RESUMO
BACKGROUNDS: Microvillus inclusion disease (MVID) characterizes as intractable life-threatening watery diarrhea malnutrition after birth. MATERIALS & METHODS: Here we describe two patients with prenatal ultrasound findings of bowel dilation or increased amniotic fluid volume presented intractable diarrhea after birth. Exome sequencing and Intestinal biopsy were performed for the patients and their parents to reveal the underlying causes. The mutations were verified by Sanger sequencing and quantitative polymerase chain reaction. RESULTS: Exome sequencing revealed that both of the patients carrying MYO5B compound heterozygote mutations that were inherited from their parents. CONCLUSION: Here we describe two cases with MVID caused by MYO5B deficiency, which was the most common caused with prenatal ultrasound findings of bowel dilation and increased amniotic fluid volume. Due to the lack of effective curative therapies, early diagnosis even in prenatal of MVID can provide parents with better genetic counseling on the fetal prognosis.
Assuntos
Síndromes de Malabsorção/etiologia , Microvilosidades/patologia , Mucolipidoses/etiologia , Cadeias Pesadas de Miosina/deficiência , Miosina Tipo V/deficiência , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Síndromes de Malabsorção/genética , Masculino , Microvilosidades/genética , Mucolipidoses/genética , Mutação/genética , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Teste Pré-Natal não Invasivo/métodos , Ultrassonografia Pré-Natal/métodos , Sequenciamento do Exoma/métodosRESUMO
Non-muscle myosin IIA plays an important role in cell adhesion, cell migration, and tissue architecture. We previously showed that low activity of the heavy chain of non-muscle myosin II Myh9 is beneficial to LGR5+ intestinal stem cell maintenance. However, the function of Myh9 in adult mouse intestinal epithelium is largely unclear. In this study, we used the inducible Villin-creERT2 knockout approach to delete Myh9 in adult mouse intestinal epithelium and observed that homozygous deletion of Myh9 causes colitis-like morphologic changes in intestine, leads to a high sensitivity to dextran sulfate sodium and promotes colitis-related adenoma formation in the colon. Myh9 deletion disturbs cell junctions and impairs intestinal lumen barrier integrity, promoting the necroptosis of epithelial cells. Consistently, these changes can be partially rescued by Ripk3 knockout. Our results indicate that Myh9 is required for the maintenance of intestinal epithelium integrity and the prevention of cell necroptosis.
Assuntos
Adenoma/patologia , Colite/patologia , Neoplasias do Colo/patologia , Homeostase , Mucosa Intestinal/patologia , Cadeias Pesadas de Miosina/metabolismo , Necroptose , Animais , Antibacterianos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Deleção de Genes , Homeostase/efeitos dos fármacos , Homozigoto , Mucosa Intestinal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cadeias Pesadas de Miosina/deficiência , Necroptose/efeitos dos fármacos , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
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.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Cardiomegalia/prevenção & controle , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dependovirus/genética , Técnicas de Introdução de Genes , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/deficiência , Animais , Proteína 9 Associada à CRISPR/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Dependovirus/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Cadeias Pesadas de Miosina/genéticaRESUMO
Hypertrophic cardiomyopathy (HCM) is an inherited disorder of the myocardium, and pathogenic mutations in the sarcomere genes myosin heavy chain 7 (MYH7) and myosin-binding protein C (MYBPC3) explain 60%-70% of observed clinical cases. The heterogeneity of phenotypes observed in HCM patients, however, suggests that novel causative genes or genetic modifiers likely exist. Here, we systemically evaluated RNA-seq data from 28 HCM patients and nine healthy controls with pathogenic variant identification, differential expression analysis, and gene co-expression and protein-protein interaction network analyses. We identified 43 potential pathogenic variants in 19 genes in 24 HCM patients. Genes with more than one variant included the following: MYBPC3, TTN, MYH7, PSEN2, and LDB3. A total of 2538 protein-coding genes, six microRNAs (miRNAs), and 1617 long noncoding RNAs (lncRNAs) were identified differentially expressed between the groups, including several well-characterized cardiomyopathy-related genes (ANKRD1, FHL2, TGFB3, miR-30d, and miR-154). Gene enrichment analysis revealed that those genes are significantly involved in heart development and physiology. Furthermore, we highlighted four subnetworks: mtDNA-subnetwork, DSP-subnetwork, MYH7-subnetwork, and MYBPC3-subnetwork, which could play significant roles in the progression of HCM. Our findings further illustrate that HCM is a complex disease, which results from mutations in multiple protein-coding genes, modulation by non-coding RNAs and perturbations in gene networks.
