Springer Science and Business Media LLCActa Medica Okayama2041-48891612025TRPV2 in muscle satellite cells is crucial for skeletal muscle remodelling888ENYanzhuChenDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKimiakiKatanosakaDepartment of Biomedical Sciences, College of Life and Health Sciences, Chubu UniversityMakotoShibuyaDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYubingDongDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityLidanZhangLaboratory of Stem Cell Regeneration and Adaptation, Graduate School of Pharmaceutical Sciences, The University of OsakaMotoiKanagawaDepartment of Cell Biology and Molecular Medicine, Ehime University Graduate School of MedicineSo-ichiroFukadaLaboratory of Stem Cell Regeneration and Adaptation, Graduate School of Pharmaceutical Sciences, The University of OsakaKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYukiKatanosakaDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversitySkeletal muscle remodelling relies on muscle stem cells (MuSCs) for regeneration after injury and hypertrophy in response to mechanical loading. However, the mechanisms that trigger MuSC activation and proliferation remain unclear. Transient receptor potential vanilloid 2 (TRPV2) ion channels respond to insulin-like growth factor-1 and mechanical stimuli to regulate the biological characteristics of various cells. Using a temporally inducible MuSC-specific conditional knockout (cKO) mouse, we show that TRPV2 regulates MuSC function and is essential for muscle remodelling. In cultured myofibre, MuSCs express TRPV2 and exhibit Ca2+ responses to the TRPV2 agonists 2-aminoethoxydiphenyl borate and probenecid, which are abolished upon TRPV2 deletion. TRPV2-deficient MuSCs exhibit reduced paired box 7 (Pax7) expression and impaired proliferation, suggesting TRPV2 is a factor that regulates the early stage of MuSC function. Myotube formation in MuSCs was enhanced by overexpression of TRPV2 and suppressed by TRPV2 deficiency, suggesting that TRPV2 is a factor that promotes myogenesis. Muscle-administered cardiotoxin promoted muscle regeneration and resulted in the appearance of numerous Pax7-positive MuSCs between myofibres. MuSC-specific TRPV2 cKO mice exhibit substantially impaired muscle regeneration after cardiotoxin-induced injury, drastically reducing Pax7-positive MuSCs between myofibres. In floxed mice, mechanical loading via synergist ablation induces hypertrophy and greatly increases the number of myonuclei per myofibre. In contrast, MuSC-specific TRPV2 cKO mice show no changes in myofibre thickness or nuclear number, either at baseline or after mechanical loading. Mechanical loading of floxed mice increased TRPV2+/Pax7+ double-positive MuSCs, but MuSC-specific TRPV2 cKO mice showed no change. Additionally, MuSCs exhibit Ca2+ responses to hypo-osmotic stimuli, which are suppressed by TRPV2 inhibitors and TRPV2 deletion, suggesting that MuSCs exhibit TRPV2-dependent mechanical responses. These results establish TRPV2 as a critical regulator of MuSC-mediated muscle remodelling, an important finding that may lead to therapeutic strategies for muscle repair and adaptation.No potential conflict of interest relevant to this article was reported.MDPI AGActa Medica Okayama1873-149X3312026Bridging the Gap Between Static Histology and Dynamic Organ-on-a-Chip Models10ENZheyiWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityFor more than a century, pathology has served as a cornerstone of modern medicine, relying primarily on static microscopic assessment of tissue morphology―such as H&E staining―which remains the “gold standard” for disease diagnosis. However, this conventional paradigm provides only a snapshot of disease states and often fails to capture their dynamic evolution and complex functional mechanisms. Moreover, animal models are constrained by marked interspecies differences, creating a persistent gap in translational research. To overcome these limitations, we propose the concept of New Pathophysiology, a research framework that transcends purely morphological descriptions and aims to resolve functional dynamics in real time. This approach integrates Organ-on-a-Chip (OOC) technology, multi-omics analyses, and artificial intelligence to reconstruct the entire course of disease initiation and to enable personalized medicine. In this review, we first outline the foundations and limitations of traditional pathology and animal models. We then systematically summarize more than one hundred existing OOC disease models across multiple organs―including the kidney, liver, and brain. Finally, we elaborate on how OOC technologies are reshaping the study of key pathological processes such as inflammation, metabolic dysregulation, and fibrosis by converting them into dynamic, mechanistic disease models, and we propose future perspectives in the field. This review adopts a relatively uncommon classification strategy based on pathological mechanisms (mechanism-based), rather than organ-based categorization, allowing readers to recognize shared principles underlying different diseases. Moreover, the focus of this work is not on emphasizing iteration or replacement of existing approaches, but on preserving past achievements from a historical perspective, with an emphasis on overcoming current limitations and enabling new advances.No potential conflict of interest relevant to this article was reported.WileyActa Medica Okayama2211-546315102025Osmotic pressure‐induced calcium response states17141722ENZidanGaoDepartment of Cardiovascular Physiology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeijiNaruseFaculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Okayama JapanMasatoshiMorimatsuFaculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Okayama JapanOsmotic pressure is essential for maintaining cellular homeostasis; however, the mechanisms by which cells sense and respond to acute osmotic stress remain incompletely understood. Here, we applied rapid osmotic pressure stimulation to cultured HEK293T cells and observed dynamic intracellular calcium responses. Acute hypotonic