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The small molecule chemical compound cinobufotalin (CB) is reported to be a potential antitumour drug that increases cisplatin (DDP) sensitivity in nasopharyngeal carcinoma. In this study, we first found that CB decreased DDP resistance, migration and invasion in lung adenocarcinoma (LUAD). Mechanistic studies showed that CB induced ENKUR expression by suppressing PI3K/AKT signalling to downregulate c-Jun, a negative transcription factor of ENKUR. Furthermore, ENKUR was shown to function as a tumour suppressor by binding to β-catenin to decrease c-Jun expression, thus suppressing MYH9 transcription. Interestingly, MYH9 is a binding protein of ENKUR. The Enkurin domain of ENKUR binds to MYH9, and the Myosin_tail of MYH9 binds to ENKUR. Downregulation of MYH9 reduced the recruitment of the deubiquitinase USP7, leading to increased c-Myc ubiquitination and degradation, decreased c-Myc nuclear translocation, and inactivation of epithelial-mesenchymal transition (EMT) signalling, thus attenuating DDP resistance. Our data demonstrated that CB is a promising antitumour drug and may be a candidate chemotherapeutic drug for LUAD patients.

Mutations in the MYH9 gene result in macrothrombocytopenia often associated with hemorrhages. Here, we studied the function and structure of platelets in three family members with a heterozygous mutation R1933X in the MYH9 gene, characteristic of closely related disorders known as the May-Hegglin anomaly and Sebastian syndrome. The examination included complete blood count, blood smear microscopy, platelet flow cytometry (expression of P-selectin and active integrin αIIbβ3 before and after activation), the kinetics of platelet-driven contraction (retraction) of blood clots, as well as scanning/transmission electron microscopy of platelets. Despite severe thrombocytopenia ranging (36–86) × 109/l, none of the patients had hemorrhages at the time of examination, although they had a history of heavy menstruation, spontaneous ecchymosis, and postpartum hemorrhage. Flow cytometry showed background platelet activation, revealed by overexpression of P-selectin and active αIIbβ3 integrin above normal levels. After TRAP-induced stimulation, the fractions of platelets expressing P-selectin in the proband and her sister were below normal response, indicating partial platelet refractoriness. The initiation of clot contraction was delayed. Electron microscopy revealed giant platelets with multiple filopodia and fusion of α-granules with dilated open canalicular system, containing filamentous and vesicular inclusions. The novel concept implies that the R1933X mutation in the MYH9 gene is associated not only with thrombocytopenia, but also with qualitative structural and functional defects in platelets. Platelet dysfunction includes impaired contractility, which can disrupt the compaction of hemostatic clots, making the clots weak and permeable, therefore predisposing patients with MYH9 gene mutations to the hemorrhagic phenotype.

Background Myosin heavy chain (MyHC) isoforms define the three major muscle fiber types in human extremity muscles. Slow beta/cardiac MyHC (MYH7) is expressed in type 1 muscle fibers. MyHC IIa (MYH2) and MyHC IIx (MYH1) are expressed in type 2A and 2B fibers, respectively. Whereas recessive MyHC IIa myopathy has been described in many cases, myopathy caused by dominant MYH2 variants is rare and has been described with clinical manifestations and muscle pathology in only one family and two sporadic cases. Methods We investigated three patients from one family with a dominantly inherited myopathy by clinical investigation, whole-genome sequencing, muscle biopsy, and magnetic resonance imaging (MRI). Results Three siblings, one woman and two men now 54, 56 and 66 years old, had experienced muscle weakness initially affecting the lower limbs from young adulthood. They have now generalized proximal muscle weakness affecting ambulation, but no ophthalmoplegia. Whole-genome sequencing identified a heterozygous MYH2 variant, segregating with the disease in the three affected individuals: c.5673 + 1G > C. Analysis of cDNA confirmed the predicted splicing defect with skipping of exon 39 and loss of residues 1860–1891 in the distal tail of the MyHC IIa, largely overlapping with the filament assembly region (aa1877–1905). Muscle biopsy in two of the affected individuals showed prominent type 1 muscle fiber predominance with only a few very small, scattered type 2A fibers and no type 2B fibers. The small type 2A fibers were frequently hybrid fibers with either slow MyHC or embryonic MyHC expression. The type 1 fibers showed variation in fiber size, internal nuclei and some structural alterations. There was fatty infiltration, which was also demonstrated by MRI. Conclusion Dominantly inherited MyHC IIa myopathy due to a splice defect causing loss of amino acids 1860–1891 in the distal tail of the MyHC IIa protein including part of the assembly competence domain. The myopathy is manifesting with slowly progressive muscle weakness without overt ophthalmoplegia and markedly reduced number and size of type 2 fibers.

Background Conditions affecting skeletal muscle, such as chronic rotator cuff tears, low back pain, dystrophies, and many others, often share changes in muscle phenotype: intramuscular adipose and fibrotic tissue increase while contractile tissue is lost. The underlying changes in cell populations and cell ratios observed with these phenotypic changes complicate the interpretation of tissue-level transcriptional data. Novel single-cell transcriptomics has limited capacity to address this problem because muscle fibers are too long to be engulfed in single-cell droplets and single nuclei transcriptomics are complicated by muscle fibers’ multinucleation. Therefore, the goal of this project was to evaluate the potential and challenges of a spatial transcriptomics technology to add dimensionality to transcriptional data in an attempt to better understand regional cellular activity in heterogeneous skeletal muscle tissue. Methods The 3′ Visium spatial transcriptomics technology was applied to muscle tissue of a rabbit model of rotator cuff tear. Healthy control and tissue collected at 2 and 16 weeks after tenotomy was utilized and freshly snap frozen tissue was compared with tissue stored for over 6 years to evaluate whether this technology is retrospectively useful in previously acquired tissues. Transcriptional information was overlayed with standard hematoxylin and eosin (H&E) stains of the exact same histological sections. Results Sequencing saturation and number of genes detected was not affected by sample storage duration. Unbiased clustering matched the underlying tissue type-based on H&E assessment. Connective-tissue-rich areas presented with lower unique molecular identifier counts are compared with muscle fibers even though tissue permeabilization was standardized across the section. A qualitative analysis of resulting datasets revealed heterogeneous fiber degeneration–regeneration after tenotomy based on (neonatal) myosin heavy chain 8 detection and associated differentially expressed gene analysis. Conclusions This protocol can be used in skeletal muscle to explore spatial transcriptional patterns and confidently relate them to the underlying histology, even for tissues that have been stored for up to 6 years. Using this protocol, there is potential for novel transcriptional pathway discovery in longitudinal studies since the transcriptional information is unbiased by muscle composition and cell type changes.

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here, we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types via the microRNA-mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells into the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.

Hearing loss (HL) is the most common sensory organ dysfunction disease. The cause is often complex, though genetics are the main factor. In this study, we investigated a Chinese family with non-syndromic delayed post-lingual deafness. Comprehensive data collection was performed on this family’s members, including basic information, audiological examinations, blood system examinations and imaging examinations. A pedigree diagram was drawn and the genetic patterns were analyzed. A new gene mutation, c.314A>T:p.Y105F in the MYH9 exon, was confirmed by next generation sequencing and Sanger sequencing. This mutation co-segregated with the phenotype in the pedigree. Patients in this family present bilateral symmetry and gradual and delayed high-frequency sensorineural hearing loss. The age of onset was approximately 30 years old. Except for hearing loss, no lesions were seen in other organs, especially the blood system. The identification and detection of a novel MYH9 mutation may be of great significance to provide the basis for gene function research and genetic consultation.

Given the controversies in the prognostic value of KIT mutations and optimal thresholds and time points of MRD monitoring for AML with CBFB-MYH11, we retrospectively evaluated 88 patients who underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT, n = 60) or autologous HSCT (Auto-HSCT, n = 28). The D816V KIT mutation was significantly associated with post-transplant relapse, contrasting with other types of mutations in KIT. Pre- and post-transplant (3 months after transplant) CBFB-MYH11 MRD assessments were useful in predicting post-transplant relapse and poor survival. The optimal threshold was determined as a 2 log reduction at both time points. In multivariate analysis, the D816V KIT mutation and CBFB-MYH11 MRD assessments were independently associated with post-transplant relapse and survival. Stratification by D816V KIT and pre-transplant CBFB-MYH11 MRD status further distinguished the risk of relapse and survival. Auto-HSCT was superior to Allo-HSCT in MRD negative patients without D816V KIT, while Allo-HSCT trended to be superior to Auto-HSCT in patients with MRD positivity or the D816V KIT mutation. In conclusion, this study demonstrated the differentiated prognostic value of the D816V KIT mutation in AML with CBFB-MYH11 and clarified optimal time points and thresholds for CBFB-MYH11 MRD monitoring in the setting of HSCT.

Muscle fibres are broadly categorised into types I and II; the fibre-type ratio determines the contractile and metabolic properties of skeletal muscle tissue. The maintenance of type I fibres is essential for the prevention of obesity and the treatment of muscle atrophy caused by type 2 diabetes or unloading. Some reports suggest that myokines are related to muscle fibre type determination. We thus explored whether a myokine determines whether satellite cells differentiate to type I fibres. By examining the fibre types separately, we identified R-spondin 3 (Rspo3) as a myokine of interest, a secreted protein known as an activator of Wnt signalling pathways. To examine whether Rspo3 induces type I fibres, primary myoblasts prepared from mouse soleus muscles were exposed to a differentiation medium containing the mouse recombinant Rspo3 protein. Expression of myosin heavy chain (MyHC) I, a marker of type I fibre, significantly increased in the differentiated myotubes compared with a control. The Wnt/β-catenin pathway was shown to be the dominant signalling pathway which induces Rspo3-induced MyHC I expression. These results revealed Rspo3 as a myokine that determines whether satellite cells differentiate to type I fibres.

Background Skeletal muscles consist of fibers of differing contractility and metabolic properties, which are primarily determined by the content of myosin heavy chain (MYH) isoforms (MYH7, MYH2, MYH1, and MYH4). The regulation of Myh genes transcription depends on three-dimensional chromatin conformation interaction, but the mechanistic details remain to be determined. Results In this study, we characterized the interaction profiles of Myh genes using 4C-seq (circular chromosome conformation capture coupled to high-throughput sequencing). The interaction profile of Myh genes changed between fast quadriceps and slow soleus muscles. Combining chromatin immunoprecipitation-sequencing (ChIP-seq) and transposase accessible chromatin with high-throughput sequencing (ATAC-seq), we found that a 38 kb intergenic region interacting simultaneously with fast Myh genes promoters controlled the coordinated expression of fast Myh genes. We also identified four active enhancers of Myh7, and revealed that binding of MYOG and MYOD increased the activity of Myh7 enhancers. Conclusions This study provides new insight into the chromatin interactions that regulate Myh genes expression.

Extramedullary infiltration (EMI), as a concomitant symptom of acute myeloid leukemia (AML), is associated with low complete remission and poor prognosis in AML. However, the mechanism of EMI remains indistinct. Clinical trials showed that increased miR-29s were associated with a poor overall survival in AML [14]. Nevertheless, they were proved to work as tumor suppressor genes by encouraging apoptosis and inhibiting proliferation in vitro. These contradictory results led us to the hypothesis that miR-29s may play a notable role in the prognosis of AML rather than leukemogenesis. Thus, we explored the specimens of AML patients and addressed this issue into miR-29c&b2 knockout mice. As a result, a poor overall survival and invasive blast cells were observed in high miR-29c&b2-expression patients, and the wildtype mice presented a shorter survival with heavier leukemia infiltration in extramedullary organs. Subsequently, we found that the miR-29c&b2 inside leukemia cells promoted EMI, but not the one in the microenvironment. The analysis of signal pathway revealed that miR-29c&b2 could target HMG-box transcription factor 1 (Hbp1) directly, then reduced Hbp1 bound to the promoter of non-muscle myosin IIB (Myh10) as a transcript inhibitor. Thus, increased Myh10 encouraged the migration of leukemia cells. Accordingly, AML patients with EMI were confirmed to have high miR-29c&b2 and MYH10 with low HBP1. Therefore, we identify that miR-29c&b2 contribute to the poor prognosis of AML patients by promoting EMI, and related genes analyses are prospectively feasible in assessment of AML outcome.

This study investigated the changes in myocardial myosin heavy chain (MHC) isoforms, MHC-α and MHC-β composition in young healthy rodents following endurance training, with and without post-exercise cold-water immersion (CWI). Male rats were either trained on a treadmill for 10 weeks with (CWI) or without (Ex) regular CWI after each running session, or left sedentary (CON). Left ventricular mRNA of MHC-α, MHC-β, thyroid receptor α1 (TR-α1) and β (TR-β) were analyzed using rt-PCR and semiquantitative PCR analysis. MHC isoform protein composition was determined using SDS-PAGE electrophoresis. MHC-α isoform protein was predominant in all groups. The relative expression of MHC-β (%MHC-β) protein was not different between groups (CWI 34.7 ± 6.9%; Ex 32 ± 5.3%; CON 35.5 ± 10%; P = 0.7). MHC-β mRNA was reduced in Ex (0.7 ± 0.3-fold) compared to CWI (1.3 ± 0.2-fold; P

Pacific oyster (Crassostrea gigas) is one of the most widely cultivated shellfish species in the world. Because of its economic value and complex life cycle involving drastic changes from a free-swimming larva to a sessile juvenile, C. gigas has been used as a model for developmental, environmental, and aquaculture research. However, due to the lack of genetic tools for functional analysis, gene functions associated with biological or economic traits cannot be easily determined. Here, we reported a successful application of CRISPR/Cas9 technology for knockout of myosin essential light chain gene (CgMELC) in C. gigas. C. gigas embryos injected with sgRNAs/Cas9 contained extensive indel mutations at the target sites. The mutant larvae showed defective musculature and reduced motility. In addition, knockout of CgMELC disrupted the expression and patterning of myosin heavy chain positive myofibers in larvae. Together, these data indicate that CgMELC is involved in larval muscle contraction and myogenesis in oyster larvae.

Human tubulin beta class IVa (TUBB4A) is a member of the β-tubulin family. In most normal tissues, expression of TUBB4A is little to none, but it is highly expressed in human prostate cancer. Here we show that high expression levels of TUBB4A are associated with aggressive prostate cancers and poor patient survival, especially for African-American men. Additionally, in prostate cancer cells, TUBB4A knockout (KO) reduces cell growth and migration but induces DNA damage through increased γH2AX and 53BP1. Furthermore, during constricted cell migration, TUBB4A interacts with MYH9 to protect the nucleus, but either TUBB4A KO or MYH9 knockdown leads to severe DNA damage and reduces the NF-κB signaling response. Also, TUBB4A KO retards tumor growth and metastasis. Functional analysis reveals that TUBB4A/GSK3β binds to the N-terminal of MYH9, and that TUBB4A KO reduces MYH9-mediated GSK3β ubiquitination and degradation, leading to decreased activation of β-catenin signaling and its relevant epithelial-mesenchymal transition. Likewise, prostate-specific deletion of Tubb4a reduces spontaneous tumor growth and metastasis via inhibition of NF-κB, cyclin D1, and c-MYC signaling activation. Our results suggest an oncogenic role of TUBB4A and provide a potentially actionable therapeutic target for prostate cancers with TUBB4A overexpression.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the sarcomere that causes otherwise unexplained cardiac hypertrophy and is associated with sudden death. While previous studies showed the role of the epigenetic modifier Brg1 in mouse models of HCM, additional work is needed to identify its role in humans. We tested the hypothesis that BRG1 expression is increased in periods of cardiac remodeling during fetal growth and in development of HCM. We employed immunohistochemical staining to evaluate protein expression of BRG1 in 796 human cardiac specimens (81 from patients with HCM) and describe elevated BRG1 expression in human fetal hearts in early development. In addition, we not only demonstrate increased expression of BRG1 in HCM, but we also show that other diseases that lead to heart failure have similar BRG1 expression to healthy controls. Inhibition of BRG1 in human induced pluripotent stem cell-derived cardiomyocytes significantly decreases MYH7 and increases MYH6, suggesting a regulatory role for BRG1 in the pathological imbalance of the two myosin heavy chain isoforms in human HCM. These data are the first demonstration of BRG1 as a specific biomarker for human HCM and provide foundation for future studies of epigenetics in human cardiac disease.

Despite the high regenerative capacity of skeletal muscle, volumetric muscle loss (VML) is an irrecoverable injury. One therapeutic approach is the implantation of engineered biologic scaffolds enriched with stem cells. The objective of this study is to investigate the synergistic effect of high-intensity interval training (HIIT) and stem cell transplantation with an amniotic membrane scaffold on innervation, vascularization and muscle function after VML injury. A VML injury was surgically created in the tibialis anterior (TA) muscle in rats. The animals were randomly assigned to three groups: untreated negative control group (untreated), decellularized human amniotic membrane bio-scaffold group (dHAM) and dHAM seeded with adipose-derived stem cells, which differentiate into skeletal muscle cells (dHAM-ADSCs). Then, each group was divided into sedentary and HIIT subgroups. The exercise training protocol consisted of treadmill running for 8 weeks. The animals underwent in vivo functional muscle tests to evaluate maximal isometric contractile force. Regenerated TA muscles were harvested for molecular analyses and explanted tissues were analyzed with histological methods. The main finding was that HIIT promoted muscle regeneration, innervation and vascularization in regenerated areas in HIIT treatment subgroups, especially in the dHAM-ADSC subgroup. In parallel with innervation, maximal isometric force also increased in vivo. HIIT upregulated neurotrophic factor gene expression in skeletal muscle. The amniotic membrane bio-scaffold seeded with differentiated ADSC, in conjunction with exercise training, improved vascular perfusion and innervation and enhanced the functional and morphological healing process after VML injury. The implications of these findings are of potential importance for future efforts to develop engineered biological scaffolds and for the use of interval training programs in rehabilitation after VML injury.

Myh7 is a classic biomarker for cardiac remodeling and a potential target to attenuate cardiomyocyte (CM) hypertrophy. This study aimed to identify the dominant function of Myh7 after birth and determine whether its removal would affect CM maturation or contribute to reversal of pathological hypertrophy phenotypes. The CASAAV (CRISPR/Cas9-AAV9-based somatic mutagenesis) technique was used to deplete Myh6 and Myh7, and an AAV dosage of 5 × 109 vg/g was used to generate a mosaic CM depletion model to explore the function of Myh7 in adulthood. CM hypertrophy was induced by transverse aortic constriction (TAC) in Rosa26Cas9-P2A-GFP mice at postnatal day 28 (PND28). Heart function was measured by echocardiography. Isolated CMs and in situ imaging were used to analyze the structure and morphology of CM. We discovered that CASAAV successfully silenced Myh6 and Myh7 in CMs, and early depletion of Myh7 led to mild adulthood lethality. However, the Myh7 PND28-knockout mice had normal heart phenotype and function, with normal cellular size and normal organization of sarcomeres and T-tubules. The TAC mice also received AAV-Myh7-Cre to produce Myh7-knockout CMs, which were also of normal size, and echocardiography demonstrated a reversal of cardiac hypertrophy. In conclusion, Myh7 has a role during the maturation period but rarely functions in adulthood. Thus, the therapeutic time should exceed the period of maturation. These results confirm Myh7 as a potential therapeutic target and indicate that its inhibition could help reverse CM hypertrophy.

Mitochondria supply ATP essential for synaptic transmission. Neurons face exceptional challenges in maintaining energy homoeostasis at synapses. Regulation of mitochondrial trafficking and anchoring is critical for neurons to meet increased energy consumption during sustained synaptic activity. However, mechanisms recruiting and retaining presynaptic mitochondria in sensing synaptic ATP levels remain elusive. Here we reveal an energy signalling axis that controls presynaptic mitochondrial maintenance. Activity-induced presynaptic energy deficits can be rescued by recruiting mitochondria through the AMP-activated protein kinase (AMPK)-p21-activated kinase (PAK) energy signalling pathway. Synaptic activity induces AMPK activation within axonal compartments and AMPK-PAK signalling triggers phosphorylation of myosin VI, which drives mitochondrial recruitment and syntaphilin-mediated anchoring on presynaptic filamentous actin. This pathway maintains presynaptic energy supply and calcium clearance during intensive synaptic activity. Disrupting this signalling cross-talk triggers local energy deficits and intracellular calcium build-up, leading to impaired synaptic efficacy during trains of stimulation and reduced recovery from synaptic depression after prolonged synaptic activity. Our study reveals a mechanistic cross-talk between energy sensing and mitochondria anchoring to maintain presynaptic metabolism, thus fine-tuning short-term synaptic plasticity and prolonged synaptic efficacy.

A 66-year-old woman had experienced abnormal bleeding since the age of 7. Thrombocytopenia was not detected until she was 48, and immune thrombocytopenia was diagnosed at age 66. She also reported experiencing hearing disturbance since the age of 30 and acute renal failure since the age of 61 but reported no visual disturbance. Her younger son, who was 40 years old, also experienced abnormal bleeding since the age of 6, but immune thrombocytopenia was diagnosed as late as age 35. He had no other associated disorders. Laboratory examinations of both mother and son revealed a low platelet count (8000 and 29,000 µL, respectively), giant platelets and Dohle body-like granulocyte inclusion bodies. The mother had a high creatinine level (15.4 mg/dL) and normal liver enzyme levels. MYH9 genetic analysis identified a heterozygous mutation, c.101T>A, p.Val34Glu at exon 2 in both patients. These clinical and laboratory findings were consistent with a diagnosis of an MYH9-related disorder with different phenotypes observed in the same family. MYH9-related disorders were recognised in 2003, but were often misdiagnosed as immune thrombocytopenia, and hence, they have rarely been reported in Taiwan.

Central nervous system (CNS) ganglioneuroblastoma is a rare neuroectodermal neoplasm and little is known about its clinical and biological features. Herein, we report a pediatric case of CNS ganglioneuroblastoma harboring MYO5A-NTRK3 fusion. The patient, a 4-year-old boy, underwent a partial resection of a supratentorial tumor that was histopathologically diagnosed as a CNS ganglioneuroblastoma. Treatment with radiotherapy was started per the St Jude Medulloblastoma 03 (SJMB03) protocol; however, the tumor progressed rapidly and radiotherapy was temporally discontinued. Meanwhile, the patient underwent a second surgery, in which a gross total resection was successfully performed, following which he completed the remaining protocol-based therapy. Although an early focal recurrence was detected for which he received additional radiotherapy and oral temozolomide, the patient remained in complete remission for 14 months after the completion of the treatment. A central pathological review and molecular analysis were performed that revealed a MYO5A-NTRK3 fusion. Interestingly, the MYO5A-NTRK3 fusion has been recurrently detected in melanocytic tumors but not in other types of tumors. Therefore, it can be speculated that our case might partly share tumorigenesis mechanisms with MYO5A-NTRK3-positive melanocytic tumors. In addition, our case may enable an improved understanding of the pathogenesis and clinical features of CNS ganglioneuroblastomas.

