular compartments. Although AGR2 is overexpressed in various human cancers and reported to promote aggressive tumor features, little is known regarding AGR2′s extracellular functions during tumorigenesis. Here, we demonstrate that secreted AGR2 promotes cell migration and metastasis of colorectal cancer (CRC) in vitro and in vivo. Mechanistically, secreted AGR2 elevated Wnt11 expression, triggering non-canonical Wnt signaling: the Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and c-jun amino-terminal kinase (JNK) pathways. Knockdown of Wnt11 or pretreatment with CaMKII and JNK inhibitors reversed the secreted AGR2′s migration-promoting effect. Further studies revealed that AGR2 antagonized canonical Wnt/β-catenin signaling via activating CaMKII. Collectively, our study uncovers a critical role of Wnt11-mediated non-canonical Wnt signaling (CaMKII and JNK pathways) in secreted AGR2′s promoted migration of CRC cells. These results raise the possibility that secreted AGR2 may be a potential therapeutic target towards inhibiting CRC metastasis.

Modulation of Murine Olivary Connexin 36 Gap Junctions by PKA and CaMKII.

The inferior olive (IO) is a nucleus located in the brainstem and it is part of the olivo-cerebellar loop. This circuit plays a fundamental role in generation and acquisition of coherent motor patterns and it relies on synchronous activation of groups of Purkinje cells (PC) in the cerebellar cortex. IO neurons integrate their intrinsic oscillatory activity with excitatory inputs coming from the somatosensory system and inhibitory feedback coming from the cerebellar nuclei. Alongside these chemical synaptic inputs, IO neurons are coupled to one another via connexin 36 (Cx36) containing gap junctions (GJs) that create a functional syncytium between neurons. Communication between olivary neurons is regulated by these GJs and their correct functioning contributes to coherent oscillations in the IO and proper motor learning. Here, we explore the cellular pathways that can regulate the coupling between olivary neurons. We combined in vitro electrophysiology and immunohistochemistry (IHC) on mouse acute brain slices to unravel the pathways that regulate olivary coupling. We found that enhancing the activity of the protein kinase A (PKA) pathway and blocking the Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway can both down-regulate the size of the coupled network. However, these two kinases follow different mechanisms of action. Our results suggest that activation of the PKA pathway reduces the opening probability of the Cx36 GJs, whereas inhibition of the CaMKII pathway reduces the number of Cx36 GJs. The low densities of Cx36 proteins and electrical synapses in βCaMKII knock-out mice point towards an essential role for this protein kinase in regulating the density of GJs in the IO. Thus, the level of olivary coupling is a dynamic process and regulated by a variety of enzymes modulating GJs expression, docking and activity.

Ca2+/Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy.

Testosterone is known to induce cardiac hypertrophy through androgen receptor (AR)-dependent and -independent pathways, but the molecular underpinnings of the androgen action remain poorly understood. Previous work has shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and myocyte-enhancer factor 2 (MEF2) play key roles in promoting cardiac myocyte growth. In order to gain mechanistic insights into the action of androgens on the heart, we investigated how testosterone affects CaMKII and MEF2 in cardiac myocyte hypertrophy by performing studies on cultured rat cardiac myocytes and hearts obtained from adult male orchiectomized (ORX) rats. In cardiac myocytes, MEF2 activity was monitored using a luciferase reporter plasmid, and the effects of CaMKII and AR signaling pathways on MEF2C were examined by using siRNAs and pharmacological inhibitors targeting these two pathways. In the in vivo studies, ORX rats were randomly assigned to groups that were administered vehicle or testosterone (125 mg⋅kg-1⋅week-1) for 5 weeks, and plasma testosterone concentrations were determined using ELISA. Cardiac hypertrophy was evaluated by measuring well-characterized hypertrophy markers. Moreover, western blotting was used to assess CaMKII and phospholamban (PLN) phosphorylation, and MEF2C and AR protein levels in extracts of left-ventricle tissue from control and testosterone-treated ORX rats. Whereas testosterone treatment increased the phosphorylation levels of CaMKII (Thr286) and phospholambam (PLN) (Thr17) in cardiac myocytes in a time- and concentration-dependent manner, testosterone-induced MEF2 activity and cardiac myocyte hypertrophy were prevented upon inhibition of CaMKII, MEF2C, and AR signaling pathways. Notably, in the hypertrophied hearts obtained from testosterone-administered ORX rats, both CaMKII and PLN phosphorylation levels and AR and MEF2 protein levels were increased. Thus, this study presents the first evidence indicating that testosterone activates MEF2 through CaMKII and AR signaling. Our findings suggest that an orchestrated mechanism of action involving signal transduction and transcription pathways underlies testosterone-induced cardiac myocyte hypertrophy.