stimulation evoked calcium response patterns, whereas hypertonic and isotonic stress did not elicit similar effects. Mechanistically, these calcium signals originated from the endoplasmic reticulum via ryanodine receptor 2 and propagated to neighboring cells through Connexin 43-mediated gap junctions. These findings reveal a previously unrecognized role for calcium signaling in the acute cellular response to osmotic stress, providing new insights into the mechanisms of intercellular communication during osmotic adaptation.No potential conflict of interest relevant to this article was reported.Springer Science and Business Media LLCActa Medica Okayama1757-65121612025Specific induction of right ventricular-like cardiomyocytes from human pluripotent stem cells519ENYukihiroSaitoDepartment of Cardiovascular Medicine, Okayama University HospitalKazufumiNakamuraDepartment of Cardiovascular Medicine, Okayama University HospitalYukiKatanosakaDepartment of Cardiovascular Physiology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesToshihiroIidaDepartment of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesDaiKusumotoDepartment of Biomedical Informatics and Molecular Biology, The Sakaguchi Laboratory, Keio University School of MedicineRyushiSatoDepartment of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of ShizuokaRikiAdachiDepartment of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of ShizuokaSatoshiShimizuDepartment of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of ShizuokaJunkoKurokawaDepartment of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of ShizuokaSatoshiAkagiDepartment of Cardiovascular Medicine, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesMasashiYoshidaDepartment of Chronic Kidney Disease and Cardiovascular Disease, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesToruMiyoshiDepartment of Cardiovascular Medicine, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesHiroshiMoritaDepartment of Cardiovascular Therapeutics, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesKeijiNaruseDepartment of Cardiovascular Physiology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesMikakoNishidaDepartment of Metabolic Immune Regulation, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesHeiichiroUdonoDepartment of Metabolic Immune Regulation, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesJianhuaZhangDepartment of Medicine, University of Wisconsin School of Medicine and Public HealthShinsukeYuasaDepartment of Cardiovascular Medicine, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesTimothy J.KampDepartment of Medicine, University of Wisconsin School of Medicine and Public HealthHiroshiItoDepartment of Cardiovascular Medicine, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesBackground Applications employing human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require well-characterized, chamber-specific hPSC-CMs. Distinct first heart field (FHF) and second heart field (SHF) cardiac progenitor populations give rise to the left ventricular (LV) and right ventricular (RV) cardiomyocytes, respectively. This developmental difference in cardiomyocyte origin suggests that chamber-specific cardiomyocytes have unique characteristics. Therefore, efficient strategies to differentiate human pluripotent stem cells (hPSCs) specifically to LV-like or RV-like cardiomyocytes are needed and it is still unknown whether there is a phenotypic difference between LV-like cardiomyocytes and RV-like cardiomyocytes derived from hPSCs.<br>
Methods An established hPSC cardiac differentiation protocol employing sequential GSK3β inhibition followed by Wnt inhibition (GiWi) was modified by addition of insulin or BMP antagonists during mesoderm formation. Cardiac progenitor populations were evaluated for FHF and SHF markers, and differentiated hPSC-CMs were characterized for chamber-specific markers.<br>
Results The GiWi protocol produced mainly FHF-like progenitor cells that gave rise to LV-like cardiomyocytes. Inhibition of endogenous BMP signaling during mesoderm induction using insulin or BMP antagonists reduced expression of FHF markers and increased expression of SHF markers in cardiac progenitor cells. hPSC-CMs arising from the SHF-like progenitor cells showed an RV-like gene expression pattern and exhibited phenotypic differences in spontaneous contraction rate, Ca2+ transients, and cell size compared to control LV-like cardiomyocytes.<br>
Conclusion This study establishes methodology to generate RV-like hPSC-CMs to support the development of disease modeling research using chamber-specific hPSC-CMs.No potential conflict of interest relevant to this article was reported.American Institute of Mathematical Sciences (AIMS)Acta Medica Okayama2377-90981232025Biophysical regulation of extracellular matrix in systemic lupus erythematosus412437ENQiweiLiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityQiangLiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityZhaoyangXiaoDepartment of Anesthesiology, The Second Affiliated Hospital of Dalian Medical UniversityKeijiNARUSEDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversitySystemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by immune dysregulation and multi-organ damage. Recent advances have underscored the critical involvement of extracellular matrix (ECM) biophysical properties in shaping immune cell behavior and metabolic states that contribute to disease progression. This review systematically delineates the pathological remodeling of ECM biophysics in SLE, with a focus on their roles in mechanotransduction, immune-metabolic interplay, and organ-specific tissue injury. By integrating current evidence, we highlight how ECM-derived mechanical cues orchestrate aberrant immune responses and propose new perspectives for targeting ECM-immune crosstalk in the development of organ-specific, mechanism-based therapies for SLE.