Publications by authors named "Seiji Takashima"

168 Publications

Visualization of Spatial Distribution of Spermatogenesis in Mouse Testes Using Creatine Chemical Exchange Saturation Transfer Imaging.

J Magn Reson Imaging 2021 May 30. Epub 2021 May 30.

Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Background: When determining treatment strategies for male infertility, it is important to evaluate spermatogenesis and its spatial distribution in the testes.

Purpose: To investigate the usefulness of creatine chemical exchange saturation transfer (CrCEST) imaging for evaluating spermatogenesis and its spatial distribution.

Study Type: Prospective.

Animal Model: C57BL/6 control mice (n = 5) and model mice of male infertility induced by whole testis X-ray irradiation (n = 11) or localized X-ray irradiation to lower regions of testes (n = 3).

Field Strength/sequence: A 11.7-T vertical-bore magnetic resonance imaging (MRI)/segmented fast low-angle shot acquisition for CEST.

Assessment: The magnetization transfer ratio for the CrCEST effect (MTR ) was calculated in each testis of the control mice and X-ray irradiation model mice at 10, 15, 20, and 30 days after irradiation. Correlation analysis was performed between MTR and Johnsen's score, a histological score for spermatogenesis. In the localized X-ray irradiation model, regional MTR and Johnsen's score were calculated for correlation analysis.

Statistical Tests: Unpaired t-test, one-way analysis of variance with Tukey's HSD test and Pearson's correlation analysis. A P value < 0.05 was considered statistically significant.

Results: In the irradiation model, CrCEST imaging revealed a significant linear decrease of MTR after irradiation (control, 8.7 ± 0.6; 10 days, 7.9 ± 0.8; 15 days, 6.5 ± 0.6; 20 days, 5.4 ± 1.0; 30 days, 4.4 ± 0.8). A significant linear correlation was found between MTR and Johnsen's score (Pearson's correlation coefficient (r) = 0.79). In the localized irradiation model, CrCEST imaging visualized a significant regional decrease of MTR in the unshielded region (shielded, 6.9 ± 0.7; unshielded, 4.9 ± 1.0), and a significant linear correlation was found between regional MTR and Johnsen's score (r = 0.78).

Data Conclusion: Testicular CrCEST effects correlated well with spermatogenesis. CrCEST imaging was useful for evaluating spermatogenesis and its spatial distribution.

Evidence Level: 2 TECHNICAL EFFICACY: Stage 2.
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http://dx.doi.org/10.1002/jmri.27734DOI Listing
May 2021

Phenotypic recapitulation and correction of desmoglein-2-deficient cardiomyopathy using human-induced pluripotent stem cell-derived cardiomyocytes.

Hum Mol Genet 2021 Jul;30(15):1384-1397

Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.

Desmoglein-2, encoded by DSG2, is one of the desmosome proteins that maintain the structural integrity of tissues, including heart. Genetic mutations in DSG2 cause arrhythmogenic cardiomyopathy, mainly in an autosomal dominant manner. Here, we identified a homozygous stop-gain mutations in DSG2 (c.C355T, p.R119X) that led to complete desmoglein-2 deficiency in a patient with severe biventricular heart failure. Histological analysis revealed abnormal deposition of desmosome proteins, disrupted intercalated disk structures in the myocardium. Induced pluripotent stem cells (iPSCs) were generated from the patient (R119X-iPSC), and the mutated DSG2 gene locus was heterozygously corrected to a normal allele via homology-directed repair (HDR-iPSC). Both isogenic iPSCs were differentiated into cardiomyocytes [induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)]. Multielectrode array analysis detected abnormal excitation in R119X-iPSC-CMs but not in HDR-iPSC-CMs. Micro-force testing of three-dimensional self-organized tissue rings (SOTRs) revealed tissue fragility and a weak maximum force in SOTRs from R119X-iPSC-CMs. Notably, these phenotypes were significantly recovered in HDR-iPSC-CMs. Myocardial fiber structures in R119X-iPSC-CMs were severely aberrant, and electron microscopic analysis confirmed that desmosomes were disrupted in these cells. Unexpectedly, the absence of desmoglein-2 in R119X-iPSC-CMs led to decreased expression of desmocollin-2 but no other desmosome proteins. Adeno-associated virus-mediated replacement of DSG2 significantly recovered the contraction force in SOTRs generated from R119X-iPSC-CMs. Our findings confirm the presence of a desmoglein-2-deficient cardiomyopathy among clinically diagnosed dilated cardiomyopathies. Recapitulation and correction of the disease phenotype using iPSC-CMs provide evidence to support the development of precision medicine and the proof of concept for gene replacement therapy for this cardiomyopathy.
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http://dx.doi.org/10.1093/hmg/ddab127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283207PMC
July 2021

Phosphorylation of MYL12 by Myosin Light Chain Kinase Regulates Cellular Shape Changes in Cochlear Hair Cells.

J Assoc Res Otolaryngol 2021 Apr 20. Epub 2021 Apr 20.

