Voltage-Sensing Phosphatases: Biophysics, Physiology, and Molecular Engineering.

Voltage-sensing phosphatase (VSP) contains a voltage sensor domain (VSD) similar to that in voltage-gated ion channels, and a phosphoinositide phosphatase region similar to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). The VSP gene is conserved from unicellular organisms to higher vertebrates. Membrane depolarization induces electrical driven conformational rearrangement in the VSD, which is translated into catalytic enzyme activity. Biophysical and structural characterization has revealed details of the mechanisms underlying the molecular functions of VSP. Coupling between the VSD and the enzyme is tight, such that enzyme activity is tuned in a graded fashion to the membrane voltage. Upon VSP activation, multiple species of phosphoinositides are simultaneously altered, and the profile of enzyme activity depends on the history of the membrane potential. VSPs have been the obvious candidate link between membrane potential and phosphoinositide regulation. However, patterns of voltage change regulating VSP in native cells remain largely unknown. This review addresses the current understanding of the biophysical biochemical properties of VSP and provides new insight into the proposed functions of VSP.

(R)-ketamine restores anterior insular cortex activity and cognitive deficits in social isolation-reared mice.

Chronic social isolation increases the risk of mental health problems, including cognitive impairments and depression. While subanesthetic ketamine is considered effective for cognitive impairments in patients with depression, the neural mechanisms underlying its effects are not well understood. Here we identified unique activation of the anterior insular cortex (aIC) as a characteristic feature in brain-wide regions of mice reared in social isolation and treated with (R)-ketamine, a ketamine enantiomer. Using fiber photometry recording on freely moving mice, we found that social isolation attenuates aIC neuronal activation upon social contact and that (R)-ketamine, but not (S)-ketamine, is able to counteracts this reduction. (R)-ketamine facilitated social cognition in social isolation-reared mice during the social memory test. aIC inactivation offset the effect of (R)-ketamine on social memory. Our results suggest that (R)-ketamine has promising potential as an effective intervention for social cognitive deficits by restoring aIC function.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Role of K364 next to the active site cysteine in voltage-dependent phosphatase activity of Ci-VSP.

Voltage-sensing phosphatase (VSP) consists of the voltage sensor domain (VSD) similar to that of voltage-gated ion channels and the cytoplasmic phosphatase region with remarkable similarity to the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Membrane depolarization activates VSD, leading to dephosphorylation of three species of phosphoinositides (phosphatidylinositol phosphates (PIPs)), PI(3,4,5)P3, PI(4,5)P2, and PI(3,4)P2. VSP dephosphorylates 3- and 5-phosphate of PIPs, unlike PTEN, which shows rigid 3-phosphate specificity. In this study, a bioinformatics search showed that some mammals have VSP orthologs with amino acid diversity in the active center motif, Cx5R, which is highly conserved among protein tyrosine phosphatases and PTEN-related phosphatases; lysine next to the active site cysteine in the Cx5R motif was substituted for methionine in VSP orthologs of Tasmanian devil, koala, and prairie deer mouse, and leucine in opossum. Since lysine at the corresponding site in PTEN is known to be critical for enzyme activities, we attempted to address the significance of amino acid diversity among VSP orthologs at this site. K364 was changed to different amino acids in sea squirt VSP (Ci-VSP), and voltage-dependent phosphatase activity in Xenopus oocyte was studied using fluorescent probes for PI(4,5)P2 and PI(3,4)P2. All mutants retained both 5-phosphatase and 3-phosphatase activity, indicating that lysine at this site is dispensable for 3-phosphatase activity, unlike PTEN. Notably, K364M mutant showed increased activity both of 5-phosphatase and 3-phosphatase compared with the wild type (WT). It also showed slower kinetics of voltage sensor motion. Malachite green assay of K364M mutant did not show significant difference of phosphatase activity from WT, suggesting tighter interaction between substrate binding and voltage sensing. Mutation corresponding to K364M in the zebrafish VSP led to enhanced voltage-dependent dephosphorylation of PI(4,5)P2. Further studies will provide clues to understanding of substrate preference in PIPs phosphatases as well as to customization of a molecular tool.