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155813722025第130回日本解剖学会／第102回日本生理学会／第98回日本薬理学会合同大会8081ENKeijiNaruseDepartment of Cardiovascular Physiology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityNo potential conflict of interest relevant to this article was reported.Springer Science and Business Media LLCActa Medica Okayama2045-23221512025Effect of mechanical stretching stimulation on maturation of human iPS cell-derived cardiomyocytes co-cultured with human gingival fibroblasts30648ENMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHarumiIdeiDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChenWangDepartment of Nursing, School of Life and Health Sciences, HuZhou CollegeYinLiangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYunLiuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYusukeMatsudaDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHiroshiKamiokaDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityIn the realm of regenerative medicine, despite the various techniques available for inducing the differentiation of induced pluripotent stem (iPS) cells into cardiomyocytes, there remains a need to enhance the maturation of the cardiomyocytes. This study aimed to improve the differentiation and subsequent maturation of iPS-derived cardiomyocytes (iPS-CMs) by incorporating mechanical stretching. Human iPS cells were co-cultured with human gingival fibroblasts (HGF) on a polydimethylsiloxane (PDMS) stretch chamber, where mechanical stretching stimulation was applied during the induction of cardiomyocyte differentiation. The maturation of iPS-CMs was assessed using qRT-PCR, immunocytochemistry, transmission electron microscopy, calcium imaging and contractility comparisons. Results indicated significantly elevated gene expression levels of cardiomyocyte markers (cTnT) and the mesodermal marker (Nkx2.5) in the stretch group compared to the control group. Fluorescent immunocytochemical staining revealed the presence of cardiac marker proteins (cTnT and MYL2) in both groups, with higher protein expression in the stretch group. Additionally, structural maturation of iPS-CMs in the stretch group was notably better than in the control group. A significant increase in the contractility and calcium cycle of iPS-CMs was observed in the stretch group. These findings demonstrate that mechanical stretching stimulation enhances the maturation of iPS-CMs co-cultured with HGF.No potential conflict of interest relevant to this article was reported.Springer Science and Business Media LLCActa Medica Okayama2045-23221512025Effect of mechanical stretching stimulation on maturation of human iPS cell-derived cardiomyocytes co-cultured with human gingival fibroblasts30648ENMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHarumiIdeiDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChenWangDepartment of Nursing, School of Life and Health Sciences, HuZhou CollegeYinLiangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYunLiuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYusukeMatsudaDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHiroshiKamiokaDepartment of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityIn the realm of regenerative medicine, despite the various techniques available for inducing the differentiation of induced pluripotent stem (iPS) cells into cardiomyocytes, there remains a need to enhance the maturation of the cardiomyocytes. This study aimed to improve the differentiation and subsequent maturation of iPS-derived cardiomyocytes (iPS-CMs) by incorporating mechanical stretching. Human iPS cells were co-cultured with human gingival fibroblasts (HGF) on a polydimethylsiloxane (PDMS) stretch chamber, where mechanical stretching stimulation was applied during the induction of cardiomyocyte differentiation. The maturation of iPS-CMs was assessed using qRT-PCR, immunocytochemistry, transmission electron microscopy, calcium imaging and contractility comparisons. Results indicated significantly elevated gene expression levels of cardiomyocyte markers (cTnT) and the mesodermal marker (Nkx2.5) in the stretch group compared to the control group. Fluorescent immunocytochemical staining revealed the presence of cardiac marker proteins (cTnT and MYL2) in both groups, with higher protein expression in the stretch group. Additionally, structural maturation of iPS-CMs in the stretch group was notably better than in the control group. A significant increase in the contractility and calcium cycle of iPS-CMs was observed in the stretch group. These findings demonstrate that mechanical stretching stimulation enhances the maturation of iPS-CMs co-cultured with HGF.No potential conflict of interest relevant to this article was reported.Nature PortfolioActa Medica Okayama2399-3642812025TRPV2 mediates stress resilience in mouse cardiomyocytes715ENYubingDongDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityGuohaoWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYoshihiroUjiharaDepartment of Electrical and Mechanical Engineering, Graduate School of Engineering, Nagoya Institute of TechnologyYanzhuChenDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMasashiYoshidaDepartment of Chronic Kidney Disease and Cardiovascular Disease, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesKazufumiNakamuraDepartment of Cardiovascular Medicine, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical SciencesKimiakiKatanosakaDepartment of Biomedical Sciences, College of Life and Health Sciences, Chubu UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYukiKatanosakaDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityThe heart dynamically compensates for haemodynamic stress, but how this resilience forms during cardiac growth is not clear. Using a temporally inducible, cardiac-specific knockout in mice we show that the Transient receptor potential vanilloid family 2 (TRPV2) channel is crucial for the maturation of cardiomyocyte stress resilience. TRPV2 defects in growing hearts lead to small morphology, abnormal intercalated discs, weak contractility, and low expression of serum response factor and Insulin-like growth factor-1 (IGF-1) signalling. Individual cardiomyocytes of TRPV2-deficient hearts show reduced contractility with abnormal Ca2+ handling. In cultured neonatal cardiomyocytes, mechanical Ca2+ response, excitation-contraction coupling, sarcoplasmic reticulum Ca2+ content, actin formation, nuclear localisation of Myocyte enhancer factor 2c, and IGF-1 expression require TRPV2. TRPV2-deficient hearts show a defective response to dobutamine stress and no compensatory hypertrophic response to phenylephrine administration, but no stress response to pressure overload. These data suggest TRPV2 mediates the maturation of cardiomyocyte stress resilience, and will advance therapeutic interventions and drug discovery for heart disease.