RESUMEN
The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle.
Asunto(s)
Corazón Fetal/citología , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Movimiento Celular , Proliferación Celular , Femenino , Corazón Fetal/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Corazón/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , EmbarazoRESUMEN
Mammalian cardiomyocytes withdraw from the cell cycle shortly after birth, although it remains unclear how cardiomyocyte cell cycles behave during development. Compared to conventional immunohistochemistry in static observation, time-lapse imaging can reveal comprehensive data in hard-to-understand biological phenomenon. However, there are no reports of an established protocol of successful time-lapse imaging in mammalian heart. Thus, it is valuable to establish a time-lapse imaging system to enable the observation of cell cycle dynamics in living murine cardiomyocytes. This study sought to establish time-lapse imaging of murine heart to study cardiomyocyte cell cycle behavior. The Fucci (fluorescent ubiquitination-based cell cycle indicator) system can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei red, green and yellow, respectively, in living mammalian cells, and could therefore be useful to visualize the real-time cell cycle transitions in living murine heart. To establish a similar system for time-lapse imaging of murine heart, we first developed an ex vivo culture system, with the culture conditions determined in terms of sample state, serum concentration, and oxygen concentration. The optimal condition (slice culture, oxygen concentration 20%, serum concentration 10%) successfully mimicked physiological cardiomyocyte proliferation in vivo. Time-lapse imaging of cardiac slices from E11.5, E14.5, E18.5, and P1 Fucci-expressing transgenic mice revealed an elongated S/G2/M phase in cardiomyocytes during development. Our time-lapse imaging of murine heart revealed a gradual elongation of the S/G2/M phase during development in living cardiomyocytes.
Asunto(s)
Ciclo Celular/fisiología , Desarrollo Embrionario/fisiología , Miocitos Cardíacos/citología , Imagen de Lapso de Tiempo , Animales , Proliferación Celular , Embrión de Mamíferos , Femenino , Colorantes Fluorescentes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente/métodos , Miocitos Cardíacos/fisiología , Embarazo , Técnicas de Cultivo de Tejidos , UbiquitinaciónRESUMEN
Glycosylation is an important mechanism regulating various biological processes, including intercellular signaling and adhesion. α-1,6-fucosyltransferase (Fut8) belongs to a family of enzymes that determine the terminal structure of glycans. Fut8 is widely conserved from Caenorhabditis elegans to humans, and its mutants have been reported in humans, mice, and zebrafish. Although mutants show various symptoms, such as spinal deformity and growth retardation, its effects on skeletal muscles are unknown. We aimed to elucidate the function of Fut8 in skeletal muscle using zebrafish and C2C12 cells for evaluation. We observed that most fut8a morphants died at 2 days post-fertilization (dpf) or in earlier developmental stages even at low concentrations of morpholino oligonucleotides (MOs). Mutant juveniles also had small body sizes, and abnormal myocepta and sarcomere structures, suggesting that Fut8a plays important roles in myogenesis. Moreover, treatment of C2C12 cells with 2-fluorofucose (2FF), a fucosylation inhibitor, during cell differentiation dramatically reduced the expression of myogenic genes, such as Myomaker and other myogenic fusion genes, and inhibited myotube formation. These results indicate that Fut8 is an important factor in myogenesis, and myofusion in particular.
Asunto(s)
Fucosiltransferasas , Pez Cebra , Humanos , Animales , Ratones , Pez Cebra/genética , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Glicosilación , Desarrollo de Músculos/genéticaRESUMEN
Alteration of the nuclear Ca2+ transient is an early event in cardiac remodeling. Regulation of the nuclear Ca2+ transient is partly independent of the cytosolic Ca2+ transient in cardiomyocytes. One nuclear membrane protein, emerin, is encoded by EMD, and an EMD mutation causes Emery-Dreifuss muscular dystrophy (EDMD). It remains unclear whether emerin is involved in nuclear Ca2+ homeostasis. The aim of this study is to elucidate the role of emerin in rat cardiomyocytes by means of hypertrophic stimuli and in EDMD induced pluripotent stem (iPS) cell-derived cardiomyocytes in terms of nuclear structure and the Ca2+ transient. The cardiac hypertrophic stimuli increased the nuclear area, decreased nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient in cardiomyocytes. Emd knockdown cardiomyocytes showed similar properties after hypertrophic stimuli. The EDMD-iPS cell-derived cardiomyocytes showed increased nuclear area, decreased nuclear invagination, and increased half-decay time of the nuclear Ca2+ transient. An autopsied heart from a patient with EDMD also showed increased nuclear area and decreased nuclear invagination. These data suggest that Emerin plays a crucial role in nuclear structure and in the nuclear Ca2+ transient. Thus, emerin and the nuclear Ca2+ transient are possible therapeutic targets in heart failure and EDMD.