Several reports demonstrated the direct contribution of cytochrome P450 1B1 (CYP1B1) enzyme and its associated cardiotoxic mid-chain, hydroxyeicosatetraenoic acid (HETEs) metabolites in the development of cardiac hypertrophy. Resveratrol is commercially available polyphenol that exerts beneficial effects in wide array of cardiovascular diseases including cardiac hypertrophy, myocardial infarction and heart failure. Nevertheless, the underlying mechanisms responsible for these effects are not fully elucidated. Since resveratrol is a well-known CYP1B1 inhibitor, the purpose of this study is to test whether resveratrol attenuates angiotensin II (Ang II)-induced cellular hypertrophy through inhibition of CYP1B1/mid-chain HETEs mechanism. RL-14 and H9c2 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 2, 10 or 50 μM resveratrol for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography–mass spectrometry (LC/MS). Hypertrophic markers and CYP1B1 gene expression and protein levels were measured using real-time PCR and Western blot analysis, respectively. Our results demonstrated that resveratrol, at concentrations of 10 and 50 μM, was able to attenuate Ang-II-induced cellular hypertrophy as evidenced by substantial inhibition of hypertrophic markers, β-myosin heavy chain (MHC)/α-MHC and atrial natriuretic peptide. Ang II significantly induced the protein expression of CYP1B1 and increased the metabolite formation rate of its associated mid-chain HETEs. Interestingly, the protective effect of resveratrol was associated with a significant decrease of CYP1B1 protein expression and mid-chain HETEs. Our results provided the first evidence that resveratrol protects against Ang II-induced cellular hypertrophy, at least in part, through CYP1B1/mid-chain HETEs-dependent mechanism.

Tumor metastasis is a terrifying characteristic of cancer. Numerous studies have been conducted to overcome metastasis by targeting tumor microenvironment (TME). However, due to complexity of tumor microenvironment, it remained difficult for accurate targeting. Dwarf-lillytruf tuber monomer-13 (DT-13) possess good potential against TME. As TME is supportive for tumor metastasis, alternatively it is a challenging for therapeutic intervention. In our present study, we explored molecular mechanism through which TME induced cell migration and how DT-13 interferes in this mechanism. We used a novel model of co-culture system which is eventually developed in our lab. Tumor cells were co-cultured with hypoxia induced cancer-associated fibroblasts (CAF) or with chemically induced cancer-associated adipocytes (CAA). The effect of hypoxia in conditioned medium for CAF was assessed through expression of α-SMA and HIF by western blotting while oil red staining was done to assess the successful chemical induction for adipocytes (CAA), the effect of TME through conditioned medium on cell migration was analyzed by trans-well cell migration, and cell motility (wound healing) analyses. The expression changes in cellular proteins were assessed through western blotting and immunofluorescent studies. Our results showed that tumor microenvironment has a direct role in promoting breast cancer cell migration by stromal cells; moreover, we found that DT-13 restricts this TME regulated cell migration via targeting stromal cells in vitro. Additionally we also found that DT-13 targets NMII-A for its effect on breast cancer cell migration for the regulation of stromal cells in TME.

Myosin heavy chain 9 (MYH9) plays an essential role in human diseases, including multiple cancers; however, little is known about its role in gliomas. In the present study, we revealed that HMGA1 and MYH9 were upregulated in gliomas and their expression correlated with WHO grade, and HMGA1 promoted the acquisition of malignant phenotypes and chemoresistance of glioma cells by regulating the expression of MYH9 through c-Jun-mediated transcription. Moreover, MYH9 interacted with GSK-3β to inhibit the expression of GSK-3β protein by promoting its ubiquitination; the downregulation of GSK-3β subsequently promoted the nuclear translocation of β-catenin, enhancing growth, invasion, migration, and temozolomide resistance in glioma cells. Expression levels of HMGA1 and MYH9 were significantly correlated with patient survival and should be considered as independent prognostic factors. Our findings provide new insights into the role of HMGA1 and MYH9 in gliomagenesis and suggest the potential application of HMGA1 and MYH9 in cancer therapy in the future.

Chronic exposure to environmental pollutants threatens human health. Arsenic, a world-wide diffused toxicant, is associated to cardiac pathology in the adult and to congenital heart defects in the foetus. Poorly known are its effects on perinatal cardiomyocytes. Here, bioinformatic image-analysis tools were coupled with cellular and molecular analyses to obtain functional and structural quantitative metrics of the impairment induced by 0.1, 0.5 or 1.0 µM arsenic trioxide exposure on the perinatal-like cardiomyocyte component of mouse embryoid bodies, within their 3D complex cell organization. With this approach, we quantified alterations to the (a) beating activity; (b) sarcomere organization (texture, edge, repetitiveness, height and width of the Z bands); (c) cardiomyocyte size and shape; (d) volume occupied by cardiomyocytes within the EBs. Sarcomere organization and cell morphology impairment are paralleled by differential expression of sarcomeric α-actin and Tropomyosin proteins and of acta2, myh6 and myh7 genes. Also, significant increase of Cx40, Cx43 and Cx45 connexin genes and of Cx43 protein expression profiles is paralleled by large Cx43 immunofluorescence signals. These results provide new insights into the role of arsenic in impairing cytoskeletal components of perinatal-like cardiomyocytes which, in turn, affect cell size, shape and beating capacity.

Background Emerging evidence reveals that the initiation and development of human cancers, including colorectal cancer (CRC), are associated with the deregulation of circular RNAs (circRNAs). Our study intended to disclose the role of circ_0026416 in the malignant behaviors of CRC. Methods The detection for circ_0026416 expression, miR-545-3p expression, and myosin VI (MYO6) mRNA expression was performed using quantitative real-time PCR (qPCR). CCK-8 assay, colony formation assay, transwell assay, and flow cytometry assay were applied for functional analysis to monitor cell proliferation, migration, invasion, and apoptosis. The protein levels of MYO6 and epithelial mesenchymal-transition (EMT) markers were detected by western blot. Mouse models were used to determine the role of circ_0026416 in vivo. The potential relationship between miR-545-3p and circ_0026416 or MYO6 was verified by dual-luciferase reporter assay and RIP assay. Results The expression of circ_0026416 was increased in CRC tumor tissues and cell lines. Circ_0026416 downregulation inhibited CRC cell proliferation, colony formation, migration, invasion, and EMT but induced cell apoptosis in vitro, and circ_0026416 knockdown also blocked tumor growth in vivo. MiR-545-3p was a target of circ_0026416, and rescue experiments indicated that circ_0026416 knockdown blocked CRC development by enriching miR-545-3p. In addition, miR-545-3p targeted MYO6 and inhibited MYO6 expression. MiR-545-3p enrichment suppressed CRC cell malignant behaviors by sequestering MYO6. Importantly, circ_0026416 knockdown depleted MYO6 expression by enriching miR-545-3p. Conclusion Circ_0026416 downregulation blocked the development of CRC through depleting MYO6 expression by enriching miR-545-3p. Highlights Circ_0026416 downregulation inhibits CRC development in vitro and in vivo. Circ_0026416 regulates the expression of MYO6 by targeting miR-545-3p. Circ_0026416 governs the miR-545-3p/MYO6 axis to regulate CRC progression.

Background Coarctation of the aorta (CoA) is a serious innate heart disease. Although surgery results are generally good, some complications such as recoarctation and aortic aneurysm or persistent hypertension were serious threats to patient’s health. To better understand the pathology of CoA and its underlying molecular mechanism is particularly important for early diagnosis and preventing the occurrence of its complications. However, the mechanisms of CoA remain unclear, especially for infants. Methods RNA sequencing (RNA-seq) was used to identify the differentially expressed genes (DEGs) in vascular tissues of 12 patients with CoA and 10 normal participants form 3- to 34-month-old infants. The characteristic of DEGs were validated by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and immunochemical staining (IHC) in vessels of patients with CoA and normal infants. Results A total of 2491 DEGs with the false discovery rate less than 0.05(> 1.5-fold, P Conclusion This study comprehensively characterized the CoA transcriptome. Migration of extracellular matrix (ECM) and smooth muscle cells (SMCs) to the subendothelial space may be the major characteristic of CoA in infants.

Hypertrophic cardiomyopathy (HCM) is characterized by phenotypic heterogeneity. We investigated the molecular basis of the cardiac phenotype in two mouse models at established disease stage (mouse-HCM), and human myectomy tissue (human-HCM). We analyzed the transcriptome in 2 mouse models with non-obstructive HCM (R403Q-MyHC, R92W-TnT)/littermate-control hearts at 24 weeks of age, and in myectomy tissue of patients with obstructive HCM/control hearts (GSE36961, GSE36946). Additionally, we examined myocyte redox, cardiac mitochondrial DNA copy number (mtDNA-CN), mt-respiration, mt-ROS generation/scavenging and mt-Ca2+ handling in mice. We identified distinct allele-specific gene expression in mouse-HCM, and marked differences between mouse-HCM and human-HCM. Only two genes (CASQ1, GPT1) were similarly dysregulated in both mutant mice and human-HCM. No signaling pathway or transcription factor was predicted to be similarly dysregulated (by Ingenuity Pathway Analysis) in both mutant mice and human-HCM. Losartan was a predicted therapy only in TnT-mutant mice. KEGG pathway analysis revealed enrichment for several metabolic pathways, but only pyruvate metabolism was enriched in both mutant mice and human-HCM. Both mutant mouse myocytes demonstrated evidence of an oxidized redox environment. Mitochondrial complex I RCR was lower in both mutant mice compared to controls. MyHC-mutant mice had similar mtDNA-CN and mt-Ca2+ handling, but TnT-mutant mice exhibited lower mtDNA-CN and impaired mt-Ca2+ handling, compared to littermate-controls. Molecular profiling reveals differences in gene expression, transcriptional regulation, intracellular signaling and mt-number/function in 2 mouse models at established disease stage. Further studies are needed to confirm differences in gene expression between mouse and human-HCM, and to examine whether cardiac phenotype, genotype and/or species differences underlie the divergence in molecular profiles.

In marine mammals, muscular development has been identified as a rate-limiting factor in achieving adult dive capacities. This study investigates the rate that myosin heavy chain (MHC) composition matures in a postural and locomotor skeletal muscle for four pinniped species with different lactation lengths: hooded seals, Cystophora cristata; harp seals, Pagophilus groenlandicus; northern fur seals, Callorhinus ursinus, and Steller sea lions, Eumetopias jubatus. The ontogeny of MHC isoform expression was compared with developmental rates of myoglobin concentrations, and aerobic (citrate synthase, β-hydroxyacyl-CoA dehydrogenase) and anaerobic (lactate dehydrogenase) enzyme activities. Within taxonomic families, species with shorter lactation periods had more mature muscles biochemically at birth, and fiber types differentiated earlier during ontogeny (Phocidae: hooded > harp seals, Otariidae: northern fur seals > Steller sea lions). Northern fur seal neonates had the most phenotypically-mature muscles in this study, with no immature MHC isoforms. The relationship between muscle biochemistry and MHC composition became more pronounced with age, and developed to reflect swimming mode and activity levels. In adults, phocids had more slow-twitch oxidative protein in their primary locomotor muscle, the Longissimus dorsi (LD), than otariids which likely reflects oxygen-sparing strategies for the phocids’ longer dives. Conversely, northern fur seal muscles had higher proportions of fast-twitch MHCs in the Pectoralis and LD, likely indicative of this species’ smaller size and higher mass-specific metabolic rates. Thus, muscle phenotype is linked with species life history, and a mismatch between muscle biochemistry and MHC composition at weaning has important implications for the first year of independent foraging in pinniped pups.

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

Changes in stiffness or extensibility of the muscle or muscle–tendon unit with aging could lead to impaired function and an increased vulnerability to injury. We aimed to investigate the passive force and viscoelastic properties of single muscle fibers in older adults. Seven older adults (mean age 79.0 ± 3.8 years) and 10 young control (mean age 25.6 ± 4.5 years) were recruited. Biopsy specimens were obtained percutaneously from m. vastus lateralis and skinned single fibers were used for the experiments. Slack tests were performed to determine maximal force and maximal unloaded shortening velocity. Passive force was measured in pCa 9.0 solution using a stepwise stretch technique with increment of sarcomere length from 2.4 to 4.2 µm. Myosin heavy chain (MHC) isoform was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Specific force was calculated as maximal force divided by cross-sectional area. Passive force, peak passive force, time to half stress relaxation (T1/2) and force decay index (a force time integral under a stress relaxation curve) were measured. No difference between the groups were found in specific force and shortening velocity. Passive force and peak passive force were greater in both MHC I and IIa fibers of older adults (p

BACKGROUND: While stress and the absence of social support during pregnancy have been linked to poor health outcomes, the underlying biological mechanisms are unclear. METHODS: We examined whether adverse experiences during pregnancy alter DNA methylation (DNAm) in maternal epigenomes. Analyses included 250 African-American mothers from the Boston Birth Cohort. Genome-wide DNAm profiling was performed in maternal blood collected after delivery, using the Infinium HumanMethylation450 Beadchip. Linear regression models, with adjustment of pertinent covariates, were applied. RESULTS: While self-reported maternal psychosocial lifetime stress and stress during pregnancy was not associated with DNAm alterations, we found that absence of support from the baby’s father was significantly associated with maternal DNAm changes in TOR3A, IQCB1, C7orf36, and MYH7B, and that lack of support from family and friends was associated with maternal DNA hypermethylation on multiple genes, including PRDM16 and BANKL. CONCLUSIONS: This study provides intriguing results suggesting biological embedding of social support during pregnancy on maternal DNAm, warranting additional investigation, and replication.

Treatment of overt form of hypertrophic cardiomyopathy (HCM) is often unsuccessful. Efforts are focused on a possible early identification in order to prevent or delaying the development of hypertrophy. Our aim was to find an echocardiographic marker able to distinguish mutation carriers without left ventricular hypertrophy (LVH) from healthy subjects. We evaluated 28 patients, members of eight families. Three types of mutation were recognized: MYBPC3 (five families), MYH7 (two families) and TNNT2 (one family). According to genetic (G) and phenotypic (Ph) features, patients were divided in three groups: Group A (10 patients), mutation carriers with LVH (G+/Ph+); Group B (9 patients), mutation carriers without LVH (G+/Ph−); Group C (9 patients), healthy subjects (G−/Ph−). Echocardiography examination was performed acquiring standard 2D, DTI and 2D-strain imaging. Global longitudinal strain (GLS) and global radial strain (GRS) at basal and mid-level were measured. GRS was significantly different between group B and C at basal level (32.18% ± 9.6 vs. 44.59% ± 12.67 respectively; p-value < 0.0001). In basal posterior and basal inferior segments this difference was particularly evident. ROC curves showed for both the involved segments good AUCs (0.931 and 0.861 for basal posterior and inferior GRS respectively) with the best predictive cut-off for basal posterior GRS at 43.65%, while it was 38.4% for basal inferior GRS. Conversely, GLS values were similar in the three group. 2D longitudinal strain is a valid technique to study HCM. Radial strain and particularly basal posterior and inferior segmental reduction could be able to identify mutation carriers in a pre-clinical phase of disease.

There is a gap in the knowledge regarding regulation of local renin-angiotensin system (RAS) in skeletal muscle during development of obesity and insulin resistance in vivo. This study evaluates the obesity- and age-related changes in the expression of local RAS components. Since RAS affects skeletal muscle remodelling, we also evaluated the muscle fibre type composition, defined by myosin heavy chain (MyHC) mRNAs and protein content. Gene expressions were determined by qPCR and/or Western blot analysis in musculus quadriceps of 3- and 8-month-old male obese Zucker rats and their lean controls. The enzymatic activity of aminopeptidase A (APA) was determined flourometrically. Activation of renin receptor (ReR)/promyelocytic leukaemia zinc finger (PLZF) negative feedback mechanism was observed in obesity. The expression of angiotensinogen and AT1 was downregulated by obesity, while neutral endopeptidase and AT2 expressions were upregulated in obese rats with aging. Skeletal muscle APA activity was decreased by obesity, which negatively correlated with the increased plasma APA activity and plasma cholesterol. The expression of angiotensin-converting enzyme (ACE) positively correlated with MyHC mRNAs characteristic for fast-twitch muscle fibres. The obesity- and age-related alterations in the expression of both classical and alternative RAS components suggest an onset of a new equilibrium between ACE/AngII/AT1 and ACE2/Ang1-7/Mas at lower level accompanied by increased renin/ReR/PLZF activation. Increased APA release from the skeletal muscle in obesity might contribute to increased plasma APA activity. There is a link between reduced ACE expression and altered muscle MyHC proportion in obesity and aging.

Cells have elaborated a complex strategy to maintain protein homeostasis under physiological as well as stress conditions with the aim to ensure the smooth functioning of vital processes and producing healthy offspring. Impairment of one of the most important processes in living cells, translation, might have serious consequences including various brain disorders in humans. Here, we describe a variant of the translation initiation factor eIF3a, Rpg1-3, mutated in its PCI domain that displays an attenuated translation efficiency and formation of reversible assemblies at physiological growth conditions. Rpg1-3-GFP assemblies are not sequestered within mother cells only as usual for misfolded-protein aggregates and are freely transmitted from the mother cell into the bud although they are of non-amyloid nature. Their bud-directed transmission and the active movement within the cell area depend on the intact actin cytoskeleton and the related molecular motor Myo2. Mutations in the Rpg1-3 protein render not only eIF3a but, more importantly, also the eIF3 core complex prone to aggregation that is potentiated by the limited availability of Hsp70 and Hsp40 chaperones. Our results open the way to understand mechanisms yeast cells employ to cope with malfunction and aggregation of essential proteins and their complexes.

The regenerative effect of Epimedium and its major bioactive flavonoid icariin (ICA) have been documented in traditional medicine, but their effect on sarcopenia has not been evaluated. The aim of this study was to investigate the effects of Epimedium extract (EE) on skeletal muscle as represented by differentiated C2C12 cells. Here we demonstrated that EE and ICA stimulated C2C12 myotube hypertrophy by activating several, including IGF-1 signal pathways. C2C12 myotube hypertrophy was demonstrated by enlarged myotube and increased myosin heavy chains (MyHCs). In similar to IGF-1, EE/ICA activated key components of the IGF-1 signal pathway, including IGF-1 receptor. Pre-treatment with IGF-1 signal pathway specific inhibitors such as picropodophyllin, LY294002, and rapamycin attenuated EE induced myotube hypertrophy and MyHC isoform overexpression. In a different way, EE induced MHyC-S overexpression can be blocked by AMPK, but not by mTOR inhibitor. On the level of transcription, EE suppressed myostatin and MRF4 expression, but did not suppress atrogenes MAFbx and MuRF1 like IGF-1 did. Differential regulation of MyHC isoform and atrogenes is probably due to inequivalent AKT and AMPK phosphorylation induced by EE and IGF-1. These findings suggest that EE/ICA stimulates pathways partially overlapping with IGF-1 signaling pathway to promote myotube hypertrophy.

Background Colorectal cancer (CRC) is a common tumor characterized by its high mortality. However, the underlying molecular mechanisms that drive CRC tumorigenesis are unclear. Clock genes have important roles in tumor development. In the present study, the expression and functions of clock gene TIMELESS (encoding the Timeless protein) in CRC were investigated. Methods Immunohistochemistry, cell proliferation, migration, invasion, EMT and xenograft tumor experiments were used to prove the function of Timeless in the tumorigenesis of CRC. Immunoprecipitation, mass spectrometry, Immunofluorescence and Chromatin immunoprecipitation (ChIP) were utilized to clarify the mechanism of Timeless in regulating CRC tumorigenesis. Results We found that Timeless was upregulated in CRC tissues compared with corresponding normal tissues and its expression was closely associated with the TNM stages and overall survival of CRC patients. Functional studies demonstrated that Timeless promoted the proliferation, invasion, and EMT of CRC cells in vitro and in vivo. Mechanistic investigations showed that Timeless activated the β-catenin signal pathway by binding to Myosin-9, which binds to β-catenin to induce its nuclear translocation. The upregulation of Timeless was attributed to CREB-binding protein (CBP)/p300-mediated H3K27 acetylation of the promoter region of Timeless. Conclusion Timeless regulates the tumorigenesis of CRC by binding to and regulating myosin-9, suggesting Timeless might be a potential prognostic biomarker and therapeutic target for CRC.

Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the αMHC-MerCreMer/loxP system. However, there is an ongoing debate on the occurrence of cardiac side effects caused by unspecific Cre activity or related to tamoxifen/oil overload. Here, we investigated potential adverse effects of DNA editing by the αMHC-MerCreMer/loxP system in combination with a low-dose treatment protocol with the tamoxifen metabolite 4-hydroxytamoxifen (OH-Txf). αMHC-MerCreMer mice received intraperitoneally OH-Txf (20 mg/kg) for 5 or 10 days. These treatment protocols were highly efficient to induce DNA editing in adult mouse hearts. Multi-parametric magnetic resonance imaging revealed neither transient nor permanent effects on cardiac function during or up to 19 days after 5 day OH-Txf treatment. Furthermore, OH-Txf did not affect cardiac phosphocreatine/ATP ratios assessed by in vivo 31P MR spectroscopy, indicating no Cre-mediated side effects on cardiac energy status. No MRI-based indication for the development of cardiac fibrosis was found as mean T1 relaxation time was unchanged. Histological analysis of myocardial collagen III content after OH-Txf confirmed this result. Last, mean T2 relaxation time was not altered after Txf treatment suggesting no pronounced cardiac lipid accumulation or tissue oedema. In additional experiments, cardiac function was assessed for up to 42 days to investigate potential delayed side effects of OH-Txf treatment. Neither 5- nor 10-day treatment resulted in a depression of cardiac function. Efficient cardiomyocyte-restricted DNA editing that is free of unwanted side effects on cardiac function, energetics or fibrosis can be achieved in adult mice when the αMHC-MerCreMer/loxP system is activated by the tamoxifen metabolite OH-Txf. Supplementary Information The online version of this article (10.1007/s00395-020-00841-9) contains supplementary material, which is available to authorized users.

DNA sequencing-based measurable residual disease (MRD) detection has shown to be clinically relevant in AML. However, the same methodology cannot be applied to fusion gene-driven subtypes of AML such as core-binding factor AML (CBF-AML). Here in this study, we evaluated the effectiveness of using DNA and RNA sequencing in MRD detection and in tracking clonal dynamics in CBF-AML. Using RNA-seq, we were able to quantify expression levels of RUNX1-RUNX1T1 and CBFB-MYH11 at diagnosis and their levels of reduction during remission (P RUNX1-RUNX1T1 as measured by RNA-seq and qPCR were highly correlated (R2 = 0.74, P RUNX1-RUNX1T1 and cKIT-D816mut at diagnosis, stratified RUNX1-RUNX1T1 AML patients into three subgroups. These three subgroups had 2-year overall survival rates at 87%, 74%, and 33% (P RUNX1-RUNX1T1 AML.

Non-muscle myosin IIA heavy chain (MYH9) has been implicated in many physiological and pathological functions including cell adhesion, polarity, motility to cancer. However, its role in melanoma remains unexplored. The aim of our study was to evaluate the role of MYH9 in melanoma tumor development and metastasis and further to find out the potential underlying mechanisms. In this study, we evaluated the in vitro migratory and invasive properties and in vivo tumor development and metastasis in C57BL/6 mice by silencing MYH9 in B16F10 melanoma cells. Knocking down MYH9 enhanced migration and invasiveness of B16F10 cells in vitro. Furthermore, MYH9 silencing accelerated tumor growth and metastasis in melanoma subcutaneous and intravenous mouse models. Next, oncogenes analysis revealed epithelial-mesenchymal transition and Erk signaling pathway are being regulated with MYH9 expression. Finally, MYH9 silencing in B16F10 cells modulates the tumor microenvironment by manipulating the leukocytes and macrophages infiltration in tumors. These findings established the opposing role of MYH9 as a tumor suppressor in melanoma suggesting specific MYH9 based approaches in therapeutics.

Background Despite its established association with chronic kidney disease (CKD) the role of myosin-9 (MYH9) gene variation on transplanted kidney function remains unknown. This study aimed at evaluating the effect of donor MYH9 nephrogenic variants on renal allograft function within the first post transplantation year. Methods In the longitudinal kidney transplant study 207 deceased donors were genotyped for previously known risk MYH9 single nucleotide polymorphisms (SNPs). The predictor was MYH9 high–risk variants status. The primary outcome was mean eGFR found in low vs. high risk MYH9 genotypes between third and twelfth post-transplant month, the secondary outcome was the risk of proteinuria. Results Distribution of genotypes remained in Hardy-Weinberg equilibrium. The T allele of rs3752462 (dominant model, TT or TC vs. CC) was associated with higher filtration rate (P = 0.05) in a multivariate analysis after adjusting for delayed graft function and donor sex. Two G alleles of rs136211 (recessive model, GG vs. GA or AA) resulted in doubling the risk of proteinuria (OR = 2.22; 95% CI = 1.18–4.37, P = 0.017) after adjusting for donor and recipient sex. Conclusion Deceased donor kidneys of European descent harboring MYH9 SNPs rs3752462 T allele show significantly superior estimated filtration rate while those of rs136211 GG genotype excessive risk of proteinuria. These findings, if replicated, may further inform and improve individualization of allocation and treatment policies.

Background Animal model studies show that reductive stress is involved in cardiomyopathy and myopathy, but the exact physiological relevance remains unknown. In addition, the microRNAs miR-143 and miR-145 have been shown to be upregulated in cardiac diseases, but the underlying mechanisms associated with these regulators have yet to be explored. Methods We developed transgenic mouse lines expressing exogenous miR-143 and miR-145 under the control of the alpha-myosin heavy chain (αMHC) promoter/enhancer. Results The two transgenic lines showed dilated cardiomyopathy-like characteristics and early lethality with markedly increased expression of miR-143. The expression of hexokinase 2 (HK2), a cardioprotective gene that is a target of miR-143, was strongly suppressed in the transgenic hearts, but the in vitro HK activity and adenosine triphosphate (ATP) content were comparable to those observed in wild-type mice. In addition, transgenic complementation of HK2 expression did not reduce mortality rates. Although HK2 is crucial for the pentose phosphate pathway (PPP) and glycolysis, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was unexpectedly higher in the hearts of transgenic mice. The expression of gamma-glutamylcysteine synthetase heavy subunit (γ-GCSc) and the in vitro activity of glutathione reductase (GR) were also higher, suggesting that the recycling of GSH and its de novo biosynthesis were augmented in transgenic hearts. Furthermore, the expression levels of glucose-6-phosphate dehydrogenase (G6PD, a rate-limiting enzyme for the PPP) and p62/SQSTM1 (a potent inducer of glycolysis and glutathione production) were elevated, while p62/SQSTM1 was upregulated at the mRNA level rather than as a result of autophagy inhibition. Consistent with this observation, nuclear factor erythroid-2 related factor 2 (Nrf2), Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) were activated, all of which are known to induce p62/SQSTM1 expression. Conclusions Overexpression of miR-143 and miR-145 leads to a unique dilated cardiomyopathy phenotype with a reductive redox shift despite marked downregulation of HK2 expression. Reductive stress may be involved in a wider range of cardiomyopathies than previously thought.

Background Spinal cord injury (SCI) is a catastrophic injury that can cause irreversible motor dysfunction with high disability. Exosomes participate in the transport of miRNAs and play an essential role in intercellular communication via transfer of genetic material. However, the miRNAs in exosomes which derived from neurons, and the underlying mechanisms by which they contribute to SCI remain unknown. Methods A contusive in vivo SCI model and a series of in vitro experiments were carried out to explore the therapeutic effects of exosomes. Then, a miRNA microarray analysis and rescue experiments were performed to confirm the role of neuron-derived exosomal miRNA in SCI. Western blot, luciferase activity assay, and RNA-ChIP were used to investigate the underlying mechanisms. Results The results indicated that neuron-derived exosomes promoted functional behavioral recovery by suppressing the activation of M1 microglia and A1 astrocytes in vivo and in vitro. A miRNA array showed miR-124-3p to be the most enriched in neuron-derived exosomes. MYH9 was identified as the target downstream gene of miR-124-3p. A series of experiments were used to confirm the miR-124-3p/MYH9 axis. Finally, it was found that PI3K/AKT/NF-κB signaling cascades may be involved in the modulation of microglia by exosomal miR-124-3p. Conclusion A combination of miRNAs and neuron-derived exosomes may be a promising, minimally invasive approach for the treatment of SCI.

Cell migration plays pivotal roles in many biological processes; however, its underlying mechanism remains unclear. Here, we find that NudC-like protein 2 (NudCL2), a cochaperone of heat shock protein 90 (Hsp90), modulates cell migration by stabilizing both myosin-9 and lissencephaly protein 1 (LIS1). Either knockdown or knockout of NudCL2 significantly increases single-cell migration, but has no significant effect on collective cell migration. Immunoprecipitation–mass spectrometry and western blotting analyses reveal that NudCL2 binds to myosin-9 in mammalian cells. Depletion of NudCL2 not only decreases myosin-9 protein levels, but also results in actin disorganization. Ectopic expression of myosin-9 efficiently reverses defects in actin disorganization and single-cell migration in cells depleted of NudCL2. Interestingly, knockdown of myosin-9 increases both single and collective cell migration. Depletion of LIS1, a NudCL2 client protein, suppresses both single and collective cell migration, which exhibits the opposite effect compared with myosin-9 depletion. Co-depletion of myosin-9 and LIS1 promotes single-cell migration, resembling the phenotype caused by NudCL2 depletion. Furthermore, inhibition of Hsp90 ATPase activity also reduces the Hsp90-interacting protein myosin-9 stability and increases single-cell migration. Forced expression of Hsp90 efficiently reverses myosin-9 protein instability and the defects induced by NudCL2 depletion, but not vice versa. Taken together, these data suggest that NudCL2 plays an important role in the precise regulation of cell migration by stabilizing both myosin-9 and LIS1 via Hsp90 pathway.

Background We report a large family with four successive generations, presenting with a complex phenotype of severe congenital neutropenia (SCN), partially penetrant monocytosis, and hearing loss of varying severity. Methods We performed whole exome sequencing to identify the causative variants. Sanger sequencing was used to perform segregation analyses on remaining family members. Results We identified and classified a pathogenic GFI1 variant and a likely pathogenic variant in MYO6 which together explain the complex phenotypes seen in this family. Conclusions We present a case illustrating the benefits of a broad screening approach that allows identification of oligogenic determinants of complex human phenotypes which may have been missed if the screening was limited to a targeted gene panel with the assumption of a syndromic disorder. This is important for correct genetic diagnosis of families and disentangling the range and severity of phenotypes associated with high impact variants.

RIPOR2 (previously known as FAM65B) localizes to stereocilia of auditory hair cells and causes deafness when its function is disturbed by mutations. Here, we demonstrate that during the morphogenesis of the hair cell bundle, absence of Ripor2 affects the orientation of this key subcellular structure. We show that Ripor2 interacts with Myh9, a protein encoded by a known deafness gene. Absence of Ripor2 is associated with low Myh9 abundance in the mouse cochlea despite increased amount of Myh9 transcripts. While Myh9 is mainly expressed in stereocilia, a phosphorylated form of Myh9 is particularly enriched in the kinocilium. In Ripor2-deficient mice, kinocilium shows an aberrant localization which associates with a reduced content of phosphorylated Myh9. Acetylated alpha tubulin, another specific kinociliary protein which contributes to microtubule stabilization, is reduced in the absence of Ripor2 as well. We propose that Ripor2 deficiency influences abundance and/or post-translational modifications of proteins expressed in both stereocilia and kinocilia. This effect may have a negative impact on the structure and function of the auditory hair cell bundle.

Ninety acute myeloid leukemia (AML) patients with inv(16) were monitored CBFβ/MYH11 transcript around allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 23 patients received HLA-matched sibling donor transplantation (MSDT) and 67 patients received unmanipulated haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were analyzed in this study. Patients were divided into four groups based on CBFβ/MYH11 expression prior to transplantation (pre-MRD): with negative (group 1)/positive (group 2) pre-MRD before MSDT; with negative (group 3)/positive (group 4) pre-MRD before haplo-HSCT. The results showed that patients in group 2 had the highest cumulative incidence of relapse (2-year CIR, 40.7%), the lowest leukemia-free survival (2-year LFS, 50.8%), and overall survival (2-year OS, 62.5%). The other three groups of patients had comparable outcomes. The patients were also classified into the other three groups according to CBFβ/MYH11 value of + 1 month after transplantation: group 5: pre- and post-transplant MRD were both negative; group 6: the value of post-transplant MRD was lower than 0.2%; group 7: the value of post-transplant MRD was higher than 0.2%. Group 7 had the highest CIR and the lowest LFS. These results indicated that AML patients with inv(16) were able to be separated into high-risk and low-risk relapse groups based on peritransplant MRD determined by RQ-PCR-based CBFβ/MYH11. Haplo-HSCT might overcome the negative impact of pre-MRD on patient outcomes compared to MSDT.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-β cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

MODY 5 and 6 have been shown to be low-penetrant MODYs. As the genetic background of unknown MODY is assumed to be similar, a new analytical strategy is applied here to elucidate genetic predispositions to unknown MODY. We examined to find whether there are major MODY gene loci remaining to be identified using SNP linkage analysis in Japanese. Whole-exome sequencing was performed with seven families with typical MODY. Candidates for novel MODY genes were examined combined with in silico network analysis. Some peaks were found only in either parametric or non-parametric analysis; however, none of these peaks showed a LOD score greater than 3.7, which is approved to be the significance threshold of evidence for linkage. Exome sequencing revealed that three mutated genes were common among 3 families and 42 mutated genes were common in two families. Only one of these genes, MYO5A, having rare amino acid mutations p.R849Q and p.V1601G, was involved in the biological network of known MODY genes through the intermediary of the INS. Although only one promising candidate gene, MYO5A, was identified, no novel, high penetrant MODY genes might remain to be found in Japanese MODY.

This study aims to improve the classification of smooth muscle types to better understand their normal and pathological functional phenotypes. Four different smooth muscle tissues (aorta, muscular arteries, intestine, urinary bladder) with a 5-fold difference in maximal shortening velocity were obtained from mice and classified according to expression of the inserted myosin heavy chain (SMHC-B). Western blotting and quantitative PCR analyses were used to determine 15 metabolic and 8 cell signaling key components in each tissue. The slow muscle type (aorta) with a 12 times lower SMHC-B had 6-fold lower expression of the phosphatase subunit MYPT1, a 7-fold higher expression of Rhokinase 1, and a 3-fold higher expression of the PKC target CPI17, compared to the faster (urinary bladder) smooth muscle. The slow muscle had higher expression of components involved in glucose uptake and glycolysis (type 1 glucose transporter, 3 times; hexokinase, 13 times) and in gluconeogenesis (phosphoenolpyruvate carboxykinase, 43 times), but lower expression of the metabolic sensing AMP-activated kinase, alpha 2 isoform (5 times). The slow type also had higher expression of enzymes involved in lipid metabolism (hormone-sensitive lipase, 10 times; lipoprotein lipase, 13 times; fatty acid synthase, 6 times; type 2 acetyl-coenzyme A carboxylase, 8 times). We present a refined division of smooth muscle into muscle types based on the analysis of contractile, metabolic, and signaling components. Slow compared to fast smooth muscle has a lower expression of the deactivating phosphatase and upregulated Ca2+ sensitizing pathways and is more adapted for sustained glucose and lipid metabolism. © 2018 The Author(s) Correspondence Address: Arner, A.; Department of Physiology and Pharmacology, Karolinska Institutet, v Eulers v 8, Sweden; email: Anders.Arner@ki.seCC BY 4.0

Vertebrate skeletal muscles consist of heterogeneous tissues containing various types of muscle fibers, where specification of the fiber type is crucial for muscle development. Fish are an attractive experimental model to study the mechanisms of such fiber type specification because of the separated localization of slow and fast muscles in the trunk myotome. We examined regulation of expression of the torafugu gene of slow/cardiac-type myosin heavy chain, MYH M5 , and isolated an operational promoter in order to force its tissue-specific expression across different fish species via the transgenic approach in zebrafish and medaka. This promoter activity was observed in adaxial cell-derived superficial slow muscle fibers under the control of a hedgehog signal. We also uncovered coordinated expression of MYH M5 and Sox6b, which is an important transcriptional repressor for specification of muscle fiber types and participates in hedgehog signaling. Sequence comparison in the 5'-flanking region identified three conserved regions, CSR1-CSR3, between torafugu MYH M5 and its zebrafish ortholog. Analysis of deletion mutants showed that CSR1 significantly stimulates gene expression in slow muscle fibers. In contrast, deletion of CSR3 resulted in ectopic expression of a reporter gene in fast muscle fibers. CSR3 was found to contain a putative Sox family protein-binding site. These results indicate that the dual mechanism causing inhibition in fast muscle fibers and activation in slow muscle fibers is essential for slow muscle fiber-specific gene expression in fish.

Microvillus inclusion disease (MVID) is a severe form of neonatal diarrhea, caused mainly by mutations in MYO5B. Inactivating mutations in MYO5B causes depolarization of enterocytes in the small intestine, which gives rise to chronic, unremitting secretory diarrhea. While the pathology of the small intestine in MVID patients is well described, little is known about extraintestinal effects of MYO5B mutation. We examined stomach, liver, pancreas, colon, and kidney in Navajo MVID patients, who share a single homozygous MYO5B-P660L (1979C>T p.Pro660Leu, exon 16). Sections were stained for markers of the apical membrane to assess polarized trafficking. Navajo MVID patients showed notable changes in H/K-ATPase-containing tubulovesicle structure in the stomach parietal cells. Colonic mucosa was morphologically normal, but did show losses in apical ezrin and Syntaxin 3. Hepatocytes in the MVID patients displayed aberrant canalicular expression of the essential transporters MRP2 and BSEP. The pancreas showed small fragmented islets and a decrease in apical ezrin in pancreatic ducts. Kidney showed normal primary cilia. These findings indicate that the effects of the P660L mutation in MYO5B in Navajo MVID patients are not limited to the small intestine, but that certain tissues may be able to compensate functionally for alterations in apical trafficking.