Neuritin Attenuates Cognitive Function Impairments in Tg2576 Mouse Model of Alzheimer's Disease

Neuritin, also known as CPG15, is a neurotrophic factor that was initially discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in the process of neural development and synaptic plasticity, although its binding receptor(s) and downstream signaling effectors remain unclear. In this study, we found that the cortical and hippocampal expression of neuritin is reduced in the brains of Alzheimer''s disease (AD) patients and demonstrated that viral-mediated expression of neuritin in the dentate gyrus of 13-month-old Tg2576 mice, an AD animal model, attenuated a deficit in learning and memory as assessed by a Morris water maze test. We also found that neuritin restored the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neuron cultures prepared from Tg2576 mice. It was also shown that viral-mediated expression of neuritin in the dentate gyrus of 7-week-old Sprague-Dawley rats increased neurogenesis in the hippocampus. Taken together, our results demonstrate that neuritin restores the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neurons from Tg2576 neurons, and also attenuates cognitive function deficits in Tg2576 mouse model of AD, suggesting that neuritin possesses a therapeutic potential for AD.     

Expression and purification of recombinant human neuritin from <Emphasis Type="Italic">Pichia pastoris</Emphasis> and a partial analysis of its neurobiological activity in vitro

Neuritin (also known as candidate plasticity gene 15 (cpg15)) is a neurotrophic factor that was recently discovered in a screen aimed at identifying genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in both neural development (Chen et al. Zhonghua Yan Ke Za Zhi 46:978–983 2010; Corriveau et al. J Neurosci 19:7999-8008 1999; Lee and Nedivi J Neurosci 22:1807-1815 2002) and synaptic plasticity (Fujino et al. Gene Dev 25:2674-2685 2011; Leslie and Nedivi Prog 14 Neurobiol 94:223-237 2011; Loebrich and Nedivi Physiol Rev 89:1079 2009). In this study, to produce bioactive, soluble recombinant human neuritin protein, a portion of NRN1 was cloned into the Pichia pastoris expression vector pPIC9K. The recombinant vector was then transformed into the methylotrophic yeast strain P. pastoris GS115, and a shaking flask method and His-tag purification strategy were utilized to express and purify neuritin protein. The resulting protein had a molecular mass of approximately 11 kDa, and subsequent functional analyses indicated that the purified neuritin promoted neurite outgrowth from embryonic chicken dorsal root ganglions, while also prolonging the survival of these ganglions, and from PC12 cells. These findings suggest that neuritin was expressed effectively in vitro and that this protein may play a role in stimulating neurite outgrowth of both dorsal root ganglions and PC12 cells. This study provides a novel strategy for the large-scale production of bioactive neuritin, which will enable further study of the biological function of this protein.     

Phospholipase C-related but Catalytically Inactive Protein Is Required for Insulin-induced Cell Surface Expression of ��-Aminobutyric Acid Type A Receptors

The γ-aminobutyric acid type A (GABA_A) receptors play a pivotal role in fast synaptic inhibition in the central nervous system. One of the key factors for determining synaptic strength is the number of receptors on the postsynaptic membrane, which is maintained by the balance between cell surface insertion and endocytosis of the receptors. In this study, we investigated whether phospholipase C-related but catalytically inactive protein (PRIP) is involved in insulin-induced GABA_A receptor insertion. Insulin potentiated the GABA-induced Cl⁻ current (I_GABA) by about 30% in wild-type neurons, but not in PRIP1 and PRIP2 double-knock-out (DKO) neurons, suggesting that PRIP is involved in insulin-induced potentiation. The phosphorylation level of the GABA_A receptor β-subunit was increased by about 30% in the wild-type neurons but not in the mutant neurons, which were similar to the changes observed in I_GABA. We also revealed that PRIP recruited active Akt to the GABA_A receptors by forming a ternary complex under insulin stimulation. The disruption of the binding between PRIP and the GABA_A receptor β-subunit by PRIP interference peptide attenuated the insulin potentiation of I_GABA. Taken together, these results suggest that PRIP is involved in insulin-induced GABA_A receptor insertion by recruiting active Akt to the receptor complex.     

