Biology subjects

Nishi, M.

Publications and source records attributed to Nishi, M..

8 recordsLinked to original sources

bioRxiv2024

Phosphodiesterase 3 inhibitors boost bone outgrowth

C-type natriuretic peptide (CNP) stimulates skeletal growth by acting on the growth plates of long bones, and a CNP variant is clinically used for achondroplasia treatment. We previously reported that CNP stimulates the autonomic Ca2+ influx mediated by TRPM7 channels in growth plate chondrocytes to facilitate extracellular matrix synthesis for bone growth. Here, we report that phosphodiesterase 3 (PDE3) inhibitors facilitate the Ca2+ influx and promote bone outgrowth. The representative PDE3 inhibitor cilostazol elevated cGMP levels and activated cell-surface K+ channels probably due to protein kinase G-mediated phosphorylation in growth-plate chondrocytes. The resulting hyperpolarization likely facilitated TRPM7-mediated Ca2+ influx by increasing the Ca2+-driving force. Moreover, cilostazol simulated the elongation of cultured bones and enlarged the body size of juvenile mice. Several PDE3 inhibitors have been used for clinical treatment of thrombosis, heart failure and asthma, while our observations suggest that the repositioning of PDE3 inhibitors would provide novel medications for syndromes involving short stature.

pharmacology and toxicology↗

bioRxiv2024

Knock out of the intracellular calcium conducting ion channel Mitsugumin 23 (MG23) protects against pressure overload induced left ventricular hypertrophy and cardiac dysfunction.

BackgroundIn cardiac dysfunction, intracellular Ca2+-dynamics are disrupted leading to leakage of Ca2+ from the sarcoplasmic reticulum (SR). This results in diminished cardiac contractility and impaired cardiac function. In cardiac tissue, the underlying molecular mechanisms responsible for RyR2-independent Ca2+ leak are poorly understood. Mitsugumin 23 (MG23) is an intracellular Ca2+-conducting ion channel located on ER/SR and nuclear membranes. We propose that MG23 contributes to regulation of intracellular Ca2+-homeostasis, and that altered MG23 function may drive progression of cardiac dysfunction. The aim of this research was to investigate the role of MG23 in SR Ca2+ leak, and whether knock out of Mg23 protects the heart against pressure-overload induced left ventricular hypertrophy. MethodsCardiac pressure-overload was induced in wild type (WT) and Mg23-knock out (KO) mice through subcutaneous Angiotensin II (AngII, 1.1 mg/kg/day) infusion via osmotic pump. After 10-days infusion, in vivo pressure-volume dynamics were measured by insertion of a pressure-volume catheter into the left ventricle. MG23 protein expression was assessed through Western blot analysis. Ventricular fibrosis and cardiomyocyte size were measured using histological and immunofluorescence approaches. Cardiomyocytes were isolated from WT and Mg23-KO hearts and intracellular Ca2+ dynamics assessed through live cell imaging using the Ca2+ indicator Fluo-4. ResultsAngII-induced cardiac pressure-overload increased expression of MG23 in WT mouse hearts. Knock out of Mg23 protected hearts against AngII-induced cardiac hypertrophy. Compared to WT animals, AngII treated Mg23-KO mice displayed a significant reduction in left ventricular fibrosis and displayed normal cardiac functioning. In Mg23-KO hearts, no alteration in expression of key Ca2+ handling proteins was identified, but cardiomyocytes displayed altered Ca2+ spark profiles consistent with a role for MG23 in SR Ca2+ leak. ConclusionMG23 plays a key role in driving Ca2+ dysregulation observed in the early pathological stages of pressure-overload induced heart failure.

physiology↗

bioRxiv2024

Non-physiologic interaction of MG53 with insulin receptor and lack of evidence for MG53's role in controlling insulin-stimulated Akt phosphorylation in muscle, heart and liver tissues.

RationaleMG53s known function in facilitating tissue repair and anti-inflammation has broad applications to regenerative medicine. There is controversy regarding MG53s role in the development of type 2 diabetes mellitus (T2DM). ObjectiveThis study aims to address this controversy - whether MG53s myokine function contributes to inhibition of insulin signaling in muscle, heart, and liver tissues. Study DesignWe determined the binding affinity of the recombinant human MG53 (rhMG53) to the insulin receptor extracellular domain (IR-ECD) and found low affinity of interaction with Kd (>480 nM). Using cultured C2C12 myotubes and HepG2 cells, we found no effect of rhMG53 on insulin-stimulated Akt phosphorylation (p-Akt). We performed in vivo assay with C57BL/6J mice subjected to insulin stimulation (1 U/kg, intraperitoneal injection) and observed no effect of rhMG53 on insulin-stimulated p-Akt in muscle, heart and liver tissues. ConclusionOverall, our data suggest that rhMG53 can bind to the IR-ECD, however has a low likelihood of a physiologic role, as the Kd for binding is [~]10,000 higher than the physiologic level of MG53 present in the serum of rodents and humans ([~]10 pM). Our findings question the notion proposed by Xiao and colleagues - whether targeting circulating MG53 opens a new therapeutic avenue for T2DM and its complications.