Department of Otorhinolaryngology-Head and Neck Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape changes of HCs are important for the development of auditory epithelia and hearing function. It was previously observed that HCs and inner sulcus cells (ISCs) demonstrate cellular shape changes similar to the apical constriction of the neural epithelia. Apical constriction is induced via actomyosin cable contraction in the apical junctional complex and necessary for the physiological function of the epithelium. Actomyosin cable contraction is mainly regulated by myosin regulatory light chain (MRLC) phosphorylation by myosin light chain kinase (MLCK). However, MRLC and MLCK isoforms expressed in HCs and ISCs are unknown. Hence, we investigated the expression patterns and roles of MRLCs and MLCKs in HCs. Droplet digital PCR revealed that HCs expressed MYL12A/B and MYL9, which are non-muscle MRLC and smooth muscle MLCK (smMLCK), respectively. Immunofluorescence staining throughout the organ of Corti demonstrated that only MYL12 was expressed in the apical portion of HCs, whereas MYL12 and MYL9 were expressed on ISCs. In addition, purified MYL12B was phosphorylated by smMLCK in vitro, and the harvested HCs contained phosphorylated MYL12. Furthermore, accompanied by the expansion of the cell area of outer HCs, MYL12 phosphorylation was reduced by ML-7, which is an inhibitor of smMLCK. In conclusion, MYL12 phosphorylation by smMLCK contributed to the apical constriction-like cellular shape change of HCs possibly relating to the development of auditory epithelia and hearing function.
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http://dx.doi.org/10.1007/s10162-021-00796-1DOI Listing
April 2021

The CR9 element is a novel mechanical load-responsive enhancer that regulates natriuretic peptide genes expression.

FASEB J 2021 04;35(4):e21495

Department of Medical Biochemistry, Graduate School of Medicine/Frontier Biosciences, Osaka University, Suita, Japan.

Enhancers regulate gene expressions in a tissue- and pathology-specific manner by altering its activities. Plasma levels of atrial and brain natriuretic peptides, encoded by the Nppa and Nppb, respectively, and synthesized predominantly in cardiomyocytes, vary depending on the severity of heart failure. We previously identified the noncoding conserved region 9 (CR9) element as a putative Nppb enhancer at 22-kb upstream from the Nppb gene. However, its regulatory mechanism remains unknown. Here, we therefore investigated the mechanism of CR9 activation in cardiomyocytes using different kinds of drugs that induce either cardiac hypertrophy or cardiac failure accompanied by natriuretic peptides upregulation. Chronic treatment of mice with either catecholamines or doxorubicin increased CR9 activity during the progression of cardiac hypertrophy to failure, which is accompanied by proportional increases in Nppb expression. Conversely, for cultured cardiomyocytes, doxorubicin decreased CR9 activity and Nppb expression, while catecholamines increased both. However, exposing cultured cardiomyocytes to mechanical loads, such as mechanical stretch or hydrostatic pressure, upregulate CR9 activity and Nppb expression even in the presence of doxorubicin. Furthermore, the enhancement of CR9 activity and Nppa and Nppb expressions by either catecholamines or mechanical loads can be blunted by suppressing mechanosensing and mechanotransduction pathways, such as muscle LIM protein (MLP) or myosin tension. Finally, the CR9 element showed a more robust and cell-specific response to mechanical loads than the -520-bp BNP promoter. We concluded that the CR9 element is a novel enhancer that responds to mechanical loads by upregulating natriuretic peptides expression in cardiomyocytes.
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http://dx.doi.org/10.1096/fj.202002111RRDOI Listing
April 2021

Accurate Estimation of the Duration of Testicular Ischemia Using Creatine Chemical Exchange Saturation Transfer (CrCEST) Imaging.

J Magn Reson Imaging 2021 05 17;53(5):1559-1567. Epub 2020 Dec 17.

Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Japan.

Background: In the management of testicular torsion, estimating the duration of testicular ischemia is essential for deciding on an appropriate surgical treatment, but there are currently limited evaluation methods.

Purpose: To perform testicular creatine chemical exchange saturation transfer (CrCEST) imaging and to evaluate its ability to accurately estimate the duration of testicular ischemia.

Study Type: Prospective.

Animal Model: C57BL/6 control mice (n = 6) and testicular ischemia models induced by clamping the spermatic cord (n = 14). Eight of testicular ischemia models were serially imaged at two or three timepoints and a total of 26 images of ischemic testis were obtained. The ischemic duration ranged from 6-42 hours.

Field Strength/sequence: 11.7T vertical-bore MRI/segment fast low-angle shot acquisition for CEST.

Assessment: CrCEST imaging was performed and the magnetization transfer ratio for the CrCEST effect (MTR ) was calculated in control mice and testicular ischemia models. Correlation analysis between the duration of testicular ischemia and MTR decline was performed.

Statistical Tests: Paired t-test, and Pearson's correlation analysis.

Results: In control mice, the CrCEST effect in testes was significantly more than five times higher than that in skeletal muscle. MTR did not differ significantly between the right and left testes (8.6 ± 0.8 vs. 8.3 ± 0.6, P = 0.96). In testicular ischemia models, MTR of ischemic testes was significantly lower than that of controls (4 ± 2 vs. 8.9 ± 0.6, P < 0.001). Correlation analysis revealed a strong linear correlation between MTR decline and the duration of ischemia (r = 0.96, P < 0.001).