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama2073-440913162024Direct Binding of Synaptopodin 2-Like Protein to Alpha-Actinin Contributes to Actin Bundle Formation in Cardiomyocytes1373ENHiroshiYamadaDepartment of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHironaOsakaGraduate School of Science, Nagoya UniversityNanamiTatsumiDepartment of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMiuArakiDepartment of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTadashiAbeDepartment of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeikoKaiharaDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityEizoTakashimaDivision of Malaria Research, Proteo-Science Center, Ehime UniversityTakayukiUchihashiGraduate School of Science, Nagoya UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKohjiTakeiDepartment of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversitySynaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric alpha-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with alpha-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of alpha-actinin and enhanced the alpha-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to alpha-actinin via its globular ends. The interaction between alpha-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with alpha-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.No potential conflict of interest relevant to this article was reported.Nature PortfolioActa Medica Okayama2045-23221412024Human heart-on-a-chip microphysiological system comprising endothelial cells, fibroblasts, and iPSC-derived cardiomyocytes18063ENYunLiuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityRumaisaKamranDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityXiaoxiaHanDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityQiangLiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityDaoyueLaiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityIn recent years, research on organ-on-a-chip technology has been flourishing, particularly for drug screening and disease model development. Fibroblasts and vascular endothelial cells engage in crosstalk through paracrine signaling and direct cell-cell contact, which is essential for the normal development and function of the heart. Therefore, to faithfully recapitulate cardiac function, it is imperative to incorporate fibroblasts and vascular endothelial cells into a heart-on-a-chip model. Here, we report the development of a human heart-on-a-chip composed of induced pluripotent stem cell (iPSC)-derived cardiomyocytes, fibroblasts, and vascular endothelial cells. Vascular endothelial cells cultured on microfluidic channels responded to the flow of culture medium mimicking blood flow by orienting themselves parallel to the flow direction, akin to in vivo vascular alignment in response to blood flow. Furthermore, the flow of culture medium promoted integrity among vascular endothelial cells, as evidenced by CD31 staining and lower apparent permeability. The tri-culture condition of iPSC-derived cardiomyocytes, fibroblasts, and vascular endothelial cells resulted in higher expression of the ventricular cardiomyocyte marker IRX4 and increased contractility compared to the bi-culture condition with iPSC-derived cardiomyocytes and fibroblasts alone. Such tri-culture-derived cardiac tissues exhibited cardiac responses similar to in vivo hearts, including an increase in heart rate upon noradrenaline administration. In summary, we have achieved the development of a heart-on-a-chip composed of cardiomyocytes, fibroblasts, and vascular endothelial cells that mimics in vivo cardiac behavior.No potential conflict of interest relevant to this article was reported.Elsevier BVActa Medica Okayama0006-3495121172022High hydrostatic pressure induces slow contraction in mouse cardiomyocytes32863294ENYoheiYamaguchiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMasayoshiNishiyamaDepartment of Physics, Faculty of Science and Engineering, Kindai UniversityHiroakiKaiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityToshiyukiKanekoDepartment of Physiology, Asahikawa Medical UniversityKeikoKaiharaDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityGentaroIribeDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityAkiraTakaiDepartment of Physiology, Asahikawa Medical UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMasatoshiMorimatsuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityCardiomyocytes are contractile cells that regulate heart contraction. Ca2+ flux via Ca2+ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca2+]i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca2+]i.No potential conflict of interest relevant to this article was reported.ELSEVIERActa Medica Okayama2215-016182021Production of TRPM4 knockout cell line using rat cardiomyocyte H9c2101404ENChenWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesMasakazuMaedaDepartment of Medicine, Okayama UniversityJianChenDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesThe method presented in this article are related to the research article entitled as "Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress" [1]. TRPM4, a non-selective monovalent cation channel, is not only involved in the generation of the action potential in cardiomyocytes, but also thought to be a key molecule in the development of the ischemia-reperfusion injury of the brain and the heart [2-5]. However, existing pharmacological inhibitors for the TRPM4 channel have problems