Prognostic Value of the Severity of Clinical Congestion in Patients Hospitalized for Decompensated Heart Failure: Findings From the Japanese KCHF Registry.

BACKGROUND: Congestion is a leading cause of hospitalization and a major therapeutic target in patients with heart failure (HF). Clinical practice in Japan is characterized by a long hospital stay, which facilitates more extensive decongestion during hospitalization. We herein examined the time course and prognostic impact of clinical congestion in a large contemporary Japanese cohort of HF. METHODS AND RESULTS: Peripheral edema, jugular venous pressure, and orthopnea were graded on a standardized 4-point scale (0-3) in 3787 hospitalized patients in a Japanese cohort of HF. Composite Congestion Scores (CCS) on admission and at discharge were calculated by summing individual scores. The primary outcome was a composite of all-cause death or HF hospitalization. The median admission CCS was 4 (interquartile range, 3-6). Overall, 255 patients died during the median hospitalization length of 16 days, and 1395 died or were hospitalized for HF over a median postdischarge follow-up of 396 days. The cumulative 1-year incidence of the primary outcome increased at higher tertiles of congestion on admission (32.5%, 39.3%, and 41.0% in the mild [CCS ≤3], moderate [CCS = 4 or 5], and severe [CCS ≥6] congestion groups, respectively, log-rank P < .001). The adjusted hazard ratios of moderate and severe congestion relative to mild congestion were 1.205 (95% confidence interval [CI], 1.065-1.365; P = .003) and 1.247 (95% CI, 1.103-1.410; P < .001), respectively. Among 3445 patients discharged alive, 85% had CCS of 0 (complete decongestion) and 15% had a CCS of 1 or more (residual congestion) at discharge. Although residual congestion predicted a risk of postdischarge death or HF hospitalization (adjusted hazard ratio, 1.314 [1.145-1.509]; P < .001), the admission CCS correlated with the risk of postdischarge death or HF hospitalization, even in the complete decongestion group. No correlation was observed for postdischarge death or HF hospitalization between residual congestion at discharge and admission CCS (P for the interaction = .316). CONCLUSIONS: In total, 85% of patients were discharged with complete decongestion in Japanese clinical practice. Clinical congestion, on admission and at discharge, was of prognostic value. The severity of congestion on admission was predictive of adverse outcomes, even in the absence of residual congestion. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02334891 (NCT02334891) https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000017241 (UMIN000015238).

Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum.

Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

Heterologous functional expression of ascidian Nav1 channels and close relationship with the evolutionary ancestor of vertebrate Nav channels.

Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary ß subunits. Detailed information on the structure-function relationships of Nav1 subunits has been obtained through heterologous expression experiments and analyses of protein structures. The basic properties of Nav1s, including their gating and ion permeation, were classically described in the squid giant axon and other invertebrates. However, heterologous functional expression of Nav1s from marine invertebrates has been unsuccessful. Ascidians belong to the Urochordata, a sister group of vertebrates, and the larval central nervous system of ascidians shows a similar plan to that of vertebrates. Here, we report the biophysical properties of ascidian Ciona Nav1 (CiNav1a) heterologously expressed in Xenopus oocytes. CiNav1a exhibited tetrodotoxin-insensitive sodium currents with rapid gating kinetics of activation and inactivation. Furthermore, consistent with the fact that the Ciona genome lacks orthologous genes to vertebrate ß subunits, the human ß1 subunit did not influence the gating properties when coexpressed with CiNav1a. Interestingly, CiNav1a contains an ankyrin-binding motif in the II-III linker, which can be targeted to the axon initial segment of mammalian cortical neurons. Our findings provide a platform to gain insight into the evolutionary and biophysical properties of Nav1s, which are important for the development of targeted therapeutics.

Starting Neurohormonal Antagonists in Patients With Acute Heart Failure With Mid-Range and Preserved Ejection Fraction.