Prevention of Remifentanil Induced Postoperative Hyperalgesia by Dexmedetomidine via Regulating the Trafficking and Function of Spinal NMDA Receptors as well as PKC and CaMKII Level In Vivo and In Vitro

Remifentanil-induced secondary hyperalgesia has been demonstrated in both animal experiments and clinical trials. Enhancement of N-methyl-D-aspartate (NMDA) receptor trafficking as well as protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII) have been reported to be involved in the induction and maintenance of central sensitization. In the current study, it was demonstrated that dexmedetomidine could prevent remifentanil-induced hyperalgesia (RIH) via regulating spinal NMDAR-PKC-Ca2+/ CaMKII pathway in vivo and in vitro. We firstly investigated the effect of dexmedetomidine, a highly selective α2-adrenergic receptor agonist, on mechanical and thermal hyperalgesia using a rat model of RIH. NMDA receptor subunits (NR1, NR2A and NR2B) expression and membrane trafficking as well as PKC and CaMKII expression in spinal cord L4–L5 segments were measured by Western blot analysis. The expression of NMDA receptor subunits (NR1, NR2A and NR2B) were also detected by immunohistochemistry. Further more, the effect of dexmedetomidine on NMDA receptor current amplitude and frequency in spinal cord slices were investigated by whole-cell patch-clamp recording. We found that remifentail infusion at 1.2 μg.kg−1.min−1 for 90 min caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Subcutaneously injection of dexmedetomidine at the dose of 50 μg/kg at 30 min before plantar incision significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was dose-dependently attenuated by dexmedetomidine. These results suggest that dexmedetomidine can significantly ameliorate RIH via modulating the expression, membrane trafficking and function of NMDA receptors as well as PKC and CaMKII level in spinal dorsal horn, which present useful insights into the mechanistic action of dexmedetomidine as a potential anti-hyperalgesic agents for treating RIH.

Contribution of NMDA, GABAA and GABAB receptors and l-arginine-NO-cGMP, MEK1/2 and CaMK-II pathways in the antidepressant-like effect of 7-fluoro-1,3-diphenylisoquinoline-1-amine in mice

It has been reported that the antidepressant-like effect of 7-fluoro-1,3-diphenylisoquinoline-1-amine (FDPI) may result from the modulation of brain monoaminergic systems. However, the mechanisms of FDPI action are not fully understood. The aim of this study was to investigate the contribution of N-methyl-d-aspartate (NMDA) and gamma-aminobutyric acid (GABA) systems as well as l-arginine-nitric oxide-(NO)-cyclic guanosine monophosphate-(cGMP), mitogen-activated protein/extracellular signal-regulated kinase (MEK1/2) and Ca2+/calmodulin-dependent protein kinase II (CaMK-II) signaling pathways in the antidepressant-like effect of FDPI in the mouse forced swimming test (FST). The levels of NO and uptake of [3H]glutamate and [3H]GABA were determined in prefrontal cortices of Swiss mice. Pretreatments with NMDA (0.1 pmol/site, i.c.v., a NMDA receptor agonist), bicuculline (1mg/kg, i.p., a GABAA receptor antagonist), phaclofen (2mg/kg, i.p., a GABAB receptor antagonist) and l-arginine (750mg/kg, i.p., a NO precursor), KN-62 (1μg/site, a CaMK-II inhibitor), U0126 (5μg/site, a MEK1/2 inhibitor) and PD09058 (5μg/site, a MEK1/2 inhibitor) blocked the antidepressant-like effect of FDPI, at a dose of 1mg/kg, in the FST. ODQ (30 pmol/site, i.c.v., a soluble guanylate cyclase (sGC) inhibitor) in combination with a sub-effective dose of FDPI (0.1mg/kg, i.g.) reduced the immobility time in the FST. The administration of FDPI (50mg/kg) to mice increased the glutamate uptake and reduced NO levels in the prefrontal cortex of mice. The results suggest a contribution of NMDA, GABAA and GABAB receptors and l-arginine-NO-cGMP pathway in the antidepressant-like action of FDPI in mice, and this effect is related to CaMK-II and MEK 1/2 activation.

Role of CaMKII and PKA in Early Afterdepolarization of Human Ventricular Myocardium Cell: A Computational Model Study.

Early afterdepolarization (EAD) plays an important role in arrhythmogenesis. Many experimental studies have reported that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and β-adrenergic signaling pathway are two important regulators. In this study, we developed a modified computational model of human ventricular myocyte to investigate the combined role of CaMKII and β-adrenergic signaling pathway on the occurrence of EADs. Our simulation results showed that (1) CaMKII overexpression facilitates EADs through the prolongation of late sodium current's (I NaL) deactivation progress; (2) the combined effect of CaMKII overexpression and activation of β-adrenergic signaling pathway further increases the risk of EADs, where EADs could occur at shorter cycle length (2000 ms versus 4000 ms) and lower rapid delayed rectifier K+ current (I Kr) blockage (77% versus 85%). In summary, this study computationally demonstrated the combined role of CaMKII and β-adrenergic signaling pathway on the occurrence of EADs, which could be useful for searching for therapy strategies to treat EADs related arrhythmogenesis.

The effect of constitutive over-expression of insulin-like growth factor 1 on the cognitive function in aged mice

The neurotrophic factor insulin-like growth factor (IGF)-1 promotes neurogenesis in the mammalian brain and provides protection against brain injury. However, studies regarding the effects of IGF-1 on cognitive function in aged mice remain limited. We investigated the effects of overexpression of IGF-1 specifically in neural stem cells of the hippocampal dentate gyrus on the recognitive function in 18-month-old transgenic mice. Immunohistocytochemistry and Nissl staining revealed the increased population of BrdU-positive cells as well as the upregulated expression of Nestin and neuronal nuclei (NeuN), respective markers for neural progenitors and neurons, in the hippocampus of the aged IGF-1 transgenic mice versus the wild-type, suggesting that IGF-1 overexpression promotes neurogenesis. In addition, the IGF-1 receptor (IGF-1R), the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) were enhanced in the transgenic mice than in the wild-type. Transgenic mice also showed superior performance in the Morris water maze and step-down memory tests to their wild-type counterparts. Moreover, the learning and memory abilities of transgenic mice were significantly undermined with the blockage of CaMKII and ERK signaling pathway. Accordingly, our findings indicated that IGF-1 may mitigate the aged-associated cognitive decline via promoting neurogenesis in the hippocampus and activating CaMKII and ERK signaling by binding with IGF-1R.

Activation of M3 cholinoceptors attenuates vascular injury after ischaemia/reperfusion by inhibiting the Ca2+/calmodulin-dependent protein kinase II pathway.

The activation of M3 cholinoceptors (M3 receptors) by choline reduces cardiovascular risk, but it is unclear whether these receptors can regulate ischaemia/reperfusion (I/R)-induced vascular injury. Thus, the primary goal of the present study was to explore the effects of choline on the function of mesenteric arteries following I/R, with a major focus on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) regulation. Rats were given choline (10 mg · kg(-1), i.v.) and then the superior mesenteric artery was occluded for 60 min (ischaemia), followed by 90 min of reperfusion. The M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), was injected (0.12 μg · kg(-1), i.v.) 5 min prior to choline treatment. Vascular function was examined in rings of mesenteric arteries isolated after the reperfusion procedure. Vascular superoxide anion production, CaMKII and the levels of Ca(2+)-cycling proteins were also assessed. Choline treatment attenuated I/R-induced vascular dysfunction, blocked elevations in the levels of reactive oxygen species (ROS) and decreased the up-regulated expression of oxidised CaMKII and phosphorylated CaMKII. In addition, choline reversed the abnormal expression of Ca(2+)-cycling proteins, including Na(+)Ca(2+) exchanger, inositol 1,4,5-trisphosphate receptor, sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban. All of these cholinergic effects of choline were abolished by 4-DAMP. Our data suggest that inhibition of the ROS-mediated CaMKII pathway and modulation of Ca(2+)-cycling proteins may be novel mechanisms underlying choline-induced vascular protection. These results represent a significant addition to the understanding of the pharmacological roles of M3 receptors in the vasculature, providing a new therapeutic strategy for I/R-induced vascular injury.