of non-specificity [6]. This article describes methods used for targeted genomic deletion in the rat cardiomyocyte H9c2 using the CRISPR-Cas9 genome editing system in order to suppress TRPM4 protein expression. Confocal microscopy, flow cytometry, Sanger sequencing, and western blotting are performed to confirm vector transfection and the subsequent knockout of the TRPM4 protein. These data provide information on the comprehensive analyses for knocking out the rat TRPM4 channel using CRISPR/Cas9. The analyses include confocal microscopy, flow cytometry, Sanger sequencing, and western blotting. This dataset will benefit biological and medical researchers studying the function of TRPM4-expressing cells including neurons, cardiomyocytes, and vascular endothelial cells. It is also useful to study the involvement of the TRPM4 channel in pathological processes such as cardiac arrhythmia and ischemia-reperfusion injury. The dataset can be used to guide the experiment of knocking out the TRPM4 gene and its subsequent application to the study of disease process caused by the gene. No potential conflict of interest relevant to this article was reported.Frontiers Media SAActa Medica Okayama2296-634X92021Meta-Analysis-Assisted Detection of Gravity-Sensitive Genes in Human Vascular Endothelial Cells689662ENYinLiangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYunLiuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChenWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityGravity affects the function and maintenance of organs, such as bones, muscles, and the heart. Several studies have used DNA microarrays to identify genes with altered expressions in response to gravity. However, it is technically challenging to combine the results from various microarray datasets because of their different data structures. We hypothesized that it is possible to identify common changes in gene expression from the DNA microarray datasets obtained under various conditions and methods. In this study, we grouped homologous genes to perform a meta-analysis of multiple vascular endothelial cell and skeletal muscle datasets. According to the t-distributed stochastic neighbor embedding (t-SNE) analysis, the changes in the gene expression pattern in vascular endothelial cells formed specific clusters. We also identified candidate genes in endothelial cells that responded to gravity. Further, we exposed human umbilical vein endothelial cells (HUVEC) to simulated microgravity (SMG) using a clinostat and measured the expression levels of the candidate genes. Gene expression analysis using qRT-PCR revealed that the expression level of the prostaglandin (PG) transporter gene SLCO2A1 decreased in response to microgravity, consistent with the meta-analysis of microarray datasets. Furthermore, the direction of gravity affected the expression level of SLCO2A1, buttressing the finding that its expression was affected by gravity. These results suggest that a meta-analysis of DNA microarray datasets may help identify new target genes previously overlooked in individual microarray analyses.No potential conflict of interest relevant to this article was reported.Elsevier BVActa Medica Okayama0006-291X5662021Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress190196ENChenWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityJianChenDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityIschemic heart disease is one of the most common causes of death worldwide. Mitochondrial
dysfunction, excessive reactive oxygen species (ROS) generation, and calcium (Ca2þ) overload are three key factors leading to myocardial death during ischemia-reperfusion (I/R) injury. Inhibition of TRPM4, a Ca2þ-activated nonselective cation channel, protects the rat heart from I/R injury, but the specific mechanism underlying this effect is unclear. In this study, we investigated the mechanism of cardioprotection against I/R injury via TRPM4 using hydrogen peroxide (H2O2), a major contributor to oxidative stress, as an I/R injury model. We knocked out the TRPM4 gene in the rat cardiomyocyte cell line H9c2 using CRISPR/Cas9. Upon H2O2 treatment, intracellular Ca2þ level and ROS production increased in wild type (WT) cells but not in TRPM4 knockout (TRPM4KO) cells. With this treatment, two indicators of mitochondrial function, mitochondrial membrane potential (DJm) and intracellular ATP levels, decreased inWT but not in TRPM4KO cells. Taken together, these findings suggest that blockade of the TRPM4 channel might protect the myocardium from oxidative stress by maintaining the mitochondrial membrane potential and intracellular ATP levels, possibly through preventing aberrant increases in intracellular Ca2þ and ROS.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1422-00672242021Treatment of Oxidative Stress with Exosomes in Myocardial Ischemia1729ENYunLiuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityYinLiangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityChenWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityA thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia-reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS.No potential conflict of interest relevant to this article was reported.NatureActa Medica Okayama2373-806572021Gravity sensing in plant and animal cells2ENKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHideyukiTakahashiGraduate School of Life Sciences, Tohoku UniversityTakuyaFuruichiFaculty of Human Life Sciences, Hagoromo University of International StudiesMasatsuguToyotaDepartment of Biochemistry and Molecular Biology, Saitama UniversityMakotoFurutani-SeikiDepartment of Systems Biochemistry in Regeneration and Pathology, Graduate School of Medicine, Yamaguchi UniversityTakeshiKobayashiDepartment of Integrative Physiology, Graduate School of Medicine, Nagoya UniversityHarukoWatanabe-TakanoDepartment of Cell Biology, National Cerebral and Cardiovascular Center Research InstituteMasahiroShinoharaDepartment of Rehabilitation for the Movement Functions, Research Institute, National Rehabilitation Center for Persons with