Data Conclusion: A decreased CrCEST effect in ischemic testes correlated well with ischemic duration. Testicular CrCEST imaging was useful for accurately estimating the duration of testicular ischemia.

Level Of Evidence: 2 TECHNICAL EFFICACY STAGE: 2.
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http://dx.doi.org/10.1002/jmri.27456DOI Listing
May 2021

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP-170.

EMBO Rep 2021 01 29;22(1):e50949. Epub 2020 Nov 29.

Department of Medical Biochemistry, Osaka University Graduate School of Frontier Biological Science, Suita, Osaka, Japan.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.
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http://dx.doi.org/10.15252/embr.202050949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788454PMC
January 2021

Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes.

Sci Rep 2020 09 18;10(1):15348. Epub 2020 Sep 18.

Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.
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http://dx.doi.org/10.1038/s41598-020-72216-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501291PMC
September 2020

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays.

J Vis Exp 2020 06 23(160). Epub 2020 Jun 23.

Department of Medical Biochemistry, Osaka University Graduate School of Medicine/Frontier Biosciences.

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 °C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 ± 0.10 mol/min/mol kinase and the average Km was around 0.5 USA µM at 25 °C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.
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http://dx.doi.org/10.3791/61168DOI Listing
June 2020

Germ cell depletion in recipient testis has adverse effects on spermatogenesis in orthotopically transplanted testis pieces via retinoic acid insufficiency.

Sci Rep 2020 07 1;10(1):10796. Epub 2020 Jul 1.

Department of Textile Science and Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, Ueda, 386-8567, Japan.

Germ cell depletion in recipient testes is indispensable for successful transplantation of spermatogonial stem cells. However, we found that such treatment had an adverse effect on spermatogenesis of orthotopically transplanted donor testis tissues. In the donor tissue, the frequency of stimulated by retinoic acid (RA) 8 (STRA8) expression was reduced in germ cells, suggesting that RA signalling indispensable for spermatogenesis was attenuated in germ cell-depleted recipient testes. In this context, germ cell depletion diminished expression of testicular Aldh1a2, which is responsible for testicular RA synthesis, while Cyp26b1, which is responsible for testicular RA metabolism, was still expressed even after germ cell depletion, suggesting an alteration of the RA synthesis/metabolism ratio. These observations suggested that RA insufficiency was one of the causes of the defective donor spermatogenesis. Indeed, repetitive RA administrations significantly improved donor spermatogenesis to produce fertile offspring without any side effects. These findings may contribute to improving fertility preservation techniques for males, especially to prevent iatrogenic infertility induced by chemotherapy in prepubertal cancer patients.
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http://dx.doi.org/10.1038/s41598-020-67595-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330030PMC
July 2020

Identification of transmembrane protein 168 mutation in familial Brugada syndrome.

FASEB J 2020 05 16;34(5):6399-6417. Epub 2020 Mar 16.

Division of Molecular Medical Biochemistry, Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Otsu, Japan.

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na current and Na 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Na 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Na 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.
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http://dx.doi.org/10.1096/fj.201902991RDOI Listing
May 2020

Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases.

Cardiovasc Res 2020 11;116(13):2116-2130

Department of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Sciences, 13-1, Takara-machi, Kanazawa, Ishikawa 920-8641, Japan.

Aims: The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them.

Methods And Results: We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as 'pathogenic' by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from 'Uncertain significance' to 'Likely pathogenic' in six probands.

Conclusion: Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.
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http://dx.doi.org/10.1093/cvr/cvaa010DOI Listing
November 2020

In vivo real-time ATP imaging in zebrafish hearts reveals G0s2 induces ischemic tolerance.

FASEB J 2020 02 8;34(2):2041-2054. Epub 2020 Jan 8.

Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Frontier Bioscience, Suita, Japan.

Most eukaryotic cells generate adenosine triphosphate (ATP) through the oxidative phosphorylation system (OXPHOS) to support cellular activities. In cultured cell-based experiments, we recently identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS, and showed that G0s2 protects cultured cardiomyocytes from hypoxia. In this study, we examined the in vivo protective role of G0s2 against hypoxia by generating both loss-of-function and gain-of-function models of g0s2 in zebrafish. Zebrafish harboring transcription activator-like effector nuclease (TALEN)-mediated knockout of g0s2 lost hypoxic tolerance. Conversely, cardiomyocyte-specific transgenic zebrafish hearts exhibited strong tolerance against hypoxia. To clarify the mechanism by which G0s2 protects cardiac function under hypoxia, we introduced a mitochondrially targeted FRET-based ATP biosensor into zebrafish heart to visualize ATP dynamics in in vivo beating hearts. In addition, we employed a mosaic overexpression model of g0s2 to compare the contraction and ATP dynamics between g0s2-expressing and non-expressing cardiomyocytes, side-by-side within the same heart. These techniques revealed that g0s2-expressing cardiomyocyte populations exhibited preserved contractility coupled with maintained intra-mitochondrial ATP concentrations even under hypoxic condition. Collectively, these results demonstrate that G0s2 provides ischemic tolerance in vivo by maintaining ATP production, and therefore represents a promising therapeutic target for hypoxia-related diseases.
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http://dx.doi.org/10.1096/fj.201901686RDOI Listing
February 2020

Higd1a improves respiratory function in the models of mitochondrial disorder.

FASEB J 2020 01 10;34(1):1859-1871. Epub 2019 Dec 10.

Department of Medical Biochemistry, Graduate School of Frontier Biological Science, Osaka University, Suita, Japan.

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP] under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease; notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal velocity of the reaction between CcO and cytochrome c without changing the affinity between them, indicating that Higd1a is a positive modulator of CcO. These results corroborate that Higd1a, or its mimic, provides therapeutic options for the treatment of mitochondrial diseases.
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http://dx.doi.org/10.1096/fj.201800389RDOI Listing
January 2020

A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G/G switch gene 2.

J Biol Chem 2019 10 1;294(40):14562-14573. Epub 2019 Aug 1.

Department of Medical Biochemistry, Osaka University Graduate School of Frontier Biosciences, Suita, Osaka 565-0871, Japan

Oxidative phosphorylation generates most of the ATP in respiring cells. ATP is an essential energy source, especially in cardiomyocytes because of their continuous contraction and relaxation. Previously, we reported that G/G switch gene 2 (G0S2) positively regulates mitochondrial ATP production by interacting with FF-ATP synthase. G0S2 overexpression mitigates ATP decline in cardiomyocytes and strongly increases their hypoxic tolerance during ischemia. Here, we show that G0S2 protein undergoes proteasomal degradation via a cytosolic molecular triage system and that inhibiting this process increases mitochondrial ATP production in hypoxia. First, we performed screening with a library of siRNAs targeting ubiquitin-related genes and identified RING finger protein 126 (RNF126) as an E3 ligase involved in G0S2 degradation. RNF126-deficient cells exhibited prolonged G0S2 protein turnover and reduced G0S2 ubiquitination. BCL2-associated athanogene 6 (BAG6), involved in the molecular triage of nascent membrane proteins, enhanced RNF126-mediated G0S2 ubiquitination both and Next, we found that Glu-44 in the hydrophobic region of G0S2 acts as a degron necessary for G0S2 polyubiquitination and proteasomal degradation. Because this degron was required for an interaction of G0S2 with BAG6, an alanine-replaced G0S2 mutant (E44A) escaped degradation. In primary cultured cardiomyocytes, both overexpression of the G0S2 E44A mutant and RNF126 knockdown effectively attenuated ATP decline under hypoxic conditions. We conclude that the RNF126/BAG6 complex contributes to G0S2 degradation and that interventions to prevent G0S2 degradation may offer a therapeutic strategy for managing ischemic diseases.
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http://dx.doi.org/10.1074/jbc.RA119.008544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779449PMC
October 2019

Mouse skeletal muscle creatine chemical exchange saturation transfer (CrCEST) imaging at 11.7T MRI.

J Magn Reson Imaging 2020 02 22;51(2):563-570. Epub 2019 Jun 22.

Laboratory of Biofunctional Imaging, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

Background: Creatine chemical exchange saturation transfer (CrCEST) imaging is expected to be a novel evaluation method of muscular energy metabolism.

Purpose: To develop CrCEST imaging of mouse skeletal muscle and to validate this technique by measuring changes in Cr concentration of ischemic hindlimbs.

Study Type: Prospective.

Animal Model: C57BL/6 mice (n = 6), mild hindlimb ischemic mice (n = 6), and severe hindlimb ischemic mice (n = 6).

Field Strength/sequence: Magnetic resonance angiography (MRA), CrCEST imaging, and phosphorus magnetic resonance spectroscopy ( P MRS) obtained at 11.7T.

Assessment: MRA and P MRS were performed to confirm the presence of ischemia following the compression by rubber tourniquet. CrCEST imaging was performed and magnetization transfer ratio asymmetry (MTR ), which reflects Cr concentration, and was calculated in severe ischemia models, mild ischemia models, and control mice. Follow-up CrCEST imaging was performed after the release of ischemia in the mild ischemia models.

Statistical Tests: Mean ± SD, one-way analysis of variance (ANOVA) with Tukey's HSD test, unpaired or paired t-test.

Results: MRA revealed the loss of blood flow of the femoral artery in the ischemic hindlimb. P MRS revealed different degrees of PCr decrease in severe and mild ischemic hindlimb (n = 3 per group, normal hindlimb: 1.0 ± 0, mild ischemic hindlimb: 0.77 ± 0.13, severe ischemic hindlimb: 0 ± 0). CrCEST imaging inversely revealed a significant stepwise increase in the MTR ratio of ischemic hindlimbs compared with controls (control, mild ischemia, and severe ischemia; 0.99 ± 0.04, 1.36 ± 0.08, and 1.59 ± 0.23, respectively, P < 0.0001). In addition, follow-up CrCEST imaging after the release of ischemia revealed normalization of the MTR ratios (recovered hindlimb: 1.01 ± 0.05).

Data Conclusion: We demonstrated an increase in the MTR of ischemic hindlimbs, along with a decrease of PCr. We demonstrated the normalization of MTR after the release of ischemia and developed CrCEST imaging of mouse skeletal muscle.

Level Of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:563-570.
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http://dx.doi.org/10.1002/jmri.26844DOI Listing
February 2020

AST-120, an Adsorbent of Uremic Toxins, Improves the Pathophysiology of Heart Failure in Conscious Dogs.

Cardiovasc Drugs Ther 2019 06;33(3):277-286

Department of Clinical Medicine and Development, National Cerebral and Cardiovascular Center, 5-7-1, Fujishirodai, Osaka, Suita, 565-8565, Japan.

Purpose: Several lines of evidence suggest that renal dysfunction is associated with cardiovascular toxicity through the action of uremic toxins. The levels of those uremic toxins can be reportedly reduced by the spherical carbon adsorbent AST-120. Because heart failure (HF) causes renal dysfunction by low cardiac output and renal edema, the removal of uremic toxins could be cardioprotective.

Method: To determine whether blood levels of the uremic toxin indoxyl sulfate (IS) increase in HF and whether AST-120 can reduce those levels and improve HF. We induced HF in 12 beagle dogs by 6 weeks of rapid right ventricular pacing at 230 beats per min. We treated six dogs with a 1-g/kg/day oral dosage of AST-120 for 14 days from week 4 after the start of rapid ventricular pacing. The other six dogs did not receive any treatment (control group).

Results: In the untreated dogs, IS levels increased as cardiac function deteriorated. In contrast, plasma IS levels in the treated dogs decreased to baseline levels, with both left ventricular fractional shortening and pulmonary capillary wedge pressure also improving when compared with untreated dogs. Finally, AST-120 treatment was shown to reduce both myocardial apoptosis and fibrosis along with decreases in extracellular signal-regulated kinase phosphorylation, the Bax/Bcl-2 ratio, and TGF-β1 expression and increases in AKT phosphorylation.

Conclusions: IS levels are increased in HF. AST-120 treatment reduces the levels of IS and improves the pathophysiology of HF in a canine model. AST-120 could be a novel candidate for the treatment of HF.
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http://dx.doi.org/10.1007/s10557-019-06875-zDOI Listing
June 2019

Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation.

Circulation 2019 04;139(18):2157-2169

Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, Japan (I.K.).

Background: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life.

Methods: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model.

Results: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( I channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of I channel function by increasing the basal current, even in the absence of m muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective I channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish.

Conclusions: The I channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant I channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective I channel blocker. Thus, the I channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the I channel.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.036761DOI Listing
April 2019

Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy.

ESC Heart Fail 2019 Apr 28;6(2):406-415. Epub 2019 Jan 28.

Department of Cardiovascular and Internal Medicine, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.

Aims: Cardiac myosin light chain kinase (cMLCK) phosphorylates ventricular myosin regulatory light chain 2 (MLC2v) and regulates sarcomere and cardiomyocyte organization. However, few data exist regarding the relationship between cMLCK mutations and MLC2v phosphorylation, particularly in terms of developing familial dilated cardiomyopathy (DCM) in whom cMLCK gene mutations were identified. The purpose of the present study was to investigate functional consequences of cMLCK mutations in DCM patients.

Methods And Results: The diagnosis of DCM was based on the patients' history and on echocardiography. We screened cMLCK gene mutations in DCM probands with high resolution melting analysis. Known DCM-causing genes mutations were excluded by exome sequencing of family members. MLC2v phosphorylation was analysed by Phos-tag sodium dodecyl sulfate-polyacrylamide gel electrophoresis assays. We also performed ADP-Glo assays for determining the total amount of adenosine triphosphate used in the kinase reaction. Unrelated DCM probands (109 males and 40 females) were enrolled in this study, of which 16 were familial and 133 sporadic. By mutation screening, a truncation variant of c1915-1 g>t (p.Pro639Valfs*15) was identified, which was not detected in 400 chromosomes of 200 healthy volunteers; it is listed in the Human Genetic Variation Database with an allele frequency < 0.001. In the proband, the presence of mutations in known DCM-causing genes was excluded with exome analysis. Familial analysis identified a 19-year-old male carrier who manifested slight left ventricular dilation with preserved systolic function. Phosphorylation assays analysed by Phos-tag SDS-PAGE revealed that the identified p.Pro639Valfs*15 mutation results in a complete lack of kinase activity, although it did not affect wild-type cMLCK activity. ADP-Glo assays confirmed that the mutant cMLCK had no kinase activity, whereas wild-type cMLCK had a Km value of 5.93 ± 1.47 μM and a V of 1.28 ± 0.03 mol/min/mol kinase.

Conclusions: These results demonstrate that a truncation mutation in the cMLCK gene p.Pro639Valfs*15 can be associated with significant impairment of MLC2v phosphorylation and possibly with development of DCM, although a larger study of DCM patients is required to determine the prevalence of this mutation and further strengthen its association with disease development.
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http://dx.doi.org/10.1002/ehf2.12410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437445PMC
April 2019

Detection of increased intracerebral lactate in a mouse model of Leigh syndrome using proton MR spectroscopy.

Magn Reson Imaging 2019 05 19;58:38-43. Epub 2019 Jan 19.

Department of Biomedical Imaging, National Cardiovascular and Cerebral Research Center, Suita, Osaka 565-8565, Japan; Department of Medical Physics and Engineering, Division of Health Sciences, Osaka University Graduate School of Medicine, Suita, Osaka 560-0871, Japan. Electronic address:

Purpose: To establish a brain proton magnetic resonance spectroscopy (H MRS) experimental system using a mouse model of Leigh syndrome for monitoring intracerebral lactate levels as a biomarker of mitochondrial disease progression.

Materials And Methods: Brain H MRS was performed in the Ndufs4 homozygous knockout (KO) mice, a mouse model of Leigh syndrome, and control mice on a horizontal 7.0-T magnetic resonance imaging system at age 5-9 weeks. In a subset of KO mice, survival analysis was performed according to the median of the intracerebral lactate levels. In addition, in KO mice alive until 9 weeks of age, both H MRS and T-weighted imaging (TWI) were longitudinally performed in the same individuals at 5, 7, and 9 weeks of age.

Results: Brain H MRS demonstrated increased lactate levels in KO mice compared with control mice (6.4 ± 1.2 mM vs. 3.3 ± 0.8 mM, p < 0.0001). The increased intracerebral lactate levels were already observed at 5 weeks of age, while no obvious abnormal findings were detected in TWI. Notably, an increased lactate level of >5.94 mM at week 5 was associated with a poor prognosis (median survival days: 24.5 vs. 42 days, log-rank p = 0.03). Longitudinal H MRS experiments revealed temporal increase of intracerebral lactate levels, peaking at week 7 (mean change: 2.6 ± 0.7 mM, p = 0.001), followed by decrease at week 9 (mean change: -3.8 ± 2.5 mM, p = 0.03), along with further disease progression, with brain lesions being detected on TWI.

Conclusion: Using brain H MRS, we demonstrated significant increase in intracerebral lactate levels in a mouse model of Leigh syndrome. Additionally, we demonstrated that intracerebral lactate is a useful biomarker of mitochondrial disease progression at stages preceding the development of brain lesions.
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http://dx.doi.org/10.1016/j.mri.2019.01.010DOI Listing
May 2019

Anti-HB-EGF Antibody-Mediated Delivery of siRNA to Atherosclerotic Lesions in Mice.

Int Heart J 2018 Nov 5;59(6):1425-1431. Epub 2018 Nov 5.

Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University.

For atherosclerotic cardiovascular diseases (ACD), gene therapy may be a potential therapeutic strategy; however, lack of effective and safe methods for gene delivery to atherosclerotic plaques have limited its potential therapeutic applications. To overcome this limitation, we developed a novel antibody-based gene delivery system (anti-HB-EGF/NA vector) by chemically crosslinking antibodies against human heparin-binding epidermal growth factor-like growth factor (HB-EGF). It has been shown to be excessively expressed in human atherosclerotic plaques and NeutrAvidin (NA) for conjugating biotinylated siRNA. Immunofluorescence staining and quantitative flow cytometry analysis using human HB-EGF-expressing cells showed both antibody-mediated selective cellular targeting and efficient intracellular delivery of conjugated biotin-fluorescence. Moreover, we demonstrated antibody-mediated significant and selective gene knockdown via conjugation with anti-HB-EGF/NA vector and biotinylated siRNA (anti-HB-EGF/NA/b-siRNA) in vitro. Furthermore, using high fat-fed human HB-EGF knock-in and apolipoprotein E-knockout (Hbegf hz/hz; Apoe-/-) mice, we demonstrated that the anti-HB-EGF/NA vector, conjugating biotin-fluorescence, increasingly accumulated within the atherosclerotic plaques of the ascending aorta in which human HB-EGF expression levels were highly elevated. Moreover, in response to a single intravenous injection of anti-HB-EGF/NA/b-siRNA in a dose-dependent manner, qPCR analysis of laser-dissected atherosclerotic plaques of the ascending aorta showed significant knockdown of the reporter gene expression. These results suggest that the anti-HB-EGF antibody-mediated siRNA delivery could be a promising delivery system for gene therapy of ACD.
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http://dx.doi.org/10.1536/ihj.17-644DOI Listing
November 2018

Biology and manipulation technologies of male germline stem cells in mammals.

Authors:
Seiji Takashima

Reprod Med Biol 2018 Oct 6;17(4):398-406. Epub 2018 Aug 6.

Faculty of Textile Science and Technology Shinshu University Ueda Japan.

Background: Spermatogonial stem cells (SSCs) are the origin of sperm and defined by their functions of "colonization in the testis" and "spermatogenesis". In vitro manipulation techniques of SSCs contribute to a wide variety of fields including reproductive medicine and molecular breeding. This review presents the recent progress of the biology and manipulation technologies of SSCs.

Methods: Research articles regarding SSC biology and technologies were collected and summarized.

Main Findings: Dr. Ralph Brinster developed the spermatogonial transplantation technique that enables SSC detection by functional markers. Using this technique, cultured SSCs, termed germline stem (GS) cells, were established from the mouse. GS cells provide the opportunity to produce genome-edited animals without using zygotes. In vitro spermatogenesis allows production of haploid germ cells from GS cells without spermatogonial transplantation. The recent advancement of pluripotent stem cell culture techniques has also achieved production of functional GS-like cells in addition to male/female germ cells.

Conclusion: Although in vitro manipulation techniques of GS cells have been developed for the mouse, it appears to be difficult to apply these techniques to other species. Understanding and control of interspecies barriers are required to extend this technology to nonrodent mammals.
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http://dx.doi.org/10.1002/rmb2.12220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194257PMC
October 2018

Author Correction: Heart Failure Phenotypes Induced by Knockdown of DAPIT in Zebrafish: A New Insight into Mechanism of Dilated Cardiomyopathy.

Sci Rep 2018 May 14;8(1):7768. Epub 2018 May 14.

Division of Cardiovascular Medicine, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-26012-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951827PMC
May 2018

Protein carbamylation exacerbates vascular calcification.

Kidney Int 2018 07 30;94(1):72-90. Epub 2018 Apr 30.

Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Protein carbamylation is a posttranslational modification that can occur non-enzymatically in the presence of high concentrations of urea. Although carbamylation is recognized as a prognostic biomarker, the contribution of protein carbamylation to organ dysfunction remains uncertain. Because vascular calcification is common under carbamylation-prone situations, we investigated the effects of carbamylation on this pathologic condition. Protein carbamylation exacerbated the calcification of human vascular smooth muscle cells (hVSMCs) by suppressing the expression of ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1), a key enzyme in the generation of pyrophosphate, which is a potent inhibitor of ectopic calcification. Several mitochondrial proteins were carbamylated, although ENPP1 itself was not identified as a carbamylated protein. Rather, protein carbamylation reduced mitochondrial membrane potential and exaggerated mitochondria-derived oxidative stress, which down-regulated ENPP1. The effects of carbamylation on ectopic calcification were abolished in hVSMCs by ENPP1 knockdown, in mitochondrial-DNA-depleted hVSMCs, and in hVSMCs treated with a mitochondria-targeted superoxide scavenger. We also evaluated the carbamylation effects using ex vivo and in vivo models. The tunica media of a patient with end-stage renal disease was carbamylated. Thus, our findings have uncovered a previously unrecognized aspect of uremia-related vascular pathology.
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http://dx.doi.org/10.1016/j.kint.2018.01.033DOI Listing
July 2018

FGF2 Has Distinct Molecular Functions from GDNF in the Mouse Germline Niche.

Stem Cell Reports 2018 06 19;10(6):1782-1792. Epub 2018 Apr 19.

Department of Textile Science and Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda 386-8567, Japan; Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan. Electronic address:

Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1 subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche.
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http://dx.doi.org/10.1016/j.stemcr.2018.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989648PMC
June 2018

Expression dynamics of self-renewal factors for spermatogonial stem cells in the mouse testis.

J Reprod Dev 2018 Jun 16;64(3):267-275. Epub 2018 Apr 16.

Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.

Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells (SSCs). Although GDNF is indispensable for the maintenance of SSCs, the role of FGF2 in the testis remains to be elucidated. To clarify this, the expression dynamics and regulatory mechanisms of Fgf2 and Gdnf in the mouse testes were analyzed. It is well known that Sertoli cells express Gdnf, and its receptor is expressed in a subset of undifferentiated spermatogonia, including SSCs. However, we found that Fgf2 was mainly expressed in the germ cells and its receptors were expressed not only in the cultured spermatogonial cell line, but also in testicular somatic cells. Aging, hypophysectomy, retinoic acid treatment, and testicular injury induced distinct Fgf2 and Gdnf expression dynamics, suggesting a difference in the expression mechanism of Fgf2 and Gdnf in the testis. Such differences might cause a dynamic fluctuation of Gdnf/Fgf2 ratio depending on the intrinsic/extrinsic cues. Considering that FGF2-cultured spermatogonia exhibit more differentiated phenotype than those cultured with GDNF, FGF2 might play a role distinct from that of GDNF in the testis, despite the fact that both factors are self-renewal factor for SSC in vitro.
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http://dx.doi.org/10.1262/jrd.2018-015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021615PMC
June 2018

Culture and transplantation of spermatogonial stem cells.

Stem Cell Res 2018 05 15;29:46-55. Epub 2018 Mar 15.

Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

The spermatogonial transplantation technique was developed by Dr. Ralph Brinster in 1994. Transplanted spermatogonial stem cells (SSCs) produce germ cell colonies after microinjection into the seminiferous tubules of infertile mice. This technique provided the first functional assay for SSCs. Although it became possible to produce transgenic animals using this transplantation technique in 2001, the lack of SSC culture systems prevented efficient genetic manipulation. To overcome this problem, a long-term SSC culture technique was developed in 2003. Cultured SSCs, designated as germline stem cells, allow drug selection of transfected SSCs, and knockout mice were produced in 2006. Using these techniques, it is now possible to address basic biological questions of SSC biology. They also open up new possibilities for male germline manipulation. In this review, we will briefly summarize our findings on SSCs and discuss unresolved issues that remain to be addressed.
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http://dx.doi.org/10.1016/j.scr.2018.03.006DOI Listing
May 2018

Genetic basis of cardiomyopathy and the genotypes involved in prognosis and left ventricular reverse remodeling.

Sci Rep 2018 01 31;8(1):1998. Epub 2018 Jan 31.

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants.
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http://dx.doi.org/10.1038/s41598-018-20114-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792481PMC
January 2018

Heat Failure Phenotypes Induced by Knockdown of DAPIT in Zebrafish: A New Insight into Mechanism of Dilated Cardiomyopathy.

Sci Rep 2017 12 12;7(1):17417. Epub 2017 Dec 12.

Division of Cardiovascular Medicine, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.

The pathogenesis of heart failure associated with dilated cardiomyopathy (DCM) may result in part from adenosine triphosphate (ATP) dysregulation in the myocardium. Under these conditions, diabetes-associated protein in insulin-sensitive tissue (DAPIT), which is encoded by the upregulated during skeletal muscle growth 5 (USMG5) gene, plays a crucial role in energy production by mitochondrial ATP synthase. To determine whether USMG5 is related to the development of heart failure, we performed clinical and experimental studies. Microarray analysis showed that the expression levels of USMG5 were positively correlated with those of natriuretic peptide precursor A in the human failed myocardium. When endogenous z-usmg5 in zebrafish was disrupted using morpholino (MO) oligonucleotides, the pericardial sac and atrial areas were larger and ventricular fractional shortening was reduced compared to in the control MO group. The expression levels of natriuretic peptides were upregulated in the z-usmg5 MO group compared to in controls. Further, microarray analysis revealed that genes in the calcium signalling pathway were downregulated in the z-usmg5 MO group. These results demonstrate that DAPIT plays a crucial role in the development of heart failure associated with DCM and thus may be a therapeutic target for heart failure.
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http://dx.doi.org/10.1038/s41598-017-17572-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727169PMC
December 2017

Slc3a2 Mediates Branched-Chain Amino-Acid-Dependent Maintenance of Regulatory T Cells.

Cell Rep 2017 Nov;21(7):1824-1838

Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan; Core Research for Evolutional Science and Technology, Japan Agency for Medical Research and Development, Tokyo, Japan. Electronic address:

Foxp3 regulatory T (Treg) cells, which suppress immune responses, are highly proliferative in vivo. However, it remains unclear how the active replication of Treg cells is maintained in vivo. Here, we show that branched-chain amino acids (BCAAs), including isoleucine, are required for maintenance of the proliferative state of Treg cells via the amino acid transporter Slc3a2-dependent metabolic reprogramming. Mice fed BCAA-reduced diets showed decreased numbers of Foxp3 Treg cells with defective in vivo proliferative capacity. Mice lacking Slc3a2 specifically in Foxp3 Treg cells showed impaired in vivo replication and decreased numbers of Treg cells. Slc3a2-deficient Treg cells showed impaired isoleucine-induced activation of the mTORC1 pathway and an altered metabolic state. Slc3a2 mutant mice did not show an isoleucine-induced increase of Treg cells in vivo and exhibited multi-organ inflammation. Taken together, these findings demonstrate that BCAA controls Treg cell maintenance via Slc3a2-dependent metabolic regulation.
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http://dx.doi.org/10.1016/j.celrep.2017.10.082DOI Listing
November 2017

Novel Synthesized Radical-Containing Nanoparticles Limit Infarct Size Following Ischemia and Reperfusion in Canine Hearts.

Cardiovasc Drugs Ther 2017 Dec;31(5-6):501-510

Department of Clinical Research and Development, National Cerebral and Cardiovascular Center, Osaka, Japan.

Purpose: Although nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) scavenge free radicals, their short half-life and considerable side effects such as systemic hypotension and bradycardia have limited their clinical application. Since a radical-containing nanoparticle (RNP) delivers nitroxyl radicals with a prolonged half-life specific to ischemic hearts, we investigated whether RNPs reduce infarct size without the occurrence of substantial side effects and whether nitric oxide (NO) contributes to the cardioprotective effects of RNPs.

Methods: The left anterior descending coronary arteries of dogs were occluded for 90 min, followed by reperfusion for 6 h. Either RNPs, micelles (not containing TEMPO) (control), or 4-hydroxy-TEMPO (TEMPOL) was injected into a systemic vein for 5 min before reperfusion. We evaluated the infarct size, myocardial apoptosis, plasma NO levels in coronary venous blood, and the RNP spectra using an electron paramagnetic resonance assay.

Results: RNPs reduced infarct size compared with the control group and TEMPOL group (19.5 ± 3.3 vs. 42.2 ± 3.7 vs. 30.2 ± 3.4%). RNPs also reduced myocardial apoptosis compared with the control and TEMPOL group. Coronary venous NO levels increased in the RNP group.

Conclusions: In conclusion, the administration of 2,2,6,6-tetramethylpiperidine-1-oxyl as a RNP exerted cardioprotective effects against ischemia and reperfusion injury in canine hearts without exerting unfavorable hemodynamic effects. RNPs may represent a promising new therapy for patients with acute myocardial infarction.
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http://dx.doi.org/10.1007/s10557-017-6758-6DOI Listing
December 2017
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