Biodentine Induces Human Dental Pulp Stem Cell Differentiation through Mitogen-activated Protein Kinase and Calcium-/Calmodulin-dependent Protein Kinase II Pathways

IntroductionBiodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. MethodshDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. ResultsBiodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. ConclusionsBiodentine is a bioactive and biocompatible material capable of inducing odontoblast differentiation of hDPSCs. Our results indicate that this induction is regulated via MAPK and CaMKII pathways.

Reducing Oxidative Stress and CaMKII Activity as an Antiarrhythmic Strategy in Woodchucks in Winter

Multiple lines of evidence have suggested that increased oxidative stress and activation of Ca2+-calmodulin-dependent protein kinase II (CaMKII) are proarrhythmic in various diseased heart models. In the present study we have evaluated the involvement of oxidative stress and CaMKII pathway in antiarrhythmic mechanisms in a unique coronary artery occlusion (CAO)-induced arrhythmia model in woodchucks. Woodchucks, are true hibernators, and in winter they survive without food or water or activity and their hearts are more resistant to cardiac arrhythmias than in summer. We conducted Western blotting analyses and compared the expression levels of key antioxidant enzymes. In control hearts (without CAO) the expression level of catalase was higher in winter than in summer, while the MnSOD levels were the same. While CAO (24 hrs) upregulated both catalase and MnSOD, the increase in catalase was greater in winter than in summer. Meanwhile, oxidized CaMKII (activated form) levels were lower in winter than in summer. The effects of the CaMKII inhibitor KN-93 on CAO-induced arrhythmias in summer were also evaluated. We carried out CAO surgeries and monitored ECG by telemetry for 24 hrs. KN-93 was administrated with an initial bolus injection followed by a continuous delivery via osmotic minipumps. Arrhythmia scores were evaluated and compared to the control group. KN-93 significantly decreased the arrhythmia score in woodchucks in summer (3.4 ± 0.4) to a lower level (1.3 ± 0.6, p<0.05), which is similar to that observed in winter (1.6 ± 0.3), suggesting the reduction of CaMKII activity may account for the adaptation for the heart's resistance to CAO-induced arrhythmias during winter in woodchucks. These results suggest that manipulation of CaMKII signaling may hold therapeutic potential for treating cardiac arrhythmias.

Oxidative activation of Ca2+/calmodulin-activated kinase II mediates ER stress-induced cardiac dysfunction and apoptosis

Endoplasmic reticulum (ER) stress elicits oxidative stress and intracellular Ca(2+) derangement via activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). This study was designed to examine the role of CaMKII in ER stress-induced cardiac dysfunction and apoptosis as well as the effect of antioxidant catalase. Wild-type FVB and transgenic mice with cardiac-specific overexpression of catalase were challenged with the ER stress inducer tunicamycin (3 mg/kg ip for 48 h). Presence of ER stress was verified using the ER stress protein markers immunoglobulin binding protein (BiP) and C/EBP homologous protein (CHOP), the effect of which was unaffected by catalase overexpression. Echocardiographic assessment revealed that tunicamycin elicited cardiac remodeling (enlarged end-systolic diameter without affecting diastolic and ventricular wall thickness), depressed fractional shortening, ejection fraction, and cardiomyocyte contractile capacity, intracellular Ca(2+) mishandling, accumulation of reactive oxygen species (superoxide production and NADPH oxidase p47phox level), CaMKII oxidation, and apoptosis (evidenced by Bax, Bcl-2/Bax ratio, and TUNEL staining), the effects of which were obliterated by catalase. Interestingly, tunicamycin-induced cardiomyocyte mechanical anomalies and cell death were ablated by the CaMKII inhibitor KN93, in a manner reminiscent of catalase. These data favored a permissive role of oxidative stress and CaMKII activation in ER stress-induced cardiac dysfunction and cell death. Our data further revealed the therapeutic potential of antioxidant or CaMKII inhibition in cardiac pathological conditions associated with ER stress. This research shows for the first time that contractile dysfunction caused by ER stress is a result of the oxidative activation of the CaMKII pathway.