DisabilitiesTakuroNumaga-TomitaDepartment of Molecular Pharmacology, Shinshu University School of MedicineAsakoSakaue-SawanoLab for Cell Function and Dynamics, CBS, RIKENAtsushiMiyawakiLab for Cell Function and Dynamics, CBS, RIKENKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityGravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space.No potential conflict of interest relevant to this article was reported.MDPIActa Medica Okayama1422-006721242020The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors9352ENManh TienTranDepartment of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYukaOkushaDepartment of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYunxiaFengDepartment of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMasatoshiMorimatsuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityPenggongWeiDepartment of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChiharuSogawaDepartment of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTakanoriEguchiDepartment of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTomokoKadowakiDepartment of Frontier Oral Science, Graduate School of Biomedical Sciences, Nagasaki UniversityEikoSakaiDepartment of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki UniversityHirohikoOkamuraDepartment of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTakayukiTsukubaDepartment of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki UniversityKuniakiOkamotoDepartment of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityRab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155813222020第59回日本生体医工学会大会開催報告110111ENKeijiNaruseDepartment of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesNo potential conflict of interest relevant to this article was reported.Journal of Visualized ExperimentsActa Medica Okayama1940-087X1592020Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytese61104ENYunLiuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYinLiangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMengxueWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChenWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University HengWeiInstitute of Laboratory Animals, Graduate School of Medicine, Kyoto UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityIschemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.No potential conflict of interest relevant to this article was reported.Frontiers MediaActa Medica Okayama2296-634X82020In vitroNeo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System307ENKensukeKataokaDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityRyotaKurimotoDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityHirokiTsutsumiDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityTomokiChibaDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityTomomiKatoDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityKanaShishidoDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityMarikoKatoDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityYoshiakiItoDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityYuichiroChoAnatomy and Physiological Science, Tokyo Medical and Dental UniversityOsamuHoshiAnatomy and Physiological Science, Tokyo Medical and Dental UniversityAyakoMimataResearch Core, Tokyo Medical and Dental UniversityYurikoSakamakiResearch Core, Tokyo Medical and Dental UniversityRyoNakamichiDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityMartin K.LotzDepartment of Molecular Medicine, The Scripps Research InstituteKeijiNaruseGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHiroshiAsaharaDepartment of Systems BioMedicine, Tokyo Medical and Dental UniversityTendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stableMkxexpression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed thatMkxexpression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.No potential conflict of interest relevant to this article was reported.Nature Publishing GroupActa Medica Okayama2041-1723102019Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure5754ENYoshihiroUjiharaDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityMotoiKanagawaDivision of Neurology/Molecular Brain Science, Kobe University Graduate School of MedicineSatoshiMohriDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversitySatomiTakatsuDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKazuhiroKobayashiDivision of Neurology/Molecular Brain Science, Kobe University Graduate School of MedicineTatsushiTodaDivision of Neurology/Molecular Brain Science, Kobe University Graduate School of MedicineKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYukiKatanosakaDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityHeart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced alpha-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.No potential conflict of interest relevant to this article was reported.Academic PressActa Medica Okayama0006291X52032019Development of a model of ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells600605ENHengWeiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityChenWangDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityRuiGuoDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKenTakahashiDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityKeijiNaruseDepartment of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityIschemic heart disease remains the largest cause of death worldwide. Accordingly, many researchers have sought curative options, often using laboratory animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart. In this study, we developed a model of ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs). After optimizing the conditions of ischemia, including the concentration of oxygen and duration of application, we evaluated the consequent damage to hiPS-CMs. Notably, exposure to 2% oxygen, 0 mg/ml glucose, and 0% fetal bovine serum increased the percentage of nuclei stained with propidium iodide, an indicator of membrane damage, and decreased cellular viability. These conditions also decreased the contractility of hiPS-CMs. Furthermore, ischemic conditioning increased the mRNA expression of IL-8, consistent with observed conditions in the in vivo heart. Taken together, these findings suggest that our hiPS-CM-based model can provide a useful platform for human ischemic heart disease research.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0014482738322019Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1111556ENTakashiOhtsukiDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityAkiraShinaokaDepartment of Human Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKanaeKumagishi-ShinaokaDepartment of Human Morphology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiichiAsanoDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOmer FarukHatipogluDepartment of Medical Technology, Graduate School of Health Sciences, Okayama UniversityJunkoInagakiDepartment of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKenTakahashiDepartment of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesToshitakaOohashiDepartment of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeiichiroNishidaDepartment of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesKeijiNaruseDepartment of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesSatoshiHirohataDepartment of Medical Technology, Graduate School of Health Sciences, Okayama University The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.No potential conflict of interest relevant to this article was reported.Nature Publishing GroupActa Medica Okayama2041-172352014TRPV2 is critical for the maintenance of cardiac structure and function in mice3932ENYukiKatanosakaKeiichiroIwasakiYoshihiroUjiharaSatomiTakatsuKokiNishitsujiMotoiKanagawaAtsushiSudoTatsushiTodaKimiakiKatanosakaSatoshiMohriKeijiNaruseThe heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca2+ handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca2+-dependent intracellular Ca2+ increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function.No potential conflict of interest relevant to this article was reported.Public Library ScienceActa Medica Okayama1932-6203972014Directed Differentiation of Patient-Specific Induced Pluripotent Stem Cells Identifies the Transcriptional Repression and Epigenetic Modification of NKX2-5, HAND1, and NOTCH1 in Hypoplastic Left Heart SyndromeENJunkoKobayashiMasashiYoshidaSuguruTaruiMasatakaHirataYusukeNagaiShingoKasaharaKeijiNaruseHiroshiItoShunjiSanoHidemasaOhThe genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.No potential conflict of interest relevant to this article was reported.Public Library ScienceActa Medica Okayama1932-6203972014The Neutral Self-Assembling Peptide Hydrogel SPG-178 as a Topical Hemostatic AgentENSeijiKomatsuYusukeNagaiKeijiNaruseYoshihiroKimataConventional self-assembling peptide hydrogels are effective as topical hemostatic agents. However, there is a possibility to harm living tissues due to their low pH. The aim of the present study was to demonstrate the efficacy of SPG-178, a neutral self-assembling peptide hydrogel, as a topical hemostatic agent. First, we measured the bleeding duration of incisions made on rat livers after application of SPG-178 (1.0% w/v), SPG-178 (1.5% w/v), RADA16 (1.0% w/v), and saline (n = 12/group). Second, we observed the bleeding surfaces by transmission electron microscopy immediately after hemostasis. Third, we measured the elastic and viscous responses (G′ and G″, respectively) of the hydrogels using a rheometer. Our results showed that bleeding duration was significantly shorter in the SPG-178 group than in the RADA16 group and that there were no significant differences in transmission electron microscopy findings between the groups. The greater the G′ value of a hydrogel, the shorter was the bleeding duration. We concluded that SPG-178 is more effective and has several advantages: it is non-biological, transparent, nonadherent, and neutral and can be sterilized by autoclaving.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812612014自己集合性ペプチドハイドロゲル内で三次元培養された細胞への機械刺激710ENYusukeNagaiHidenoriYokoiKeikoKaiharaKeijiNaruseNo potential conflict of interest relevant to this article was reported.InTechActa Medica Okayama2012Use of Silicone Elastomer-Based Microfluidic Devices and Systems in Reproductive Technologies243262ENKojiMatsuuraKeijiNaruseNo potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama1472-64832412012Screening of sperm velocity by fluid mechanical characteristics of a cyclo-olefin polymer microfluidic sperm-sorting device109115ENKojiMatsuuraMamiTakenamiYukaKurodaToruHyakutakeShinichiroYanaseKeijiNaruseThe microfluidic sperm-sorting (MFSS) device is a promising advancement for assisted reproductive technology. Previously, poly(dimethylsiloxiane) and quartz MFSS devices were developed and used for intracytoplasmic sperm injection. However, these disposable devices were not clinically suitable for assisted reproduction, so a cyclo-olefin polymer MFSS (COP-MFSS) device was developed. By micromachining, two microfluidic channels with different heights and widths (chip A: 0.3 x 0.5 mm; chip B: 0.1 x 0.6 mm) were prepared. Sorted sperm concentrations were similar in both microfluidic channels. Linear-velocity distribution using the microfluidic channel of chip B was higher than that of chip A. Using confocal fluorescence microscopy, it was found that the highest number of motile spermatozoa swam across the laminar flow at the bottom of the microfluidic channel. The time required to swim across the laminar flow was longer at the bottom and top of the microfluidic channels than in the middle because of the low fluid velocity. These results experimentally demonstrated that the width of microfluidic channels should be increased in the region of laminar flow from the semen inlet to the outlet for unsorted spermatozoa to selectively recover spermatozoa with high linear velocity.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0015-02829432010Blastocyst quality scoring based on morphologic grading correlates with cell number11351137ENKojiMatsuuraNobuyoshiHayashiChisatoTakiueReiHirataToshihiroHabaraKeijiNaruseBlastocyst quality score (BQS), first reported by Rehman et al., is a numerical blastocyst-morphology grading system based on the criteria established by Gardner and Schoolcraft. We demonstrate a positive correlation between the calculated BQS score and cell number by staining thawed human embryos and suggest that BQS can be applied to evaluate culture systems clinically.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama0093-691X7452010Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration863870ENHikaruSanoKojiMatsuuraKeijiNaruseHiroakiFunahashiThe objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 x 10(8) with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 +/- 0.5 x 10(4) to 575.0 +/- 56.3 x 10(4) cells/mL; mean +/- SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 +/- 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 +/- 4.8%) than those at 7.5 mm (53.1 +/- 6.0%) or further (41.9 +/- 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 +/- 0.040) than both standard IVF and transient IVF (0.222 +/- 0.028 and 0.189 +/- 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 +/- 2.3%) than in either standard or transient IVF (22.6 +/- 1.4 and 33.7 +/- 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.No potential conflict of interest relevant to this article was reported.Society for Reproduction and DevelopmentActa Medica Okayama0916-88185652010In-vitro Culture with a Tilting Device in Chemically Defined Media During Meiotic Maturation and Early Development Improves the Quality of Blastocysts Derived from In-vitro Matured and Fertilized Porcine Oocytes552557ENTakayukiKoikeKojiMatsuuraKeijiNaruseHiroakiFunahashiUnder physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P<0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence.No potential conflict of interest relevant to this article was reported.Springer USActa Medica Okayama1387-21761112009Application of a numerical simulation to improve the separation efficiency of a sperm sorter2533ENToruHyakutakeYukiHashimotoShinichiroYanaseKojiMatsuuraKeijiNaruseThis paper describes a study in which numerical simulations were applied to improve the separation efficiency of a microfluidic-based sperm sorter. Initially, the motion of 31 sperm were modeled as a sinusoidal wave. The modeled sperm were expected to move while vibrating in the fluid within the microchannel. In this analysis, the number of sperm extracted at the outlet channel and the rate of movement of the highly motile sperm were obtained for a wide range of flow velocities within the microchannel. By varying the channel height, and the width and the position of the sperm-inlet channel, we confirmed that the separation efficiency was highly dependent on the fluid velocity within the channel. These results will be valuable for improving the device configuration, and might help to realize further improvements in efficiency in the future.No potential conflict of interest relevant to this article was reported.The Royal Society of ChemistryActa Medica Okayama1473-01979222009Fabricating small-scale, curved, polymeric structures with convex and concave menisci through interfacial free energy equilibrium33063309ENChao-MinChengKojiMatsuuraI-JanWangYukaKurodaPhilip R.LeDucKeijiNarusePolymeric curved structures are widely used in imaging systems including optical fibers and microfluidic channels. Here, we demonstrate that small-scale, poly(dimethylsiloxane) (PDMS)-based, curved structures can be fabricated through controlling interfacial free energy equilibrium. Resultant structures have a smooth, symmetric, curved surface, and may be convex or concave in form based on surface tension balance. Their curvatures are controlled by surface characteristics (i.e., hydrophobicity and hydrophilicity) of the molds and semi-liquid PDMS. In addition, these structures are shown to be biocompatible for cell culture. Our system provides a simple, efficient and economical method for generating integrateable optical components without costly fabrication facilities.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama1472-64832032010Improved development of mouse and human embryos using a tilting embryo culture system358364ENKojiMatsuuraNobuyoshiHayashiYukaKurodaChisatoTakiueReiHirataMamiTakenamiYokoAoiNanakoYoshiokaToshihiroHabaraTetsunoriMukaidaKeijiNaruseMammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression and friction force in the Fallopian tube before nidation. In order to apply mechanical stimuli to embryos in a conventional IVF culture system, the tilting embryo culture system (TECS) was developed. The observed embryo images from the TECS suggest that the velocities and shear stresses of TECS embryos are similar to those experienced in the oviduct. Use of TECS enhanced the development rate to the blastocyst stage and significantly increased the cell number of mouse blastocysts (P < 0.05). Although not statistically significant, human thawed embryos showed slight improvement in development to the blastocyst stage following culture in TECS compared with static controls. Rates of blastocyst formation following culture in TECS were significantly improved in low-quality embryos and those embryos cultured under suboptimal conditions (P < 0.05). The TECS is proposed as a promising approach to improve embryo development and blastocyst formation by exposing embryos to mechanical stimuli similar to those in the Fallopian tube.No potential conflict of interest relevant to this article was reported.Elsevier Sci Ltd.Acta Medica Okayama0021-929042132009Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells20972103ENTomonoriTetsunagaTakayukiFurumatsuNobuhiroAbeKeiichiroNishidaKeijiNaruseToshifumiOzakiBiomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin αVβ3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin αVβ3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin αVβ3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin αVβ3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030155811812006メカノバイオロジー―基礎から臨床まで―2331ENNo potential conflict of interest relevant to this article was reported.