Assuntos
Redes Reguladoras de Genes , Estudos de Associação Genética , Heterogeneidade Genética , Miocárdio/química , Análise de Sequência de RNA , Adulto , Miosinas Cardíacas/deficiência , Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Éxons/genética , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes/genética , Humanos , Mutação INDEL , Masculino , Pessoa de Meia-Idade , Mutação , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Polimorfismo de Nucleotídeo Único , RNA não Traduzido/genética , Fumar , Adulto JovemRESUMO
Congenital titinopathies are myopathies with variable phenotypes and inheritance modes. Here, we fully characterized, using an integrated approach (deep phenotyping, muscle morphology, mRNA and protein evaluation in muscle biopsies), two siblings with congenital multicore myopathy harboring three TTN variants predicted to affect titin stability and titin-myosin interactions. Muscle biopsies showed multicores, type 1 fiber uniformity and sarcomeric structure disruption with some thick filament loss. Immunohistochemistry and Western blotting revealed a marked reduction of fast myosin heavy chain isoforms. This is the first observation of a titinopathy suggesting that titin defect leads to secondary loss of fast myosin heavy chain isoforms.
Assuntos
Conectina/genética , Doenças Musculares , Cadeias Pesadas de Miosina/deficiência , Adolescente , Criança , Músculo Deltoide/patologia , Feminino , Humanos , Masculino , Doenças Musculares/congênito , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Linhagem , IrmãosRESUMO
Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.
Assuntos
Matriz Extracelular/metabolismo , Pneumopatias/metabolismo , Cadeias Pesadas de Miosina/deficiência , Miosina não Muscular Tipo IIB/deficiência , Sequência de Aminoácidos , Animais , Regulação para Baixo/genética , Enfisema/patologia , Etilnitrosoureia , Feminino , Pneumopatias/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Mutagênese/genética , Mutação de Sentido Incorreto/genética , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/química , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Organogênese , Fenótipo , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/metabolismo , Regulação para Cima/genéticaRESUMO
Genetic cholestasis has been dissected through genetic investigation. The major PFIC genes are now described. ATP8B1 encodes FIC1, ABCB11 encodes BSEP, ABCB4 encodes MDR3, TJP2 encodes TJP2, NR1H4 encodes FXR, and MYO5B encodes MYO5B. The full spectra of phenotypes associated with mutations in each gene are discussed, along with our understanding of the disease mechanisms. Differences in treatment response and targets for future treatment are emerging.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/metabolismo , Metabolismo dos Lipídeos , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/deficiência , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Colestase Intra-Hepática/diagnóstico , Homeostase , Humanos , Metabolismo dos Lipídeos/genética , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/deficiência , Miosina Tipo V/genética , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Proteína da Zônula de Oclusão-2/deficiência , Proteína da Zônula de Oclusão-2/genéticaRESUMO
Smooth muscle myosin heavy chain (SM-MHC) is exclusively expresses in smooth muscle, which takes part in smooth muscle cell contraction. Here, we used an insertional mutation mouse whose heavy polypeptide 11 (Myh11) gene has been disrupted and no SM-MHC protein has been detected. Compared to the wild-type and SM-MHC+/- mice, the SM-MHC-/- neonates had large round bellies, thin-walled giant bladders, and large stomachs with huge gas bubbles. Most of it died within 10 h and the rest within 20 h after birth. Further analysis of the developing foetuses from 16.5 days postcoitum (dpc) stage to newborn showed no significant (P<0.05) difference in the ratio of Mendelian inheritance and average body weight among SM-MHC+/+ , SM-MHC+/- and SM-MHC-/- mice, whereas the abnormal exterior appearance was observed in each SM-MHC-/- bladders from 16.5 dpc. Histological analysis showed no difference in stomach tissues but evidently thin-walled smooth muscle layer and a giant cavity in bladders of SM-MHC-/- foetuses at various stages from 15.5 dpc to newborn. The results indicated that the defect of SM-MHC lead to the bladder developing lesions initially at 15.5 dpc stage in mouse and also implied that the SM-MHC loss might result in the gas bubbles in stomach. The study should facilitate further detailed analyses of the potential role of SM-MHC in bladder and stomach development.
Assuntos
Desenvolvimento Fetal/genética , Mucosa Gástrica/metabolismo , Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Bexiga Urinária/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Feminino , Masculino , Camundongos , Camundongos Knockout , Músculo Liso/embriologia , Cadeias Pesadas de Miosina/deficiência , Estômago/embriologia , Fatores de Tempo , Bexiga Urinária/embriologiaRESUMO
Ronin (THAP11), a DNA-binding protein that evolved from a primordial DNA transposon by molecular domestication, recognizes a hyperconserved promoter sequence to control developmentally and metabolically essential genes in pluripotent stem cells. However, it remains unclear whether Ronin or related THAP proteins perform similar functions in development. Here, we present evidence that Ronin functions within the nascent heart as it arises from the mesoderm and forms a four-chambered organ. We show that Ronin is vital for cardiogenesis during midgestation by controlling a set of critical genes. The activity of Ronin coincided with the recruitment of its cofactor, Hcf-1, and the elevation of H3K4me3 levels at specific target genes, suggesting the involvement of an epigenetic mechanism. On the strength of these findings, we propose that Ronin activity during cardiogenesis offers a template to understand how important gene programs are sustained across different cell types within a developing organ such as the heart.