The A-type potassium channel Kv4.2 is a substrate for the mitogen-activated protein kinase ERK

The mitogen-activated protein kinase ERK has recently become a focus of studies of synaptic plasticity and learning and memory. Due to the prominent role of potassium channels in regulating the electrical properties of membranes, modulation of these channels by ERK could play an important role in mediating learning-related synaptic plasticity in the CNS. Kv4.2 is a Shal-type potassium channel that passes an A-type current and is localized to dendrites and cell bodies in the hippocampus. The sequence of Kv4.2 contains several consensus sites for ERK phosphorylation. In the present studies, we tested the hypothesis that Kv4.2 is an ERK substrate. We determined that the Kv4.2 C-terminal cytoplasmic domain is an effective ERK2 substrate, and that it is phosphorylated at three sites: Thr(602), Thr(607), and Ser(616). We used this information to develop antibodies that recognize Kv4.2 phosphorylated by ERK2. One of our phospho-site-selective antibodies was generated using a triply phosphorylated peptide as the antigen. We determined that this antibody recognizes ERK-phosphorylated Kv4.2 in COS-7 cells transfected with Kv4.2 and native ERK-phosphorylated Kv4.2 in the rat hippocampus. These observations indicate that Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERK may regulate potassium-channel function by direct phosphorylation of the pore-forming alpha subunit.     

Kv 1.1 is associated with neuronal apoptosis and modulated by protein kinase C in the rat cerebellar granule cell

Previously, we reported that apoptosis of cerebellar granular neurons induced by low‐K⁺ and serum‐free (LK‐S) was associated with an increase in the A‐type K⁺ channel current (I_A), and an elevated expression of main α‐subunit of the I_A channel, which is known as Kv4.2 and Kv4.3. Here, we show, as assessed by quantitative RT‐PCR and whole‐cell recording, that besides Kv4.2 and Kv4.3, Kv1.1 is very important for I_A channel. The expression of Kv1.1 was elevated in the apoptotic neurons, while silencing Kv1.1 expression by siRNA reduced the I_A amplitude of the apoptotic neuron, and increased neuron viability. Inhibiting Kv1.1 current by dendrotoxin‐K evoked a similar effect of reduction of I_A amplitude and protection of neurons. Applying a protein kinase C (PKC) activator, phorbol ester acetate A (PMA) mimicked the LK‐S‐induced neuronal apoptotic effect, enhanced the I_A amplitude and reduced the granule cell viability. The PKC inhibitor, bisindolylmaleimide I and Gö6976 protected the cell against apoptosis induced by LK‐S. After silencing the Kv1.1 gene, the effect of PMA on the residual K⁺ current was reduced significantly. Quantitative RT‐PCR and Western immunoblot techniques revealed that LK‐S treatment and PMA increased the level of the expression of Kv1.1, in contrast, bisindolylmaleimide I inhibited Kv1.1 expression. In addition, the activation of the PKC isoform was identified in apoptotic neurons. We thus conclude that in the rat cerebellar granule cell, the I_A channel associated with apoptotic neurons is encoded mainly by the Kv1.1 gene, and that the PKC pathway promotes neuronal apoptosis by a brief modulation of the I_A amplitude and a permanent increase in the levels of expression of the Kv1.1 α‐subunit.     

Co-assembly of Kv4 α Subunits with K+ Channel-interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes

Members of the K⁺ channel-interacting protein (KChIP) family bind the distal N termini of members of the Shal subfamily of voltage-gated K⁺ channel (Kv4) pore-forming (α) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I_A) and cardiac transient outward (I_to) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 α subunits and Kv4 current densities, findings interpreted to suggest that Kv4·KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (∼40-fold) by an order of magnitude more than the increase in total protein (∼2-fold) or in current densities (∼3-fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2·KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2 but also KChIP2 protein expression. This reciprocal protein stabilization and Kv4·KChIP2 complex formation are lost with deletion of the distal (10 amino acids) Kv4.2 N terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.     

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways: Akt ACTIVATION THROUGH PHOSPHATIDYLINOSITOL 3-KINASE �� AND VESICLE ENDOCYTOSIS FOR NEURONAL SURVIVAL*

MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC₁ receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC₁HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC₁HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC₁HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gα_q/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC₁HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.     

Multi-Walled Carbon Nanotubes Impair Kv4.2/4.3 Channel Activities,Delay Membrane Repolarization and Induce Bradyarrhythmias in the Rat

Purpose

The potential hazardous effects of multi-walled carbon nanotubes (MWCNTs) on cardiac electrophysiology are seldom evaluated. This study aimed to investigate the impacts of MWCNTs on the Kv4/I _to channel, action potential and heart rhythm and the underlying mechanisms.

Methods

HEK293 cells were engineered to express Kv4.2 or Kv4.3 with or without KChIP2 expression. A series of approaches were introduced to analyze the effects of MWCNTs on Kv4/I _to channel kinetics, current densities, expression and trafficking. Transmission electron microscopy was performed to observe the internalization of MWCNTs in HEK293 cells and rat cardiomyocytes. Current clamp was employed to record the action potentials of isolated rat cardiomyocytes. Surface ECG and epicardial monophasic action potentials were recorded to monitor heart rhythm in rats in vivo. Vagal nerve discharge monitoring and H&E staining were also performed.

Results

Induction of MWCNTs into the cytosole through pipette solution soon accelerated the decay of I _Kv4 in HEK293 cells expressing Kv4.2/4.3 and KChIP2, and promoted the recovery from inactivation when Kv4.2 or Kv4.3 was expressed alone. Longer exposure (6 h) to MWCNTs decreased the I _Kv4.2 density, Kv4.2/Kv4.3 (but not KChIP2) expression and trafficking towards the plasma membrane in HEK293 cells. In acutely isolated rat ventricular myocytes, pipette MWCNTs also quickly accelerated the decay of I _Kv4 and prolonged the action potential duration (APD). Intravenous infusion of MWCNTs (2 mg/rat) induced atrioventricular (AV) block and even cardiac asystole. No tachyarrhythmia was observed after MWCNTs administration. MWCNTs did not cause coronary clot but induced myocardial inflammation and increased vagus discharge.

Conclusions

MWCNTs suppress Kv4/I _to channel activities likely at the intracellular side of plasma membrane, delay membrane repolarization and induce bradyarrhythmia. The delayed repolarization, increased vagus output and focal myocardial inflammation may partially underlie the occurrence of bradyarrhythmias induced by MWCNTs. The study warns that MWCNTs are hazardous to cardiac electrophysiology.     

Bis(propyl)-cognitin protects against glutamate-induced neuro-excitotoxicity via concurrent regulation of NO,MAPK/ERK and PI3-K/Akt/GSK3β pathways

We have previously reported that bis(propyl)-cognitin (B3C), similar to memantine (MEM), is an uncompetitive N-methyl-d-aspartate receptor antagonist with fast off-rate property. In the current study, we further demonstrated that in primary cultures of rat cerebellar granule neurons (CGNs), 2 h pretreatment of B3C (IC₅₀, 0.45 μM) prevented glutamate-induced excitotoxicity 10 times more potently than memantine (IC₅₀, 4.58 μM), as evidenced by cell viability and lactate dehydrogenase release assays. Additionally, B3C pretreatment could inhibit the increase of intracellular nitric oxide (NO) and the activation of phosphorylated ERK, and reverse the suppression of phosphorylated Akt and GSK3β caused by glutamate. Furthermore, the neuroprotection of B3C was abolished by phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. Meanwhile, pharmacological inhibition showed that neither the single specific inhibitors of nitric oxide synthase (L-NMMA), MEK1/2 (U0126) and GSK3β (SB415286 and LiCl) nor the combinations of any two of them could fully protect against glutamate-induced apoptosis. However, the co-application of these three inhibitors produced nearly 100% inhibition of glutamate-induced apoptosis. These results taken together suggest that B3C elicits neuroprotection against glutamate-induced neurotoxicity in CGNs via concurrent inhibition of NO, MAPK/ERK pathways and activation of PI3-K/Akt/GSK3β pathway. Combining these and our previous publications, it is conjectured that the dimer might be an ideal candidate drug in delaying the course of neurodegeneration related with Alzheimer’s disease.     

Bidirectional regulation of dendritic voltage-gated potassium channels by the fragile X mental retardation protein

How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.     

The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with K+ Channel-interacting Protein (KChIP) Subunits Are Variable,Depending on the Relative Expression Level

Kv4 is a voltage-gated K⁺ channel, which underlies somatodendritic subthreshold A-type current (I_SA) and cardiac transient outward K⁺ (I_to) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K⁺ channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4·KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2·KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4·KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.     

The Role of Neurotrophins in Brain Aging: A Perspective in Honor of Regino Perez-Polo

During brain aging and progression of Alzheimer’s disease, the levels of Aβ and proinflammatory cytokines accumulate very early in the pathogenic process prior to any major degenerative changes. Accumulation of these molecules may impair with signal transduction pathways critical for neuronal health. Neurotrophin signaling is a critical mechanism involved in synaptic plasticity, learning and memory and neuronal health. We have recently shown that exposure to low levels of Aβ impairs BDNF trkB signal transduction, suppressing the Ras/ERK, and the PI3-K/Akt pathways but not the PLCγ pathway. As a result, downstream regulation of gene expression and neuronal viability are impaired. Recently, we have found that at least three agents – Aβ, TNFα, Il-1β – suppress TrkB signaling and act via a common and novel mechanism. These factors all regulate the docking proteins (e.g., IRS and Shc) that link the activated Trk receptor to downstream effectors. While this is a novel mechanism underlying regulation of Trk signaling, such a mechanism has been identified for the insulin/IGF-1 receptor in the presence of proinflammatory cytokines and is one of the mechanisms for insulin/IGF-resistance, which is a key risk factor for type II diabetes (1). We suggest that accumulation of AB and proinflammatory cytokines during aging generates in the brain a “neurotrophin resistance” state that places the brain at risk for cognitive decline and dementia.     

Characterization of neuritin as a novel angiogenic factor

Neuritin (NRN1), a neurotrophic factor, plays an important role in neurite growth and neuronal survival. In this study, we identify a new function of neuritin as a novel angiogenic factor in vitro and in vivo. Recombinant neuritin protein had no effect on the proliferation and adhesion of human umbilical vein endothelial cells (HUVEC), but it dose-dependently increased endothelial cell migration. Furthermore, overexpression of neuritin significantly promoted tumor angiogenesis, and surprisingly, it inhibited tumor growth in a xenograft tumor model. Thus, our results indicate that neuritin may act as an important angiogenic factor and serve as a potential target for cancer therapy.     

Angiotensin II upregulates Kv1.5 expression through ROS-dependent transforming growth factor-beta1 and extracellular signal-regulated kinase 1/2 signalings in neonatal rat atrial myocytes

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin–angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β₁)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β₁, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT₁) receptor antagonist losartan, an anti-TGF-β₁ antibody and the ERK 1/2 inhibitor PD98059 but not by the AT₂ receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT₁ receptor/TGF-β₁/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β₁ antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT₁ receptor/TGF-β₁/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression.     

Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons

Voltage-gated A-type K+ channel Kv4.2 subunits are highly expressed in the dendrites of hippocampal CA1 neurons. However, little is known about the subcellular distribution and trafficking of Kv4.2-containing channels. Here we provide evidence for activity-dependent trafficking of Kv4.2 in hippocampal spines and dendrites. Live imaging and electrophysiological recordings showed that Kv4.2 internalization is induced rapidly upon glutamate receptor stimulation. Kv4.2 internalization was clathrin mediated and required NMDA receptor activation and Ca2+ influx. In dissociated hippocampal neurons, mEPSC amplitude depended on functional Kv4.2 expression level and was enhanced by stimuli that induced Kv4.2 internalization. Long-term potentiation (LTP) induced by brief glycine application resulted in synaptic insertion of GluR1-containing AMPA receptors along with Kv4.2 internalization. We also found evidence of Kv4.2 internalization upon synaptically evoked LTP in CA1 neurons of hippocampal slice cultures. These results present an additional mechanism for synaptic integration and plasticity through the activity-dependent regulation of Kv4.2 channel surface expression.     

Effects of Candesartan on Electrical Remodeling in the Hearts of Inherited Dilated Cardiomyopathy Model Mice

Inherited dilated cardiomyopathy (DCM) is characterized by dilatation and dysfunction of the ventricles, and often results in sudden death or heart failure (HF). Although angiotensin receptor blockers (ARBs) have been used for the treatment of HF, little is known about the effects on postulated electrical remodeling that occurs in inherited DCM. The aim of this study was to examine the effects of candesartan, one of the ARBs, on cardiac function and electrical remodeling in the hearts of inherited DCM model mice (TNNT2 ΔK210). DCM mice were treated with candesartan in drinking water for 2 months from 1 month of age. Control, non-treated DCM mice showed an enlargement of the heart with prolongation of QRS and QT intervals, and died at t_1/2 of 70 days. Candesartan dramatically extended the lifespan of DCM mice, suppressed cardiac dilatation, and improved the functional parameters of the myocardium. It also greatly suppressed prolongation of QRS and QT intervals and action potential duration (APD) in the left ventricular myocardium and occurrence of ventricular arrhythmia. Expression analysis revealed that down-regulation of Kv4.2 (I_to channel protein), KChIP2 (auxiliary subunit of Kv4.2), and Kv1.5 (I_Kur channel protein) in DCM was partially reversed by candesartan administration. Interestingly, non-treated DCM heart had both normal-sized myocytes with moderately decreased I_to and I_Kur and enlarged cells with greatly reduced K⁺ currents (I_to, I_Kur I_K1 and I_ss). Treatment with candesartan completely abrogated the emergence of the enlarged cells but did not reverse the I_to, and I_Kur in normal-sized cells in DCM hearts. Our results indicate that candesartan treatment suppresses structural remodeling to prevent severe electrical remodeling in inherited DCM.     

Involvement of PI3K/Akt signaling in PTTH-stimulated ecdysteroidogenesis by prothoracic glands of the silkworm, Bombyx mori

The prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by prothoracic gland in larval insects. Previous studies showed that Ca²⁺, cAMP, extracellular signal-regulated kinase (ERK), and tyrosine kinase are involved in PTTH-stimulated ecdysteroidogenesis by the prothoracic glands of both Bombyx mori and Manduca sexta. In the present study, the involvement of phosphoinositide 3-kinase (PI3K)/Akt signaling in PTTH-stimulated ecdysteroidogenesis by B. mori prothoracic glands was further investigated. The results showed that PTTH-stimulated ecdysteroidogenesis was partially blocked by LY294002 and wortmannin, indicating that PI3K is involved in PTTH-stimulated ecdysteroidogenesis. Akt phosphorylation in the prothoracic glands appeared to be moderately stimulated by PTTH in vitro. PTTH-stimulated Akt phosphorylation was inhibited by LY294002. An in vivo PTTH injection into day 6 last instar larvae also increased Akt phosphorylation of the prothoracic glands. In addition, PTTH-stimulated ERK phosphorylation of the prothoracic glands was not inhibited by either LY294002 or wortmannin, indicating that PI3K is not involved in PTTH-stimulated ERK signaling. A23187 and thapsigargin, which stimulated B. mori prothoracic gland ERK phosphorylation and ecdysteroidogenesis, could not activate Akt phosphorylation. PTTH-stimulated ecdysteroidogenesis was not further activated by insulin, indicating the absence of an additive action of insulin and PTTH on the prothoracic glands. The present study, together with the previous demonstration that insulin stimulates B. mori ecdysteroidogenesis through PI3K/Akt signaling, suggests that crosstalk exists in B. mori prothoracic glands between insulin and PTTH signaling, which may play a critical role in precisely regulated ecdysteroidogenesis during development.     

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K⁺ current (I_to) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K⁺ channel is an important component of I_to. The function and expression of Kv4.3 K⁺ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K⁺ channel in heart diseases and discussed the potential role of Kv4.3 K⁺ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K⁺ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca²⁺]_i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K⁺ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K⁺ channel/APD/[Ca²⁺]_i pathway, there exits another mechanism of Kv4.3 K⁺ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K⁺ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K⁺ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K⁺ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K⁺ channel might be potentially harmful or beneficial to hearts through CaMKII.