BACKGROUND: The clinical benefits of neurohormonal antagonists for patients with heart failure (HF) with mid-range and preserved ejection fraction (HFmrEF and HFpEF) are uncertain.Methods and Results: This study analyzed 858 consecutive patients with HFmrEF (EF: 40-49%) or HFpEF (EF ≥50%), who were hospitalized for acute HF, and who were discharged alive, and were not taking angiotensin-converting enzyme inhibitors (ACE)-I/ angiotensin II receptor blockers (ARB) or ß-blockers at admission. The study population was classified into 4 groups according to the status of prescription of ACE-I/ARB and ß-blocker at discharge: no neurohormonal antagonist (n=342, 39.9%), ACE-I/ARB only (n=128, 14.9%), ß-blocker only (n=189, 22.0%), and both ACE-I/ARB and ß-blocker (n=199, 23.2%) groups. The primary outcome measure was a composite of all-cause death or HF hospitalization. The cumulative 1-year incidence of the primary outcome measure was 41.2% in the no neurohormonal antagonist group, 34.0% in the ACE-I/ARB only group, 28.6% in the ß-blocker only group, and 16.4% in the both ACE-I/ARB and ß-blocker group (P<0.001). Compared with the no neurohormonal antagonist group, both the ACE-I/ARB and ß-blocker groups were associated with a significantly lower risk for a composite of all-cause death or HF hospitalization (HR: 0.46, 95% CI: 0.28-0.76, P=0.002). CONCLUSIONS: In hospitalized patients with HFmrEF and HFpEF, starting both ACE-I/ARB and a ß-blocker was associated with a reduced risk of the composite of all-cause death or HF hospitalization compared with patients not starting on an ACE-I/ARB or ß-blocker.

Polarized PtdIns(4,5)P2 distribution mediated by a voltage-sensing phosphatase (VSP) regulates sperm motility.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.

Heterogeneity of microglial proton channel in different brain regions and its relationship with aging.

The properties of microglia largely differ depending on aging as well as on brain regions. However, there are few studies that investigated the functional importance of such heterogeneous properties of microglia at the molecular level. Voltage-gated proton channel, Hv1/VSOP, could be one of the candidates which confers functional heterogeneity among microglia since it regulates brain oxidative stress in age-dependent manner. In this study, we found that Hv1/VSOP shows brain region-dependent heterogeneity of gene expression with the highest level in the striatum. We studied the importance of Hv1/VSOP in two different brain regions, the cerebral cortex and striatum, and examined their relationship with aging (using mice of different ages). In the cortex, we observed the age-dependent impact of Hv1/VSOP on oxidative stress, microglial morphology, and gene expression profile. On the other hand, we found that the age-dependent significance of Hv1/VSOP was less obvious in the striatum than the cortex. Finally, we performed a battery of behavioral experiments on Hv1/VSOP-deficient mice both at young and aged stages to examine the effect of aging on Hv1/VSOP function. Hv1/VSOP-deficient mice specifically showed a marked difference in behavior in light/dark transition test only at aged stages, indicating that anxiety state is altered in aged Hv1/VSOP mice. This study suggests that a combination of brain region heterogeneity and animal aging underscores the functional importance of Hv1/VSOP in microglia.

Regulation of hepatic oxidative stress by voltage-gated proton channels (Hv1/VSOP) in Kupffer cells and its potential relationship with glucose metabolism.

Voltage-gated proton channels (Hv1/VSOP), encoded by Hvcn1, are important regulator of reactive oxygen species (ROS) production in many types of immune cells. While in vitro studies indicate that Hv1/VSOP regulates ROS production by maintaining pH homeostasis, there are few studies investigating the functional importance of Hv1/VSOP in vivo. In the present study, we first show that Hv1/VSOP is functionally expressed in liver resident macrophage, Kupffer cells, regulating the hepatic oxidative stress in vivo. Our immunocytochemistry and electrophysiology data showed that Hvcn1 is specifically expressed in Kupffer cells, but not in hepatocytes. Furthermore, Hvcn1-deficiency drastically altered the hepatic oxidative stress. The Hvcn1-deficient mice showed high blood glucose and serum insulin but normal insulin sensitivity, indicating that these phenotypes were not linked to insulin resistance. Transcriptome analysis indicated that the gene expression of glycogen phosphorylase (Pygl) and Glucose-6-phosphatase, catalytic subunit (G6pc) were upregulated in Hvcn1-deficient liver tissues, and quantitative PCR confirmed the result for Pygl. Furthermore, we observed higher amount of glucose-6-phosphate, a key sugar intermediate for glucose in Hvcn1-deficient liver than WT, suggesting that glucose production in liver is accelerated in Hvcn1-deficient mice. The present study sheds light on the functional importance of Kupffer cells in hepatic oxidative stress and its potential relationship with glucose metabolism.

Real-time functional analysis of Hv1 channel in neutrophils: a new approach from zebrafish model.

Voltage-gated proton channel (Hv1) has been studied in various immune cells, including neutrophils. However, most studies have taken an in vitro approach using isolated cells or primary cultured cells of mammals; therefore, limited evidence is available on the function of Hv1 in a physiological context. In this study, we have developed the in vivo system that enables real-time functional analysis of Hv1 using zebrafish embryos (Danio rerio). Hvcn1-deficiency (hvcn1-/-) in zebrafish completely abolished voltage-gated proton current, which is typically observed in wild-type neutrophils. Importantly, hvcn1-deficiency significantly reduced reactive oxygen species production and calcium response of zebrafish neutrophils, comparable to the results observed in mammalian models. These findings verify zebrafish Hv1 (DrHv1) as the primary contributor for native Hv1-derived proton current in neutrophils and suggest the conserved function of Hv1 in the immune cells across vertebrate animals. Taking advantage of Hv1 zebrafish model, we compared real-time behaviors of neutrophils between wild-type and hvcn1-/- zebrafish in response to tissue injury and acute bacterial infection. Notably, we observed a significant increase in the number of phagosomes in hvcn1-/- neutrophils, raising a possible link between Hv1 and phagosomal maturation. Furthermore, survival analysis of zebrafish larvae potentially supports a protective role of Hv1 in the innate immune response against systemic bacterial infection. This study represents the influence of Hv1 on neutrophil behaviors and highlights the benefits of in vivo approach toward the understanding of Hv1 in a physiological context.

Demographics, Management, and In-Hospital Outcome of Hospitalized Acute Heart Failure Syndrome Patients in Contemporary Real Clinical Practice in Japan　- Observations From the Prospective, Multicenter Kyoto Congestive Heart Failure (KCHF) Registry.

BACKGROUND: There is a scarcity of reports on the clinical characteristics and management practice in contemporary all-comer patients with acute decompensated heart failure (ADHF). Methods and Results: The Kyoto Congestive Heart Failure (KCHF) registry is a prospective observational cohort study enrolling 4,056 consecutive patients who had hospital admission due to ADHF without any exclusion criteria between October 2014 and March 2016 in the 19 participating hospitals in Japan. Baseline characteristics, clinical presentations, management, and in-hospital outcomes were compared between heart failure (HF) with reduced left ventricular ejection fraction (LVEF; HFrEF, LVEF <40%), HF with mid-range LVEF (HFmrEF, LVEF 40-49%), and HF with preserved LVEF (HFpEF, LVEF ≥50%). Of the 4,041 patients with documented LVEF, 1,744 (43%) had HFpEF; 746 (19%), HFmrEF; and 1,551 (38%), HFrEF. The median age was 80 years (IQR, 72-86 years) in the entire population, and was higher with increasing LVEF (P<0.001). The in-hospital mortality rate was higher in the HFrEF than in the HFmrEF and HFpEF groups (9.2%, 4.8%, and 5.1%, respectively, P<0.001). CONCLUSIONS: This registry elucidated the clinical features and clinically relevant in-hospital outcomes in contemporary consecutive patients with ADHF in real-world clinical practice in Japan. When classified by LVEF, significant differences in characteristics and in-hospital outcomes existed between patients with HFrEF, HFmrEF, and HFpEF.

Unconventional role of voltage-gated proton channels (VSOP/Hv1) in regulation of microglial ROS production.

It has been established that voltage-gated proton channels (VSOP/Hv1), encoded by Hvcn1, support reactive oxygen species (ROS) production in phagocytic activities of neutrophils (El Chemaly et al. ) and antibody production in B lymphocytes (Capasso et al. ). VSOP/Hv1 is a potential therapeutic target for brain ischemia, since Hvcn1 deficiency reduces microglial ROS production and protects brain from neuronal damage (Wu et al. ). In the present study, we report that VSOP/Hv1 has paradoxical suppressive role in ROS production in microglia. Extracellular ROS production was lower in neutrophils of Hvcn1-/- mice than WT mice as reported. In contrast, it was drastically enhanced in isolated Hvcn1-/- microglia as compared with cells from WT mice. Actin dynamics was altered in Hvcn1-/- microglia and intracellular distribution of cytosolic NADPH oxidase subunit, p67, was changed. When expression levels of oxidative stress responsive antioxidant genes were compared between WT and Hvcn1-/- in cerebral cortex at different ages of animals, they were slightly decreased in Hvcn1-/- mice at younger stage (1 day, 5 days, 3 weeks old), but drastically increased at aged stage (6 months old), suggesting that the regulation of microglial ROS production by VSOP/Hv1 is age-dependent. We also performed brain ischemic stroke experiments and found that the neuroprotective effect of VSOP/Hv1deficiency on infarct volume depended on the age of animals. Taken together, regulation of ROS production by VSOP/Hv1 is more complex than previously thought and significance of VSOP/Hv1 in microglial ROS production depends on age.

Development of interpretable machine learning models to predict in-hospital prognosis of acute heart failure patients.

AIMS: In recent years, there has been remarkable development in machine learning (ML) models, showing a trend towards high prediction performance. ML models with high prediction performance often become structurally complex and are frequently perceived as black boxes, hindering intuitive interpretation of the prediction results. We aimed to develop ML models with high prediction performance, interpretability, and superior risk stratification to predict in-hospital mortality and worsening heart failure (WHF) in patients with acute heart failure (AHF). METHODS AND RESULTS: Based on the Kyoto Congestive Heart Failure registry, which enrolled 4056 patients with AHF, we developed prediction models for in-hospital mortality and WHF using information obtained on the first day of admission (demographics, physical examination, blood test results, etc.). After excluding 16 patients who died on the first or second day of admission, the original dataset (n = 4040) was split 4:1 into training (n = 3232) and test datasets (n = 808). Based on the training dataset, we developed three types of prediction models: (i) the classification and regression trees (CART) model; (ii) the random forest (RF) model; and (iii) the extreme gradient boosting (XGBoost) model. The performance of each model was evaluated using the test dataset, based on metrics including sensitivity, specificity, area under the receiver operating characteristic curve (AUC), Brier score, and calibration slope. For the complex structure of the XGBoost model, we performed SHapley Additive exPlanations (SHAP) analysis, classifying patients into interpretable clusters. In the original dataset, the proportion of females was 44.8% (1809/4040), and the average age was 77.9 ± 12.0. The in-hospital mortality rate was 6.3% (255/4040) and the WHF rate was 22.3% (900/4040) in the total study population. In the in-hospital mortality prediction, the AUC for the XGBoost model was 0.816 [95% confidence interval (CI): 0.815-0.818], surpassing the AUC values for the CART model (0.683, 95% CI: 0.680-0.685) and the RF model (0.755, 95% CI: 0.753-0.757). Similarly, in the WHF prediction, the AUC for the XGBoost model was 0.766 (95% CI: 0.765-0.768), outperforming the AUC values for the CART model (0.688, 95% CI: 0.686-0.689) and the RF model (0.713, 95% CI: 0.711-0.714). In the XGBoost model, interpretable clusters were formed, and the rates of in-hospital mortality and WHF were similar among each cluster in both the training and test datasets. CONCLUSIONS: The XGBoost models with SHAP analysis provide high prediction performance, interpretability, and reproducible risk stratification for in-hospital mortality and WHF for patients with AHF.

Burst generation mediated by cholinergic input in terminal nerve-gonadotrophin releasing hormone neurones of the goldfish.

Peptidergic neurones play a pivotal role in the neuromodulation of widespread areas in the nervous system. Generally, it has been accepted that the peptide release from these neurones is regulated by their firing activities. The terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones, which are one of the well-studied peptidergic neurones in vertebrate brains, are characterised by their spontaneous regular pacemaker activities, and GnRH has been suggested to modulate the sensory responsiveness of animals. Although many peptidergic neurones are known to exhibit burst firing activities when they release the peptides, TN-GnRH neurones show spontaneous burst firing activities only infrequently. Thus, it remains to be elucidated whether the TN-GnRH neurones show burst activities and, if so, how the mode switching between the regular pacemaking and bursting modes is regulated in these neurones. In this study, we found that only a single pulse electrical stimulation of the neuropil surrounding the TN-GnRH neurones reproducibly induces transient burst activities in TN-GnRH neurones. Our combined physiological and morphological data suggest that this phenomenon occurs following slow inhibitory postsynaptic potentials mediated by cholinergic terminals surrounding the TN-GnRH neurones. We also found that the activation of muscarinic acetylcholine receptors induces persistent opening of potassium channels, resulting in a long-lasting hyperpolarisation. This long hyperpolarisation induces sustained rebound depolarisation that has been suggested to be generated by a combination of persistent voltage-gated Na(+) channels and low-voltage-activated Ca(2+) channels. These new findings suggest a novel type of cholinergic regulation of burst activities in peptidergic neurones, which should contribute to the release of neuropeptides.

Glucose absorption activity and gene expression of sugar transporters in the trophotaenia of the viviparous teleost Xenotoca eiseni.

In viviparous reproductive systems, nutrient transfer from mother to embryo plays a critical role in the generation of offspring. Herein, we investigated the mother-to-embryo nutrient transfer machinery in the viviparous teleost Xenotoca eiseni, which belongs to the family Goodeidae. The intraovarian embryo absorbs maternal supplements via the hindgut-derived placental structure termed the trophotaenia. Tracer analysis indicated that the trophotaenia can take up glucose analogs in ex vivo cultured embryos. The candidate genes for absorption, sglt1, glut2, atp1a, and atp1b, were determined from published transcriptomes. These genes were expressed in the trophotaenia of X. eiseni embryos. Fluorescent immunohistochemistry of Na+/K+ ATPase indicated the polarity of epithelial cells in the trophotaenia. The presented evidence suggests that the epithelial cell layer transports monosaccharides from the apical membrane of epithelial cells in a basolateral direction. Taken together, this study provides insight into how maternal fish maintain their offspring during gestation and will aid in the development of strategies to improve offspring generation in these fish.

Association between changes in loop diuretic dose and outcomes in acute heart failure.

AIMS: Little is known about the association between the starting of or dose changes in loop diuretics during acute heart failure (AHF) hospitalization and post-discharge outcomes. We investigated the clinical impact of starting loop diuretics and changing the loop diuretics dose during hospitalization on post-discharge outcomes. METHODS AND RESULTS: From the Kyoto Congestive Heart Failure registry, 3665 consecutive patients hospitalized for HF and discharged alive were included in this study. We analysed 1906 patients without loop diuretics on admission and were discharged alive and 1759 patients who received loop diuretics on admission and were discharged alive. The primary outcome measure was all-cause death. Of the 1906 patients without loop diuretics on admission, 1366 (71.7%) patients started loop diuretics during the index AHF hospitalization. Starting loop diuretics was not associated with lower post-discharge mortality [adjusted hazard ratio (HR) 0.92, 95% confidence interval (CI) 0.68-1.25]. Of the 1759 patients who received loop diuretics on admission, loop diuretic dose was decreased in 23.8%, unchanged in 44.6%, and increased in 31.6% of the patients. Changes in the dose at discharge compared with no change in dose were not associated with lower risk of post-discharge mortality (decrease relative to no change: adjusted HR 0.98, 95% CI 0.76-1.28; increase relative to no change: adjusted HR 1.00, 95% CI 0.78-1.27). Compared with no loop diuretics at discharge, a loop diuretics dose of ≥80 mg at discharge was associated with higher post-discharge mortality risk. CONCLUSIONS: In patients with AHF, we found no association between the starting of loop diuretics and post-discharge outcomes and between dose changes and post-discharge outcomes.

The characteristics and outcomes in patients with acute heart failure who used tolvaptan: from KCHF registry.

AIMS: The use of tolvaptan is increasing in clinical practice in Japan. However, the characteristics of patients who used tolvaptan and the timing of its use in patients with acute heart failure (AHF) are not fully elucidated. METHODS AND RESULTS: Among consecutive 4056 patients in the Kyoto Congestive Heart Failure registry, we analysed 3802 patients after excluding patients on dialysis, prior or unknown tolvaptan use at admission, and unknown timing of tolvaptan use, and we divided them into two groups: tolvaptan use (N = 773) and no tolvaptan use (N = 3029). The prevalence of tolvaptan use varied widely from 48.7% to 0% across the participating centres. Factors independently associated with tolvaptan use were diabetes, poor medical adherence, oedema, pleural effusion, hyponatraemia, estimated glomerular filtration rate < 30 mL/min/1.73 m2 , moderate/severe tricuspid regurgitation, dobutamine infusion within 24 h, and additional inotropes infusion beyond 24 h after admission. The mortality rate at 90 days after admission was significantly higher in the tolvaptan use group than in the no tolvaptan use group (14.3% vs. 8.6%, P = 0.049). However, after adjustment, the excess mortality risk of tolvaptan use relative to no tolvaptan use was no longer significant (hazard ratio = 1.53, 95% confidence interval = 0.77-3.02, P = 0.22). Patients with tolvaptan use had a longer hospital stay [median (interquartile range): 22 (15-34) days vs. 15 (11-21) days, P < 0.0001] and a higher prevalence of worsening renal failure (47.0% vs. 31.8%, P < 0.0001) and worsening heart failure (24.8% vs. 14.4%, P < 0.0001) than those without. CONCLUSIONS: AHF patients with tolvaptan use had more congestive status with poorer in-hospital outcomes and higher short-term mortality than those without tolvaptan use. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02334891 (NCT02334891) and https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000017241 (UMIN000015238).

Tricuspid regurgitation in elderly patients with acute heart failure: insights from the KCHF registry.

AIMS: Several studies demonstrated that tricuspid regurgitation (TR) is associated with poor clinical outcomes. However, data on patients with TR who experienced acute heart failure (AHF) remains scarce. The purpose of this study is to evaluate the association between TR and clinical outcomes in patients admitted with AHF, using a large-scale Japanese AHF registry. METHODS AND RESULTS: The current study population consisted of 3735 hospitalized patients due to AHF in the Kyoto Congestive Heart Failure (KCHF) registry. TR grades were assessed according to the routine clinical practice at each participating centre. We compared the baseline characteristics and outcomes according to the severity of TR. The primary outcome was all-cause death. The secondary outcome was hospitalization for heart failure (HF). The median age of the entire study population was 80 (interquartile range: 72-86) years. One thousand two hundred five patients (32.3%) had no TR, while mild, moderate, and severe TR was found in 1537 patients (41.2%), 776 patients (20.8%), and 217 patients (5.8%), respectively. Pulmonary hypertension, significant mitral regurgitation, and atrial fibrillation/flutter were strongly associated with the development of moderate/severe of TR, while left ventricular ejection fraction <50% was inversely associated with it. Among 993 patients with moderate/severe TR, the number of patients who underwent surgical intervention for TR within 1 year was only 13 (1.3%). The median follow-up duration was 475 (interquartile range: 365-653) days with 94.0% follow-up at 1 year. As the TR severity increased, the cumulative 1 year incidence of all-cause death and HF admission proportionally increased ([14.8%, 20.3%, 23.4%, 27.0%] and [18.9%, 23.0%, 28.5%, 28.4%] in no, mild, moderate, and severe TR, respectively). Compared with no TR, the adjusted risks of patients with mild, moderate, and severe TR were significant for all-cause death (hazard ratio [95% confidence interval]: 1.20 [1.00-1.43], P = 0.0498, 1.32 [1.07-1.62], P = 0.009, and 1.35 [1.00-1.83], P = 0.049, respectively), while those were not significant for hospitalization for HF (hazard ratio [95% confidence interval]: 1.16 [0.97-1.38], P = 0.10, 1.19 [0.96-1.46], P = 0.11, and 1.20 [0.87-1.65], P = 0.27, respectively). The higher adjusted HRs of all the TR grades relative to no TR were significant for all-cause death in patients aged <80 years, but not in patients aged ≥80 years with significant interaction. CONCLUSIONS: In a large Japanese AHF population, the grades of TR could successfully stratify the risk of all-cause death. However, the association of TR with mortality was only modest and attenuated in patients aged 80 or more. Further research is warranted to evaluate how to follow up and manage TR in this elderly population.