Assuntos
Coração/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Animais , Bradicardia/metabolismo , Bradicardia/fisiopatologia , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Imunoprecipitação da Cromatina , Ecocardiografia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Coração/diagnóstico por imagem , Histonas/genética , Histonas/metabolismo , Proteína Homeobox Nkx-2.5/deficiência , Proteína Homeobox Nkx-2.5/genética , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/metabolismo , Metilação , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética , Sítio de Iniciação de TranscriçãoRESUMO
BACKGROUND: Oral squamous cell carcinoma (OSCC) accounts for 95% of all oral cancer with higher mortality and morbidity rates worldwide. However, the potential molecular mechanism of OSCC remains largely unclear. Myosin VI (MYO6) is a unique actin motor and reported to be overexpressed in several cancers. This study aims to examine the functional relationship between OSCC and MYO6. METHODS: The mRNA expression of MYO6 was firstly investigated by analyzing data derived from Oncomine database. On the basis of the results, the expression of MYO6 was knocked down using lentivirus-delivered RNA interference in human OSCC cell line CAL27, as confirmed by qPCR and Western blot analysis. Stable MYO6 knockdown cells were employed to determine the effects of MYO6-silencing on cell growth by MTT, colony formation and cell cycle distribution and apoptosis by flow cytometry assay. Moreover, the expressions of cell apoptotic proteins were examined by Western blot analysis. RESULTS: We first observed MYO6 was overexpressed in tongue squamous cell carcinoma TSCC belongs to OSCC, compared with normal tissues. For cellular analysis, shRNA sequences against MYO6 could efficiently reduce its expression in CAL27 cells. Knockdown of MYO6 significantly decreased cell proliferation, caused cell cycle arrest at G2/M phase, and promoted cell apoptosis. Moreover, cell apoptosis-associated proteins, caspase-3 and PARP, were obviously upregulated in CAL27 after MYO6-silencing. CONCLUSION: MYO6 could play an essential role in the growth of OSCC cells via regulation of cell cycle progression and apoptosis.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias da Língua/genética , Neoplasias da Língua/patologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Sequência de Bases , Carcinoma de Células Escamosas/metabolismo , Caspase 3/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Neoplasias da Língua/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05µm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1µm per 0.35µm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0µm where TFL was ~1.05µm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.
Assuntos
Conectina/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Sarcômeros/fisiologia , Animais , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos , Microscopia , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Miofibrilas , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genéticaRESUMO
MYH14 is a member of the myosin family, which has been implicated in many motile processes such as ion-channel gating, organelle translocation, and the cytoskeleton rearrangement. Mutations in MYH14 lead to a DFNA4-type hearing impairment. Further evidence also shows that MYH14 is a candidate noise-induced hearing loss (NIHL) susceptible gene. However, the specific roles of MYH14 in auditory function and NIHL are not fully understood. In the present study, we used CRISPR/Cas9 technology to establish a Myh14 knockout mice line in CBA/CaJ background (now referred to as Myh14-/- mice) and clarify the role of MYH14 in the cochlea and NIHL. We found that Myh14-/- mice did not exhibit significant hearing loss until five months of age. In addition, Myh14-/- mice were more vulnerable to high intensity noise compared to control mice. More significant outer hair cell loss was observed in Myh14-/- mice than in wild type controls after acoustic trauma. Our findings suggest that Myh14 may play a beneficial role in the protection of the cochlea after acoustic overstimulation in CBA/CaJ mice.
Assuntos
Limiar Auditivo/fisiologia , Cóclea/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Perda Auditiva Provocada por Ruído/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Animais , Genótipo , Perda Auditiva Provocada por Ruído/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Cadeias Pesadas de Miosina/deficiência , Miosina Tipo II/deficiênciaAssuntos
Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Deleção de Genes , Homeostase/fisiologia , Ferro/sangue , Miócitos Cardíacos/metabolismo , Animais , Camundongos , Camundongos Knockout , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genéticaRESUMO
The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.
Assuntos
Fundo Gástrico/fisiologia , Relaxamento Muscular , Músculo Liso/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Óxido Nítrico/metabolismo , Pênis/fisiologia , Animais , Fundo Gástrico/efeitos dos fármacos , Fundo Gástrico/inervação , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Cadeias Pesadas de Miosina/deficiência , Miosina Tipo V/deficiência , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Pênis/efeitos dos fármacos , Pênis/inervaçãoRESUMO
RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.