physiology↗

bioRxiv2023

Atypical cell death in the growth plate chondrocytes of Tric-b-knockout mice

TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model. Here we report irregular cell death in proliferating growth plate chondrocytes in developing Tric-b-knockout bones. In the knockout chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, the PERK pathway was preferentially hyperactivated in the knockout chondrocytes, and most likely altered gene expression to induce apoptosis-related proteins including CHOP and caspase 12. In Ca2+ imaging experiments, the knockout chondrocytes exhibited aberrant Ca2+ handling; ER Ca2+ release was impaired, and intracellular Ca2+ concentration was elevated. Our data suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic stages by compromising cellular Ca2+-handling and exacerbating ER stress, leading to atypical apoptotic cell death.

developmental biology↗

bioRxiv2022

Analysis of splicing abnormalities in the white matter of myotonic dystrophy type 1 brain using RNA-sequence

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by the genomic expansions of CTG repeats, in which RNA-binding proteins, such as muscleblind-like protein, are sequestered in the nucleus, and abnormal splicing is observed in various genes. Although abnormal splicing reportedly occurs in the brains of patients with DM1, it is relation to the central nervous system symptoms is unknown. Several imaging studies have indicated substantial white matter (WM) defects in patients with DM1. Here, we performed RNA-sequencing and analysis of CTG repeat lengths in the frontal lobe of patients with DM1, separating the grey matter (GM) and WM, to investigate the splicing abnormalities in the DM1 brain, especially in the WM. The results demonstrated the number of repeats in the GM tended to be increase, with several genes showing similar levels of splicing abnormalities in the GM and WM, suggesting that the WM defects in DM1 are not only caused by aberrant splicing of GM RNA but also of WM RNA, which could be attributed to abnormal splicing of glial cell RNAs.

molecular biology↗

bioRxiv2022

A Checking Model of Obsessive-Compulsive Disorder Based on the Hypothalamic Defensive System Against a Potential Threat

BACKGROUNDObsessive-compulsive disorder (OCD) is a psychiatric disorder characterized by obsessive thoughts and compulsive behavior. While some theories imply that OCD patients have cognitive biases and dysfunctional motivation regarding a potential threat, these views are not adequately supported by neurological evidence. Hypothalamic perifornical (PeF) urocortin-3 (UCN3) neurons are involved in defensive responses to a potential threat, and the activation of these neurons in mice induces repetitive and excessive checking and burying of novel objects. In this study, we evaluated the hypothesis that mice in which PeF UCN3 neurons are activated can serve as an OCD model. METHODSPeF UCN3 neurons were chemogenetically activated with clozapine-N oxide (CNO) in Ucn3-Cre mice. Marble-burying activity, repetitive/stereotypic behaviors in the homecage, and excessive responses to a novel object were measured as OCD-like behaviors. The effects of clinically used drugs for treating OCD on these behaviors were evaluated. The effect of CNO on neural activity in the cortico-striato-thalamo-cortical loop (which is regarded as an OCD circuit) was assessed with c-Fos immunolabeling. RESULTSCNO increased marble-burying activity, evoked homecage-specific repetitive/stereotypic behaviors that probably aimed to seal entrances, and induced repetitive and excessive checking and burying of novel objects. These behaviors were suppressed by selective serotonin-reuptake inhibitors but not by diazepam. CNO increased neural activity in the cortico-striato-thalamo-cortical loop. CONCLUSIONSThese results indicated that mice whose PeF UCN3 neurons are activated can serve as a model of OCD, particularly as a checking model. This supports theories concerning the role of potential threats in the pathophysiology of OCD.

animal behavior and cognition↗

bioRxiv2021

C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide experimental evidence indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

cell biology↗

bioRxiv2019

Differences in splicing defects between the grey and white matter in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multi-system disorder caused by CTG repeats in the myotonic dystrophy protein kinase (DMPK) gene. This leads to sequestration of the splicing factor, muscleblind-like 2 (MBNL2), and aberrant splicing, mainly in the central nervous system. We investigated the splicing patterns of MBNL1/2 and genes controlled by MBNL2 in several regions of the brain and between the grey matter (GM) and white matter (WM) in DM1 patients using RT-PCR. Compared with the control, the percentage of spliced-in parameter (PSI) for most of the examined exons were significantly altered in most of the brain regions of DM1 patients, except for the cerebellum. The splicing of many genes was differently regulated between the GM and WM in both DM1 and control. The level of change in PSI between DM1 and control was higher in the GM than in the WM. The differences in alternative splicing between the GM and WM may be related to the effect of DM1 on the WM of the brain. We hypothesize that in DM1, aberrantly spliced isoforms in the neuronal cell body of the GM may not be transported to the axon. This might affect the WM as a consequence of Wallerian degeneration secondary to cell body damage. Our findings may have implications for analysis of the pathological mechanisms and exploring potential therapeutic targets.

molecular biology↗