Akira Hanashima, Misaki Kimoto, Yoshihiro Ujihara, Ken Hashimoto, Yuu Usui, Momoko Ohira, Masato Hoshino, Kentaro Uesugi, Stephanie Witt, Dittmar Labeit, Sumiko Kimura, Takashi Murayama, Takashi Sakurai, Siegfried Labeit and Satoshi Mohri : CCDC141 is a Connectin/Titin and Nesprin-1 binding protein that adapts cardiomyocytes to mechanical stress, Communications Biology, 8, 1, 1693, 2025.
(Summary)
The heart adapts to mechanical stresses such as hypertension, yet the underlying mechanisms remain poorly understood. We identify CCDC141 as a mechanoadaptive factor in cardiomyocytes. CCDC141 interacts with the sarcomeric connectin/titin filament system and the nuclear envelope protein nesprin-1, and localizes to the costameres in adult cardiomyocytes. CCDC141-KO mouse cardiomyocytes exhibit hypertrophy. Their mitochondria display abnormal alignment and fusion, are burdened by elevated ATP production, and show reduced spare respiratory capacity. Increased SERCA2a expression enhances Ca²+ handling within the sarcoplasmic reticulum of CCDC141-KO cardiomyocytes. During pressure overload-induced heart failure in mice, CCDC141 relocalizes to the nuclear envelope, suggesting a stress-responsive role in maintaining nuclear integrity. CCDC141-KO hearts show low tolerance to mechanical stress, and despite only moderate pressure overload induced by transverse aortic constriction, they are more susceptible to fatal arrhythmias and progressive heart failure with disrupted nuclear morphology. These findings provide new insights into the molecular basis of cardiac mechanoadaptation and disease.
Akira Hanashima, Yuu Usui, Ken Hashimoto and Satoshi Mohri : The Ancestor and Evolution of the Giant Muscle Protein Connectin/Titin, Journal of Molecular Evolution, 93, 3, 306-321, 2025.
(Summary)
The emergence of connectin, also called titin, a muscular spring and the largest protein in living organisms, is critical in metazoan evolution as it enables striated muscle-based locomotion. However, its evolutionary history remains unclear. Here, we investigated the evolutionary process using genomic analysis and deduced the ancestor of connectin. The region between the HOX and WNT clusters in the human genome, where the connectin gene (CON (TTN)) is located, was quadrupled by two rounds of whole-genome duplication (WGD) in the ancestor of jawed vertebrates. However, connectin ohnologs were deleted before the advent of jawed vertebrates, resulting in a single connectin gene. Additionally, one of the connectin ohnologs created by the third round of teleost WGD disappeared, while the other was duplicated on the same chromosome. We also discovered that the connectin and connectin family genes consistently underwent local duplication on the same chromosome, though the underlying mechanism remains unknown. Using synteny analysis, we identified KALRN and its ohnolog TRIO as putative ancestral paralogs of the connectin gene. TRIO/KALRN has a connected structure of SESTD1-CCDC141-CON (TTN), and its synteny is conserved in the Drosophila genome. Furthermore, we confirmed that this connected structure, termed 'connectitin,' (connected-connectin/titin) is conserved in cnidarians and placozoans. Molecular phylogenetic analysis revealed that it diverged from TRIO/KALRN prior to the emergence of these animals, suggesting that metazoan muscle may have a single origin. These findings enhance our understanding of the evolutionary processes of striated muscles in the animal kingdom.
Yuu Usui, Naoki Yamane, Akira Hanashima, Ken Hashimoto, Yuji Kanaoka and Satoshi Mohri : Precaudal Vertebrae in the Postcranial Region of Moray Eels Form Ventral Processes., Journal of Morphology, 285, 10, e21776, 2024.
(Summary)
Fish vertebrae are primarily morphologically classified into precaudal vertebrae jointed to the ribs and caudal vertebrae with hemal spines, through which the caudal artery and veins pass. Moray eels (family Muraenidae) capture prey by directly biting, combining oral and pharyngeal jaw. During feeding motions, they exhibit various head manipulations, such as neurocranial elevation, ventral flexion, and horizontal shaking, with their postcranial region acting like the neck of amniotes. However, the bone morphology supporting these movements remains unclear. In this study, the vertebral morphologies of the Kidako moray (Gymnothorax kidako), starry moray (Echidna nebulosa), pink-lipped moray (Echidna rhodochilus), tidepool snake moray (Uropterygius micropterus), and Seychelles moray (Anarchias seychellensis) were investigated using X-ray computed tomography. These five species exhibited longitudinal ventral processes in the second to approximately 12th precaudal vertebrae with canals for blood vessels, structurally similar to hemal spines. In addition, the morphology of the precaudal vertebrae in three Anguilliformes species closely related to moray eels and two Gasterosteiformes species, including a seahorse that flexes its head ventrally as a feeding motion, was compared with that of moray eels. However, no remarkable ventral processes were observed in their precaudal vertebrae in the postcranial region, suggesting that these structural features in the postcranial vertebrae were preserved in Muraenidae but not necessarily required for the fish to bend its head ventrally. Although the functional significance of the ventral process has yet to be determined, our findings highlight a novel aspect of fish vertebral morphology.
Ken Hashimoto, Momoko Ohira, Aya Kodama, Misaki Kimoto, Mariko Inoue, Shigenobu Toné, Yuu Usui, Akira Hanashima, Takato Goto, Yuhei Ogura, Yoshihiro Ujihara and Satoshi Mohri : Loss of connectin novex-3 leads to heart dysfunction associated with impaired cardiomyocyte proliferation and abnormal nuclear mechanics, Scientific Reports, 14, 1, 13727, 2024.
(Summary)
Connectin (also known as titin) is a giant striated muscle protein that functions as a molecular spring by providing elasticity to the sarcomere. Novex-3 is a short splice variant of connectin whose physiological function remains unknown. We have recently demonstrated using in vitro analyses that in addition to sarcomere expression, novex-3 was also expressed in cardiomyocyte nuclei exclusively during fetal life, where it provides elasticity/compliance to cardiomyocyte nuclei and promotes cardiomyocyte proliferation in the fetus, suggesting a non-sarcomeric function. Here, we analyzed novex-3 knockout mice to assess the involvement of this function in cardiac pathophysiology in vivo. Deficiency of novex-3 compromised fetal cardiomyocyte proliferation and induced the enlargement of individual cardiomyocytes in neonates. In adults, novex-3 deficiency resulted in chamber dilation and systolic dysfunction, associated with Ca2+ dysregulation, resulting in a reduced life span. Mechanistic analyses revealed a possible association between impaired proliferation and abnormal nuclear mechanics, including stiffer nuclei positioned peripherally with stabilized circumnuclear microtubules in knockout cardiomyocytes. Although the underlying causal relationships were not fully elucidated, these data show that novex-3 has a vital non-sarcomeric function in cardiac pathophysiology and serves as an early contributor to cardiomyocyte proliferation.
Yuu Usui, Akira Hanashima, Ken Hashimoto, Misaki Kimoto, Momoko Ohira and Satoshi Mohri : Comparative analysis of ventricular stiffness across species., Physiological Reports, 12, 8, e16013, 2024.
(Summary)
Investigating ventricular diastolic properties is crucial for understanding the physiological cardiac functions in organisms and unraveling the pathological mechanisms of cardiovascular disorders. Ventricular stiffness, a fundamental parameter that defines ventricular diastolic functions in chordates, is typically analyzed using the end-diastolic pressure-volume relationship (EDPVR). However, comparing ventricular stiffness accurately across chambers of varying maximum volume capacities has been a long-standing challenge. As one of the solutions to this problem, we propose calculating a relative ventricular stiffness index by applying an exponential approximation formula to the EDPVR plot data of the relationship between ventricular pressure and values of normalized ventricular volume by the ventricular weight. This article reviews the potential, utility, and limitations of using normalized EDPVR analysis in recent studies. Herein, we measured and ranked ventricular stiffness in differently sized and shaped chambers using ex vivo ventricular pressure-volume analysis data from four animals: Wistar rats, red-eared slider turtles, masu salmon, and cherry salmon. Furthermore, we have discussed the mechanical effects of intracellular and extracellular viscoelastic components, Titin (Connectin) filaments, collagens, physiological sarcomere length, and other factors that govern ventricular stiffness. Our review provides insights into the comparison of ventricular stiffness in different-sized ventricles between heterologous and homologous species, including non-model organisms.
(Keyword)
Animals / Rats / Diastole / Heart Ventricles / Species Specificity / Ventricular Function / Turtles / Salmon
Kehao Wang, Yutian Pu, Leran Chen, Masato Hoshino, Kentaro Uesugi, Naoto Yagi, Xiaoyong Chen, Yuu Usui, Akira Hanashima, Ken Hashimoto, Satoshi Mohri and K Barbara Pierscionek : Optical development in the murine eye lens of accelerated senescence-prone SAMP8 and senescence-resistant SAMR1 strains., Experimental Eye Research, 241, 2024.
(Summary)
The eye lens is responsible for focusing objects at various distances onto the retina and its refractive power is determined by its surface curvature as well as its internal gradient refractive index (GRIN). The lens continues to grow with age resulting in changes to the shape and to the GRIN profile. The present study aims to investigate how the ageing process may influence lens optical development. Murine lenses of accelerated senescence-prone strain (SAMP8) aged from 4 to 50 weeks; senescence-resistant strain (SAMR1) aged from 5 to 52 weeks as well as AKR strain (served as control) aged from 6 to 70 weeks were measured using the X-ray interferometer at the SPring-8 synchrotron Japan within three consecutive years from 2020 to 2022. Three dimensional distributions of the lens GRIN were reconstructed using the measured data and the lens shapes were determined using image segmentation in MatLab. Variations in the parameters describing the lens shape and the GRIN profile with age were compared amongst three mouse strains. With advancing age, both the lens anterior and posterior surface flattens and the lens sagittal thickness increase in all three mouse strains (Anterior radius of curvature increase at 0.008 mm/week, 0.007 mm/week and 0.002 mm/week while posterior radius of curvature increase at 0.002 mm/week, 0.007 mm/week and 0.003 mm/week respectively in AKR, SAMP8 and SAMR1 lenses). Compared with the AKR strain, the SAMP8 samples demonstrate a higher rate of increase in the posterior curvature radius (0.007 mm/week) and the thickness (0.015 mm/week), whilst the SAMR1 samples show slower increases in the anterior curvature radius (0.002 mm/week) and its thickness (0.013 mm/week). There are similar age-related trends in GRIN shape in the radial direction (in all three types of murine lenses nr2 and nr6 increase with age while nr4 decrease with age consistently) but not in the axial direction amongst three mouse strains (nz1 of AKR lens decrease while of SAMP8 and SAMR1 increase with age; nz2 of all three models increase with age; nz3 of AKR lens increase while of SAMP8 and SAMR1 decrease with age). The ageing process can influence the speed of lens shape change and affect the GRIN profile mainly in the axial direction, contributing to an accelerated decline rate of the optical power in the senescence-prone strain (3.5 D/week compared to 2.3 D/week in the AKR control model) but a retardatory decrease in the senescence-resistant strain (2.1 D/week compared to the 2.3D/week in the AKR control model).
(Keyword)
Mice / Animals / Aging / Lens, Crystalline / Japan
Yuu Usui, Misaki Kimoto, Akira Hanashima, Ken Hashimoto and Satoshi Mohri : Cardiac hemodynamics and ventricular stiffness of sea-run cherry salmon (Oncorhynchus masou masou) differ critically from those of landlocked masu salmon., PLoS ONE, 17, 11, 2022.
(Summary)
Ventricular diastolic mechanical properties are important determinants of cardiac function and are optimized by changes in cardiac structure and physical properties. Oncorhynchus masou masou is an anadromous migratory fish of the Salmonidae family, and several ecological studies on it have been conducted; however, the cardiac functions of the fish are not well known. Therefore, we investigated ventricular diastolic function in landlocked (masu salmon) and sea-run (cherry salmon) types at 29-30 months post fertilization. Pulsed-wave Doppler echocardiography showed that the atrioventricular inflow waveforms of cherry salmon were biphasic with early diastolic filling and atrial contraction, whereas those of masu salmon were monophasic with atrial contraction. In addition, end-diastolic pressure-volume relationship analysis revealed that the dilatability per unit myocardial mass of the ventricle in cherry salmon was significantly suppressed compared to that in masu salmon, suggesting that the ventricle of the cherry salmon was relatively stiffer (relative ventricular stiffness index; p = 0.0263). Contrastingly, the extensibility of cardiomyocytes, characterized by the expression pattern of Connectin isoforms in their ventricles, was similar in both types. Histological analysis showed that the percentage of the collagen accumulation area in the compact layer of cherry salmon increased compared with that of the masu salmon, which may contribute to ventricle stiffness. Although the heart mass of cherry salmon was about 11-fold greater than that of masu salmon, there was no difference in the morphology of the isolated cardiomyocytes, suggesting that the heart of the cherry salmon grows by cardiomyocyte proliferation, but not cell hypertrophy. The cardiac physiological function of the teleosts varies with differences in their developmental processes and life history. Our multidimensional analysis of the O. masou heart may provide a clue to the process by which the heart acquires a biphasic blood-filling pattern, i.e., a ventricular diastolic suction.
Ken Hashimoto, Aya Kodama, Momoko Ohira, Misaki Kimoto, Reiko Nakagawa, Yuu Usui, Yoshihiro Ujihara, Akira Hanashima and Satoshi Mohri : Postnatal expression of cell cycle promoter Fam64a causes heart dysfunction by inhibiting cardiomyocyte differentiation through repression of Klf15., iScience, 25, 5, 2022.
(Summary)
Introduction of fetal cell cycle genes into damaged adult hearts has emerged as a promising strategy for stimulating proliferation and regeneration of postmitotic adult cardiomyocytes. We have recently identified Fam64a as a fetal-specific cell cycle promoter in cardiomyocytes. Here, we analyzed transgenic mice maintaining cardiomyocyte-specific postnatal expression of Fam64a when endogenous expression was abolished. Despite an enhancement of cardiomyocyte proliferation, these mice showed impaired cardiomyocyte differentiation during postnatal development, resulting in cardiac dysfunction in later life. Mechanistically, Fam64a inhibited cardiomyocyte differentiation by repressing Klf15, leading to the accumulation of undifferentiated cardiomyocytes. In contrast, introduction of Fam64a in differentiated adult wildtype hearts improved functional recovery upon injury with augmented cell cycle and no dedifferentiation in cardiomyocytes. These data demonstrate that Fam64a inhibits cardiomyocyte differentiation during early development, but does not induce de-differentiation in once differentiated cardiomyocytes, illustrating a promising potential of Fam64a as a cell cycle promoter to attain heart regeneration.
Yuu Usui and Masakatsu Watanabe : Role of the Connexin C-terminus in skin pattern formation of Zebrafish., BBA Advances, 1, 2021.
(Summary)
These results provide evidence for the interactions between Cx39.4 and Cx41.8 in pigment cells of zebrafish and suggest that at least one connexin must have a CT domain.
Yuu Usui, Toshihiro Aramaki, Shigeru Kondo and Masakatsu Watanabe : The minimal gap-junction network among melanophores and xanthophores required for stripe pattern formation in zebrafish., Development, 146, 22, 2019.
(Summary)
Here, by arranging Cx39.4 and Cx41.8 expression in pigment cells, we have identified the simplest gap-junction network required for stripe generation: Cx39.4 expression in melanophores is required but expression in xanthophores is not necessary for stripe patterning, whereas Cx41.8 expression in xanthophores is sufficient for the patterning, and Cx41.8 expression in melanophores might stabilize the stripes. Moreover, patch-clamp recordings revealed that Cx39.4 gap junctions exhibit spermidine-dependent rectification property. Our results suggest that Cx39.4 facilitates the crucial cell-cell interactions between melanophores and xanthophores that mediate a unidirectional activation-signal transfer from xanthophores to melanophores, which is essential for melanophore survival.
Yuu Usui, Shigeru Kondo and Masakatsu Watanabe : Melanophore multinucleation pathways in zebrafish., Development Growth & Differentiation, 60, 7, 454-459, 2018.
(Summary)
In zebrafish, apart from mononuclear melanophores, bi- and trinuclear melanophores are frequently observed; however, the manner in which multinucleation of these cells occurs during fish development remains unknown. Here, we analyzed the processes underlying multinucleation of zebrafish melanophores. Transgenic zebrafish in which melanophore nuclei were labeled with a histone H2B-red fluorescent reporter protein were used to evaluate the distribution of mono-, bi-, and trinuclear melanophores in both the trunk and fin. Half of the melanophores examined were binuclear and approximately 1% were trinuclear. We compared cell size, cell motility, and survival rate between mono- and binuclear melanophores grown in a culture dish, and we found that cell size and survival rate were significantly larger in binuclear melanophores. We then analyzed the behavior of melanoblasts and melanophores from transgenic zebrafish using in vivo and in vitro live-cell imaging. We detected division and differentiation of melanoblasts, as well as melanoblast nuclear division without subsequent cellular division. In addition, we observed cellular and nuclear division in melanophores, although these events were very infrequent in vitro. On the basis of our findings, we present a scheme for melanophore multinucleation in zebrafish.
Masatoshi Kanda, Hiroyuki Yamanaka, Satoshi Kojo, Yuu Usui, Hiroaki Honda, Yusuke Sotomaru, Michishige Harada, Masaru Taniguchi, Nao Suzuki, Tatsuya Atsumi, Haruka Wada, Muhammad Baghdadi and Ken-Ichiro Seino : Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFN-γ production in iNKT cells., Proceedings of the National Academy of Sciences of the United States of America, 113, 24, E3394-402, 2016.
(Summary)
Invariant natural killer T (iNKT) cells are a subset of innate-like T cells that act as important mediators of immune responses. In particular, iNKT cells have the ability to immediately produce large amounts of IFN-γ upon activation and thus initiate immune responses in various pathological conditions. However, molecular mechanisms that control IFN-γ production in iNKT cells are not fully understood. Here, we report that basic helix-loop-helix transcription factor family, member e40 (Bhlhe40), is an important regulator for IFN-γ production in iNKT cells. Bhlhe40 is highly expressed in stage 3 thymic iNKT cells and iNKT1 subsets, and the level of Bhlhe40 mRNA expression is correlated with Ifng mRNA expression in the resting state. Although Bhlhe40-deficient mice show normal iNKT cell development, Bhlhe40-deficient iNKT cells show significant impairment of IFN-γ production and antitumor effects. Bhlhe40 alone shows no significant effects on Ifng promoter activities but contributes to enhance T-box transcription factor Tbx21 (T-bet)-mediated Ifng promoter activation. Chromatin immunoprecipitation analysis revealed that Bhlhe40 accumulates in the T-box region of the Ifng locus and contributes to histone H3-lysine 9 acetylation of the Ifng locus, which is impaired without T-bet conditions. These results indicate that Bhlhe40 works as a cofactor of T-bet for enhancing IFN-γ production in iNKT cells.
Hirotake Abe, Haruka Wada, Muhammad Baghdadi, Sayaka Nakanishi, Yuu Usui, Takahiro Tsuchikawa, Toshiaki Shichinohe, Satoshi Hirano and Ken-Ichiro Seino : Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S., Human Cell, 29, 2, 58-66, 2016.
(Summary)
Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1-designated as 4T1-Sapporo (4T1-S)-which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.
Shintaro Takeuchi, Muhammad Baghdadi, Takahiro Tsuchikawa, Haruka Wada, Toru Nakamura, Hirotake Abe, Sayaka Nakanishi, Yuu Usui, Kohtaro Higuchi, Mizuna Takahashi, Kazuho Inoko, Syoki Sato, Hironobu Takano, Toshiaki Shichinohe, Ken-ichiro Seino and Satoshi Hirano : Chemotherapy-Derived Inflammatory Responses Accelerate the Formation of Immunosuppressive Myeloid Cells in the Tissue Microenvironment of Human Pancreatic Cancer., Cancer Research, 75, 13, 2629-2640, 2015.
(Summary)
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic malignancies. PDAC builds a tumor microenvironment that plays critical roles in tumor progression and metastasis. However, the relationship between chemotherapy and modulation of PDAC-induced tumor microenvironment remains poorly understood. In this study, we report a role of chemotherapy-derived inflammatory response in the enrichment of PDAC microenvironment with immunosuppressive myeloid cells. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a major cytokine associated with oncogenic KRAS in PDAC cells. GM-CSF production was significantly enhanced in various PDAC cell lines or PDAC tumor tissues from patients after treatment with chemotherapy, which induced the differentiation of monocytes into myeloid-derived suppressor cells (MDSC). Furthermore, blockade of GM-CSF with monoclonal antibodies helped to restore T-cell proliferation when cocultured with monocytes stimulated with tumor supernatants. GM-CSF expression was also observed in primary tumors and correlated with poor prognosis in PDAC patients. Together, these results describe a role of GM-CSF in the modification of chemotherapy-treated PDAC microenvironment and suggest that the targeting of GM-CSF may benefit PDAC patients' refractory to current anticancer regimens by defeating MDSC-mediated immune escape.
Molecular Basis of Tension Sensing Transduction System in Cardiomyocytes and Its Application to Heart Failure (Project/Area Number: 24K03254 )
Biomedical engineering multi-scale ventricular diastolic function analysis to elucidate the pathogenesis of diastolic heart failure (Project/Area Number: 23H00556 )