Assuntos
Membrana Celular/metabolismo , Canal de Potássio Kv1.5/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Transporte Proteico/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Linhagem Celular , Conexina 43/análise , Canal de Potássio ERG1 , Endocitose , Canais de Potássio Éter-A-Go-Go/análise , Junções Comunicantes , Genes Reporter , Sistema de Condução Cardíaco/fisiopatologia , Transporte de Íons , Canal de Potássio Kv1.5/genética , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Cardiovasculares , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/deficiência , Miosina Tipo V/genética , Miosinas/deficiência , Miosinas/genética , Potássio/metabolismo , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
BACKGROUND: The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. METHODOLOGY/PRINCIPAL FINDINGS: Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a(-/-) platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin α(IIb)ß(3) activation, Ca(2+) signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a(-/-) platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. CONCLUSION: Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets.
Assuntos
Plaquetas/citologia , Plaquetas/metabolismo , Cadeias Pesadas de Miosina/deficiência , Miosina Tipo V/deficiência , Fenótipo , Animais , Sinalização do Cálcio , Forma Celular , Feminino , Masculino , Camundongos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Vesículas Secretórias/metabolismoRESUMO
Patients with a low platelet count are prone to bleeding. The occurrence of a thrombotic event in congenital thrombocytopenic patients is rare and puzzling. At least nine patients with Glanzmann thrombasthenia have been reported to have had a thrombotic event, eight venous and one arterial (intracardiac, in the left ventricle). On the contrary, three patients with Bernard-Soulier syndrome have been shown to have had arterial thrombosis (myocardial infarction) but no venous thrombosis. Finally, seven patients with the familiar macrothrombocytopenia due to alterations of the MYH9 gene have been reported to have had thrombosis (five myocardial infractions, one ischemic stroke, one deep vein thrombosis and one portal vein thrombosis). The significance of these findings is discussed with particular emphasis on the discrepancy between venous and arterial thrombosis seen in patients with Glanzmann thrombasthenia and Bernard-Soulier syndrome.
Assuntos
Síndrome de Bernard-Soulier/complicações , Trombastenia/complicações , Trombocitopenia/congênito , Trombofilia/genética , Trombose/etiologia , Adulto , Idoso , Isquemia Encefálica/epidemiologia , Isquemia Encefálica/etiologia , Criança , Pré-Escolar , Feminino , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Proteínas Motores Moleculares/deficiência , Proteínas Motores Moleculares/genética , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/etiologia , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Trombocitopenia/complicações , Trombose/epidemiologia , Trombose Venosa/epidemiologia , Trombose Venosa/etiologiaRESUMO
Cytokinesis, the final stage of cell division, bisects the cytoplasm into two daughter cells. In mitotic cells, this process depends on the activity of non-muscle myosin II (NMII), a family of actin-binding motor-proteins that participate in the formation of the cleavage furrow. The relevance of NMII for meiotic cell division, however, is poorly understood. The NMII family consists of three members, NMIIA, NMIIB, and NMIIC, containing different myosin heavy chains (MYH9, MYH10, and MYH14, respectively). We find that a single non-muscle myosin II, NMIIB, is required for meiotic cytokinesis in male but not female mice. Specifically, NMIIB-deficient spermatocytes exhibit cytokinetic failure in meiosis I, resulting in bi-nucleated secondary spermatocytes. Additionally, cytokinetic failure at meiosis II gives rise to bi-nucleated or even tetra-nucleated spermatids. These multi-nucleated spermatids fail to undergo normal differentiation, leading to male infertility. In spite of the presence of multiple non-muscle myosin II isoforms, we demonstrate that a single member, NMIIB, plays an essential and non-redundant role in cytokinesis during meiotic cell divisions of the male germline.
Assuntos
Citocinese/fisiologia , Meiose/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Espermatogênese/fisiologia , Animais , Divisão Celular/genética , Divisão Celular/fisiologia , Citocinese/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIB/deficiência , Miosina não Muscular Tipo IIB/genética , Espermátides/metabolismo , Espermátides/ultraestrutura , Espermatócitos/metabolismo , Espermatócitos/ultraestrutura , Espermatogênese/genética , Espermatogônias/metabolismo , Espermatogônias/ultraestrutura , Testículo/citologia , Testículo/metabolismoRESUMO
Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, we show that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with â¼100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabeling revealed that Arr2 in the cell body colocalized with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. We propose that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction.
Assuntos
Arrestinas/metabolismo , Cálcio/fisiologia , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Cadeias Pesadas de Miosina/deficiência , Miosina Tipo III/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Visão Ocular/fisiologia , Animais , Animais Geneticamente Modificados , Arrestinas/genética , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Proteínas do Olho/genética , Feminino , Masculino , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/genética , Células Fotorreceptoras de Invertebrados/citologia , Transporte Proteico/fisiologiaRESUMO
UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin.