Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca2+ Homeostasis.

BACKGROUND: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however. METHODS: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. RESULTS: We found that cardiac expression of Gαo, an inhibitory G protein encoded in humans by GNAO1, is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gαo expression increased surface sarcolemmal L-type Ca2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca2+ handling in ventricular myocytes, which led to cardiac dysfunction. CONCLUSIONS: These findings shed light on a novel function of Gαo in the regulation of cardiac Ca2+ homeostasis and systolic function and suggest Gαo may be an effective therapeutic target for the treatment of heart failure.

Cytokine receptor gp130 promotes postnatal proliferation of cardiomyocytes required for the normal functional development of the heart.

Mammalian ventricular cardiomyocytes are premature at birth and exhibit substantial phenotypic changes before weaning. Mouse ventricular myocytes undergo cell division several times after birth; however, the regulatory mechanisms and roles of cardiomyocyte division in postnatal heart development remain unclear. Here, we investigated the physiological role of glycoprotein 130 (gp130), the main subunit of multifunctional receptors for the IL-6 family of cytokines, in postnatal cardiomyocyte proliferation. Pharmacological inhibition of gp130 within the first month after birth induced significant systolic dysfunction of the left ventricle in mice. Consistently, mice with postnatal cardiomyocyte-specific gp130 depletion exhibited impaired left ventricular contractility compared with control mice. In these mice, cardiomyocytes exhibited a moderately decreased size and dramatically inhibited proliferation in the left ventricle but not in the right ventricle. Stereological analysis revealed that this change significantly decreased the number of cardiomyocytes in the left ventricle. Furthermore, IL-6 was mainly responsible for promoting ventricular cardiomyocyte proliferation by activating the JAK/STAT3 pathway. Taken together, the IL-6/gp130/JAK/STAT3 axis plays a crucial role in the physiological postnatal proliferation and hypertrophy of left ventricular cardiomyocytes to ensure normal cardiac functional development.NEW & NOTEWORTHY Although cardiomyocytes undergo proliferation in the early postnatal period, the regulatory mechanisms and physiological importance of this process have not been clarified. We found that the pharmacological and genetic depletion of gp130 in preweaning mice resulted in significant impairment of cardiomyocyte proliferation, thinning of the myocardium, and systolic dysfunction of the left but not right ventricle by perturbing JAK/STAT3 signaling. Thus, the IL-6/gp130/JAK/STAT3 axis is crucial for the postnatal functional development of the left ventricle.

GAD67-mediated GABA Synthesis and Signaling Impinges on Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum.

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD67. GABA may act as a trophic factor during brain development, but its contribution to the development and maturation of cerebellar neural circuits is not known. To understand the roles of GABA in cerebellar organization and associated functions in motor coordination and balance, we examined GAD65 conventional knock out (KO) mice and mice in which GAD67 was eliminated in parvalbumin-expressing neurons (PV-Cre; GAD67flox/flox mice). We found aberrant subcellular localization of the Shaker-type K channel Kv1.1 in basket cell collaterals of PV-Cre; GAD67 flox/flox mice and abnormal projections from basket cells to Purkinje cells in both mouse strains. We also found that altered synaptic properties of basket cell terminals to Purkinje cells in PV-Cre; GAD67flox/flox mice. Furthermore, PV-Cre; GAD67 flox/flox mice exhibited abnormal motor coordination in the rotarod test. These results indicate that GABA signaling in the cerebellum is critical for establishing appropriate connections between basket cells and Purkinje cells and is associated with motor coordination in mice.

Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction.

Close physical association of CaV1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is unexplored. Junctophilin 1 (JP1) and JP2 stabilize the JM by bridging the sarcolemmal and SR membranes. Here, we examined the roles of JPs in localization and function of LTCCs. Knockdown of JP1 or JP2 in cultured myotubes inhibited LTCC clustering at the JM and suppressed evoked Ca2+ transients without disrupting JM structure. Coimmunoprecipitation and GST pull-down assays demonstrated that JPs physically interacted with 12-aa residues in the proximal C terminus of the CaV1.1. A JP1 mutant lacking the C terminus including the transmembrane domain (JP1ΔCT) interacted with the sarcolemmal/T-tubule membrane but not the SR membrane. Expression of this mutant in adult mouse muscles in vivo exerted a dominant-negative effect on endogenous JPs, impairing LTCC-RyR coupling at triads without disrupting JM morphology, and substantially reducing Ca2+ transients without affecting SR Ca2+ content. Moreover, the contractile force of the JP1ΔCT-expressed muscle was dramatically reduced compared with the control. Taken together, JPs recruit LTCCs to the JM through physical interaction and ensure robust ECC at triads in skeletal muscle.

Early life stress from allergic dermatitis causes depressive-like behaviors in adolescent male mice through neuroinflammatory priming.

Allergic dermatitis (AD), associated with pruritus and itchiness, is one of the major stressful conditions early in life. AD also influences the incidence of neuropsychiatric disorders and developmental disorders through neuro-immune interactions. To the best of our knowledge, there is no report that assesses the effects of early childhood dermatitis on psychiatric disorders later in life using an animal model. Here, we developed an oxazolone (Ox)-induced AD model in the early life of male C57BL/6J mice whose ears were challenged by Ox repeatedly from postnatal days (PD) 2 to PD30. On PD30, the Ox-treated ears were remarkably thickened and showed epidermal hyperplasia coupled with increased expression of T helper 2 cytokines, interleukin (IL)-4, and IL-13 in the ear tissue. Additionally, serum immunoglobulin E levels and serum corticosterone levels were higher in the Ox-treated mice than those in the control mice. Although Ox-treated PD40 mice showed neither behavioral abnormalities nor increases in pro-inflammatory cytokine expression in the brain, this study revealed that they experienced downregulation of CD200R1 expression in the amygdala under basal conditions and that additional lipopolysaccharide (LPS) administration induced enhanced neuroinflammatory reaction as the priming effect was accompanied by an increase of Iba-1-positive microglia in the amygdala and hippocampus. Furthermore, the Ox-treated PD40 mice showed depressive-like behaviors 24 h after LPS administration, whereas the control mice did not. Interestingly, the expression of indoleamine 2,3-dioxygenase and kynurenine 3-monooxygenase, key rate-limiting enzymes of the kynurenine metabolism pathway, was upregulated in the hippocampus, prefrontal cortex, and amygdala of the Ox-treated mice 4 h after LPS administration. Based on these results, we suggest that early life stress from AD aggravates susceptibility to systemic inflammation in the adolescent brain, leading to depressive behaviors with abnormal kynurenine metabolism.

Benzothiazepines, diltiazem and JTV-519, exert an anxiolytic-like effect via neurosteroid biosynthesis in mice.

We investigated whether benzothiazepines could produce anxiolytic effects via allopregnanolone (ALLO) biosynthesis in mice. We compared the behavioral effects caused by benzothiazepines to those caused by carbamazepine and sodium valproate. We found that a pretreatment with finasteride (a 5 alpha-reductase inhibitor) suppressed carbamazepine-induced anxiolytic effects but not the effects of sodium valproate. Similar to carbamazepine, diltiazem and JTV-519 displayed anxiolytic effects that were suppressed by a pretreatment with finasteride. We clearly demonstrate that the benzothiazepines, diltiazem and JTV-519, exert an anxiolytic-like effect via ALLO biosynthesis in mice.

Two-day assertive-case-management educational program for medical personnel to prevent suicide attempts: A multicenter pre-post observational study.

AIM: Suicide attempters have a high risk of repeated suicide attempts and completed suicide. There is evidence that assertive case management can reduce the incidence of recurrent suicidal behavior among suicide attempters. This study evaluated the effect of an assertive-case-management training program. METHODS: This multicenter, before-and-after study was conducted at 10 centers in Japan. Participants were 274 medical personnel. We used Japanese versions of the Attitudes to Suicide Prevention Scale, the Gatekeeper Self-Efficacy Scale, the Suicide Intervention Response Inventory (SIRI), and the Attitudes Toward Suicide Questionnaire. We evaluated the effects with one-sample t-tests, and examined prognosis factors with multivariable analysis. RESULTS: There were significant improvements between pre-training and post-training in the Attitudes to Suicide Prevention Scale (mean: -3.07, 95% confidence interval [CI]: -3.57 to -2.57, P < 0.001), the Gatekeeper Self-Efficacy Scale (mean: 10.40, 95%CI: 9.48 to 11.32, P < 0.001), SIRI-1 (appropriate responses; mean: 1.15, 95%CI: 0.89 to 1.42, P < 0.001), and SIRI-2 (different to the expert responses; mean: -4.78, 95%CI: -6.18 to -3.38, P < 0.001). Significant improvements were found on all Attitudes Toward Suicide Questionnaire subscale scores, except Unjustified Behavior. The effect of training was influenced by experience of suicide-prevention training and experience of working with suicidal patients. CONCLUSION: The training program (which was developed to implement and disseminate evidence-based suicide-prevention measures) improved attitudes, self-efficacy, and skills for suicide prevention among medical personnel. Specialized suicide-prevention training and experience with suicidal patients are valuable for enhancing positive attitudes and self-efficacy; furthermore, age and clinical experience alone are insufficient for these purposes.

Prostanoid EP4 Receptor-Mediated Augmentation of I h Currents in Aß Dorsal Root Ganglion Neurons Underlies Neuropathic Pain.

An injury of the somatosensory system causes neuropathic pain, which is usually refractory to conventional analgesics, thus warranting the development of novel drugs against this kind of pain. The mechanism of neuropathic pain in rats that had undergone left L5 spinal nerve transection was analyzed. Ten days after surgery, these rats acquired neuropathic pain. The patch-clamp technique was used on the isolated bilateral L5 dorsal root ganglion neurons. The current-clamped neurons on the ipsilateral side exhibited significantly higher excitability than those on the contralateral side. However, only neurons with diameters of 40-50 µm on the ipsilateral side exhibited significantly larger voltage sags in response to hyperpolarizing current pulses than those on the contralateral side. Under the voltage clamp, only these neurons on the ipsilateral side showed a significantly larger density of an inward current at < -80 mV [hyperpolarization-activated nonselective cation (I h) current] with a rightward-shifted activation curve than that on the contralateral side. Ivabradine-an I h current inhibitor-inhibited I h currents in these neurons on both sides in a similar concentration-dependent manner, with an IC50 value of ∼3 µM. Moreover, the oral administration of ivabradine significantly alleviated the neuropathic pain on the ipsilateral side. An inhibitor of adenylyl cyclase or an antagonist of prostanoid EP4 receptors (CJ-023423) inhibited ipsilateral, but not contralateral I h, currents in these neurons. Furthermore, the intrathecal administration of CJ-023423 significantly attenuated neuropathic pain on the ipsilateral side. Thus, ivabradine and/or CJ-023423 may be a lead compound for the development of novel therapeutics against neuropathic pain.

Prediction of remission in pharmacotherapy of untreated major depression: development and validation of multivariable prediction models.

BACKGROUND: Depression is increasingly recognized as a chronic and relapsing disorder. However, an important minority of patients who start treatment for their major depressive episode recover to euthymia. It is clinically important to be able to predict such individuals. METHODS: The study is a secondary analysis of a recently completed pragmatic megatrial examining first- and second-line treatments for hitherto untreated episodes of non-psychotic unipolar major depression (n = 2011). Using the first half of the cohort as the derivation set, we applied multiply-imputed stepwise logistic regression with backward selection to build a prediction model to predict remission, defined as scoring 4 or less on the Patient Health Quetionnaire-9 at week 9. We used three successively richer sets of predictors at baseline only, up to week 1, and up to week 3. We examined the external validity of the derived prediction models with the second half of the cohort. RESULTS: In total, 37.0% (95% confidence interval 34.8-39.1%) were in remission at week 9. Only the models using data up to week 1 or 3 showed reasonable performance. Age, education, length of episode and depression severity remained in the multivariable prediction models. In the validation set, the discrimination of the prediction model was satisfactory with the area under the curve of 0.73 (0.70-0.77) and 0.82 (0.79-0.85), while the calibration was excellent with non-significant goodness-of-fit χ2 values (p = 0.41 and p = 0.29), respectively. CONCLUSIONS: Patients and clinicians can use these prediction models to estimate their predicted probability of achieving remission after acute antidepressant therapy.

Impact of chronic kidney dysfunction on serum Sulfatides and its metabolic pathway in mice.

Serum sulfatides are critical glycosphingolipids that are present in lipoproteins and exert anticoagulant effects. A previous study reported decreased levels of serum sulfatides in hemodialysis patients and suggested an association with cardiovascular disease. However, the mechanism of changes in serum sulfatides in chronic kidney dysfunction has not been well investigated. The current study examined whether a chronic kidney disease (CKD) state could decrease serum sulfatide levels using 5/6 nephrectomy (5/6NCKD) mice, an established CKD murine model, and studied the mechanisms contributing to diminished sulfatides. 5/6NCKD mice and sham operation control mice were sacrificed at the 4th or 12th postoperative week (POW) for measurement of serum sulfatide levels. Hepatic sulfatide content, which is the origin of serum sulfatides, and the expression of sulfatide metabolic enzymes in liver tissue were assessed as well. The 5/6NCKD mice developed CKD and showed increased serum creatinine and indoxyl sulfate. The serum levels and hepatic amounts of sulfatides were significantly decreased in 5/6NCKD mice at both 4 and 12 POW, while the degradative enzymes of sulfatides arylsulfatase A and galactosylceramidase were significantly increased. In a Hepa1-6 murine liver cell line, indoxyl sulfate addition caused intracellular levels of sulfatides to decrease and degradative enzymes of sulfatides to increase in a manner comparable to the changes in 5/6NCKD mice liver tissue. In conclusion, chronic kidney dysfunction causes degradation of sulfatides in the liver to decrease serum sulfatide levels. One explanation of these results is that indoxyl sulfate, a uremic toxin, accelerates the degradation of sulfatides in liver tissue.

Systemic administration of a delta opioid receptor agonist, KNT-127, facilitates extinction learning of fear memory in rats.

Strategies to facilitate extinction of fear memory have attracted increasing attention for enhancing the effectiveness of exposure therapy for anxiety disorders. Previously, we demonstrated that systemic administration of a delta opioid receptor agonist, KNT-127, has clear anxiolytic-like effects in rats, without impairing memory. These observations led us to hypothesize that KNT-127 might be an appropriate therapeutic agent for anxiety disorders when combined with exposure therapy. In the present study, we demonstrate that KNT-127 (3 mg/kg) facilitates extinction learning of fear memory using the contextual fear conditioning test. As expected, a partial agonist at the glycine-binding site on the glutamatergic N-methyl-d-aspartate receptor, d-cycloserine (15 mg/kg), facilitated extinction learning of contextual fear in rats. In contrast, a benzodiazepine anxiolytic, diazepam (1 mg/kg), impaired the fear extinction learning. Interestingly, the facilitatory effect of KNT-127 on extinction learning was observed not only after a 10-min re-exposure, but also after a much shorter (2-min) re-exposure to the context, while d-cycloserine was ineffective at facilitating extinction when a short-duration exposure was given. Our findings may suggest that administration of a delta opioid receptor agonist might have therapeutic efficacy when combined with exposure therapy for treating a range of anxiety disorders.

Active contact and follow-up interventions to prevent repeat suicide attempts during high-risk periods among patients admitted to emergency departments for suicidal behavior: a systematic review and meta-analysis.

BACKGROUND: There is evidence that several intervention types, including psychotherapy, reduce repeat suicide attempts. However, these interventions are less applicable to the heterogeneous patients admitted to emergency departments (EDs). The risk of a repeat suicide attempt is especially high in the first 6 months after the initial attempt. Therefore, it is particularly important to develop effective ED interventions to prevent repeat suicide attempts during this 6-month period. METHODS: We systematically reviewed randomized controlled trials of ED-initiated interventions for suicidal patients admitted to EDs using the databases MEDLINE, PsychoINFO, CINAHL, and EMBASE up to January 2015 in accordance with an a priori published protocol (PROSPERO: CRD42013005463). Interventions were categorized into four types, including active contact and follow-up interventions (intensive care plus outreach, brief interventions and contact, letter/postcard, telephone, and composite of letter/postcard and telephone), and a meta-analysis was conducted to determine pooled relative risks (RRs) and 95% confidence intervals (CIs) of a repeat suicide attempt within 6 months. RESULTS: Of the 28 selected trials, 14 were active contact and follow-up interventions. Two of these trials (n = 984) reported results at 6 months (pooled RR = 0.48; 95% CI: 0.31 to 0.76). There were not enough trials of other interventions to perform meta-analysis. Some trials included in the meta-analysis were judged as showing risk of bias. CONCLUSION: Active contact and follow-up interventions are recommended for suicidal patients admitted to an ED to prevent repeat suicide attempts during the highest-risk period of 6 months. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42013005463 (27 August 2013).

Sonic hedgehog signaling regulates the mammalian cardiac regenerative response.

Certain organisms, including zebrafish, are capable of complete cardiac regeneration in response to injury. This response has also been observed in newborn mice, although in this case, the regenerative capacity is lost at approximately one week of age. The mechanisms regulating this short temporal window of cardiac regeneration in mice are not well understood. Here, we show that sonic hedgehog (Shh) signaling modulates the neonatal mouse regenerative response. In particular, we demonstrate that following apical resection of the heart on postnatal day 1, mice activate Shh ligand expression and downstream signaling. This response is largely absent when surgery is performed on non-regenerative, postnatal day 7 pups. Furthermore, an enhanced cardiac regeneration response was detected in ptch heterozygous mice which have a genetically-based constitutive increase in Shh signaling. We further show that Shh ligand is produced in the myocardium by non-myocytes and appears to regulate cardiomyocyte proliferation, as well as the recruitment of monocytes/macrophages to the regenerating area. Finally, we demonstrate that a small molecule activator of Shh signaling promotes heart regeneration, whereas an inhibitor of Shh signaling impairs the regenerative response. Together, these results implicate Shh signaling as a regulator of mammalian heart regeneration and suggest that modulating this pathway may lead to new potential therapies for cardiovascular diseases.

PDGF-induced migration of synthetic vascular smooth muscle cells through c-Src-activated L-type Ca2+ channels with full-length CaV1.2 C-terminus.

In atherosclerosis, vascular smooth muscle cells (VSMC) migrate from the media toward the intima of the arteries in response to cytokines, such as platelet-derived growth factor (PDGF). However, molecular mechanism underlying the PDGF-induced migration of VSMCs remains unclear. The migration of rat aorta-derived synthetic VSMCs, A7r5, in response to PDGF was potently inhibited by a CaV1.2 channel inhibitor, nifedipine, and a Src family tyrosine kinase (SFK)/Abl inhibitor, bosutinib, in a less-than-additive manner. PDGF significantly increased CaV1.2 channel currents without altering CaV1.2 protein expression levels in A7r5 cells. This reaction was inhibited by C-terminal Src kinase, a selective inhibitor of SFKs. In contractile VSMCs, the C-terminus of CaV1.2 is proteolytically cleaved into proximal and distal C-termini (PCT and DCT, respectively). Clipped DCT is noncovalently reassociated with PCT to autoinhibit the channel activity. Conversely, in synthetic A7r5 cells, full-length CaV1.2 (CaV1.2FL) is expressed much more abundantly than truncated CaV1.2. In a heterologous expression system, c-Src activated CaV1.2 channels composed of CaV1.2FL but not truncated CaV1.2 (CaV1.2Δ1763) or CaV1.2Δ1763 plus clipped DCT. Further, c-Src enhanced the coupling efficiency between the voltage-sensing domain and activation gate of CaV1.2FL channels by phosphorylating Tyr1709 and Tyr1758 in PCT. Compared with CaV1.2Δ1763, c-Src could more efficiently bind to and phosphorylate CaV1.2FL irrespective of the presence or absence of clipped DCT. Therefore, in atherosclerotic lesions, phenotypic switching of VSMCs may facilitate pro-migratory effects of PDGF on VSMCs by suppressing posttranslational CaV1.2 modifications.

Optimising first- and second-line treatment strategies for untreated major depressive disorder - the SUN☺D study: a pragmatic, multi-centre, assessor-blinded randomised controlled trial.

BACKGROUND: For patients starting treatment for depression, current guidelines recommend titrating the antidepressant dosage to the maximum of the licenced range if tolerated. When patients do not achieve remission within several weeks, recommendations include adding or switching to another antidepressant. However, the relative merits of these guideline strategies remain unestablished. METHODS: This multi-centre, open-label, assessor-blinded, pragmatic trial involved two steps. Step 1 used open-cluster randomisation, allocating clinics into those titrating sertraline up to 50 mg/day or 100 mg/day by week 3. Step 2 used central randomisation to allocate patients who did not remit after 3 weeks of treatment to continue sertraline, to add mirtazapine or to switch to mirtazapine. The primary outcome was depression severity measured with the Patient Health Questionnaire-9 (PHQ-9) (scores between 0 and 27; higher scores, greater depression) at week 9. We applied mixed-model repeated-measures analysis adjusted for key baseline covariates. RESULTS: Between December 2010 and March 2015, we recruited 2011 participants with hitherto untreated major depression at 48 clinics in Japan. In step 1, 970 participants were allocated to the 50 mg/day and 1041 to the 100 mg/day arms; 1927 (95.8%) provided primary outcomes. There was no statistically significant difference in the adjusted PHQ-9 score at week 9 between the 50 mg/day arm and the 100 mg/day arm (0.25 point, 95% confidence interval (CI), - 0.58 to 1.07, P = 0.55). Other outcomes proved similar in the two groups. In step 2, 1646 participants not remitted by week 3 were randomised to continue sertraline (n = 551), to add mirtazapine (n = 537) or to switch to mirtazapine (n = 558): 1613 (98.0%) provided primary outcomes. At week 9, adding mirtazapine achieved a reduction in PHQ-9 scores of 0.99 point (0.43 to 1.55, P = 0.0012); switching achieved a reduction of 1.01 points (0.46 to 1.56, P = 0.0012), both relative to continuing sertraline. Combination increased the percentage of remission by 12.4% (6.1 to 19.0%) and switching by 8.4% (2.5 to 14.8%). There were no differences in adverse effects. CONCLUSIONS: In patients with new onset depression, we found no advantage of titrating sertraline to 100 mg vs 50 mg. Patients unremitted by week 3 gained a small benefit in reduction of depressive symptoms at week 9 by switching sertraline to mirtazapine or by adding mirtazapine. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01109693 . Registered on 23 April 2010.

Increase in phospholamban content in mouse skeletal muscle after denervation.

It is well-known that denervation of motor nerves induces atrophy and decreases contractile force of the skeletal muscle. However, it is not completely understood how denervation alters calcium handling in the skeletal muscle. We investigated the effect of denervation on the expression and function of proteins involved in calcium handling. Two weeks after denervation of the right sciatic nerve in mice, we observed a significant decrease in mass and cross-sectional area of the ipsilateral tibialis anterior (TA) and flexor digitorum brevis (FDB) muscles. Also, we observed a significant decrease in the specific tetanus contractile force in the ipsilateral TA muscle. Calcium imaging of the ipsilateral FDB showed that the peak twitch and tetanus calcium concentrations were significantly decreased due to a decrease in calcium content of the sarcoplasmic reticulum (SR). Denervation reduced the maximum rate of sarcoplasmic/endoplasmic calcium ATPase (SERCA) activity. Interestingly, the amount of phospholamban (PLN), but not its transcripts, was increased in the ipsilateral vs. contralateral side after denervation, suggesting that denervation impairs constitutive regulation of PLN. Immunohistochemical analysis revealed increased PLN in all major fiber types in TA with IIx fibers showing a threefold higher expression than the contralateral side. These results suggest that the abnormal increase in PLN in the ipsilateral fast-twitch fibers may be involved in decreased SERCA activity, SR calcium content, peak calcium transients, and contractile forces of denervated muscles.

A delta opioid receptor agonist, KNT-127, in the prelimbic medial prefrontal cortex attenuates glial glutamate transporter blocker-induced anxiety-like behavior in mice.

We previously reported that systemic administration of a delta opioid receptor (DOP) agonist, KNT-127, produced a potent anxiolytic-like effect in rats. Interestingly, DOPs are highly distributed in the prelimbic medial prefrontal cortex (PL-PFC). In the present study, we investigated the effect of KNT-127 co-perfusion in the PL-PFC on anxiety-like behavior in mice, induced by a glial glutamate transporter inhibitor, (3S)-3-[[3-[[4-(Trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA). Extracellular glutamate levels were measured in male C57BL/6N mice by in vivo microdialysis high-performance liquid chromatography/electrochemical detection, with behavior simultaneously assessed in the open field test. As expected, extracellular glutamate levels were significantly increased, and anxiety-like behavior was induced after local perfusion of TFB-TBOA in the PL-PFC. Uniquely, co-perfusion of KNT-127 in the PL-PFC diminished anxiety-like behavior induced by TFB-TBOA without affecting extracellular glutamate levels. Further, the effect of KNT-127 on anxiety-like behavior was antagonized by a selective DOP antagonist, naltrindole, suggesting that KNT-127 acts via DOPs. These findings do not support our preconceived hypothesis that KNT-127 in PL-PFC produces an anxiolytic-like effect via suppression of glutamatergic transmission. Hence, further studies are necessary to understand the mechanisms of DOP agonist-induced anxiolytic-like effects in the PL-PFC.

Angiotensin II activates CaV 1.2 Ca2+ channels through ß-arrestin2 and casein kinase 2 in mouse immature cardiomyocytes.

KEY POINTS: Angiotensin II (AngII) is crucial in cardiovascular regulation in perinatal mammalians. Here we show that AngII increases twitch Ca2+ transients of mouse immature but not mature cardiomyocytes by robustly activating CaV 1.2 L-type Ca2+ channels through a novel signalling pathway involving angiotensin type 1 (AT1 ) receptors, ß-arrestin2 and casein kinase 2. A ß-arrestin-biased AT1 receptor agonist, TRV027, was as effective as AngII in activating L-type Ca2+ channels. Our results help understand the molecular mechanism by which AngII regulates the perinatal circulation and also suggest that ß-arrestin-biased AT1 receptor agonists may be valuable therapeutics for paediatric heart failure. ABSTRACT: Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system, plays important roles in cardiovascular regulation in the perinatal period. Despite the well-known stimulatory effect of AngII on vascular contraction, little is known about regulation of contraction of the immature heart by AngII. Here we found that AngII significantly increased the peak amplitude of twitch Ca2+ transients by robustly activating L-type CaV 1.2 Ca2+ (CaV 1.2) channels in mouse immature but not mature cardiomyocytes. This response to AngII was mediated by AT1 receptors and ß-arrestin2. A ß-arrestin-biased AT1 receptor agonist was as effective as AngII in activating CaV 1.2 channels. Src-family tyrosine kinases (SFKs) and casein kinase 2α'ß (CK2α'ß) were sequentially activated when AngII activated CaV 1.2 channels. A cyclin-dependent kinase inhibitor, p27Kip1 (p27), inhibited CK2α'ß, and AngII removed this inhibitory effect through phosphorylating tyrosine 88 of p27 via SFKs in cardiomyocytes. In a human embryonic kidney cell line, tsA201 cells, overexpression of CK2α'ß but not c-Src directly activated recombinant CaV 1.2 channels composed of C-terminally truncated α1C , the distal C-terminus of α1C , ß2C and α2 δ1 subunits, by phosphorylating threonine 1704 located at the interface between the proximal and the distal C-terminus of CaV 1.2α1C subunits. Co-immunoprecipitation revealed that CaV 1.2 channels, CK2α'ß and p27 formed a macromolecular complex. Therefore, stimulation of AT1 receptors by AngII activates CaV 1.2 channels through ß-arrestin2 and CK2α'ß, thereby probably exerting a positive inotropic effect in the immature heart. Our results also indicated that ß-arrestin-biased AT1 receptor agonists may be used as valuable therapeutics for paediatric heart failure in the future.

Post-reexposure administration of D-cycloserine facilitates reconsolidation of contextual conditioned fear memory in rats.

The effect of D-cycloserine, a N-methyl-D-aspartate receptor partial agonist, on the reconsolidation of conditioned fear memory is not precisely understood. In this study, we clarified the effects of D-cycloserine on the reconsolidation in rats, by performing contextual fear conditioning with a mild fear-conditioning procedure and with post-reexposure administration of the drug. The D-cycloserine (15 mg/kg subcutaneously)-treated rats showed a persistent and greater fear response during the test session compared with the control group. In conclusion, we have confirmed that post-reexposure administration of D-cycloserine facilitates the reconsolidation of fear memory in rats.