Asunto(s)
Calcio/metabolismo , Cardiomegalia/genética , Proteínas de la Membrana/genética , Distrofia Muscular de Emery-Dreifuss/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Angiotensina II/farmacología , Compuestos de Anilina/química , Animales , Remodelación Atrial , Cardiomegalia/metabolismo , Cardiomegalia/patología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Citoplasma/ultraestructura , Modelos Animales de Enfermedad , Endotelina-1/farmacología , Colorantes Fluorescentes/química , Regulación de la Expresión Génica , Compuestos Heterocíclicos con 3 Anillos/química , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/ultraestructura , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fenilefrina/farmacología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Remodelación Ventricular , Xantenos/químicaRESUMEN
SCN5A is abundant in heart and has a major role in INa. Loss-of-function mutation in SCN5A results in Brugada syndrome (BrS), which causes sudden death in adults. It remains unclear why disease phenotype does not manifest in the young even though mutated SCN5A is expressed in the young. The aim of the present study is to elucidate the timing of the disease manifestation in BrS. A gain-of-function mutation in SCN5A also results in Long QT syndrome type 3 (LQTS3), leading to sudden death in the young. Induced pluripotent stem cells (iPSCs) were generated from a patient with a mixed phenotype of LQTS3 and BrS with the E1784K SCN5A mutation. Here we show that electrophysiological analysis revealed that LQTS3/BrS iPSC-derived cardiomyocytes recapitulate the phenotype of LQTS3 but not BrS. Each ß-subunit of the sodium channel is differentially expressed in embryonic and adult hearts. SCN3B is highly expressed in embryonic hearts and iPSC-derived cardiomyocytes. A heterologous expression system revealed that INa of mutated SCN5A is decreased and SCN3B augmented INa of mutated SCN5A. Knockdown of SCN3B in LQTS3/BrS iPSC-derived cardiomyocytes successfully unmasked the phenotype of BrS. Isogenic control of LQTS3/BrS (corrected-LQTS3/BrS) iPSC-derived cardiomyocytes gained the normal electrophysiological properties.
RESUMEN
Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS-iPSCs can be models for MELAS but we should carefully select MELAS-iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling.
RESUMEN
Duchenne muscular dystrophy (DMD) is a chronic and life-threatening disease that is initially supported by muscle regeneration but eventually shows satellite cell exhaustion and muscular dysfunction. The life-long maintenance of skeletal muscle homoeostasis requires the satellite stem cell pool to be preserved. Asymmetric cell division plays a pivotal role in the maintenance of the satellite cell pool. Here we show that granulocyte colony-stimulating factor receptor (G-CSFR) is asymmetrically expressed in activated satellite cells. G-CSF positively affects the satellite cell population during multiple stages of differentiation in ex vivo cultured fibres. G-CSF could be important in developing an effective therapy for DMD based on its potential to modulate the supply of multiple stages of regenerated myocytes. This study shows that the G-CSF-G-CSFR axis is fundamentally important for long-term muscle regeneration, functional maintenance and lifespan extension in mouse models of DMD with varying severities.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/farmacología , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Regeneración/efectos de los fármacos , Células Satélite del Músculo Esquelético/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Longevidad/efectos de los fármacos , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Células Musculares/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Proteína MioD/genética , Proteína MioD/metabolismo , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Cultivo Primario de Células , Receptores de Factor Estimulante de Colonias de Granulocito/deficiencia , Regeneración/genética , Células Satélite del Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/patología , Transducción de Señal , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
After skeletal muscle injury, neutrophils, monocytes, and macrophages infiltrate the damaged area; this is followed by rapid proliferation of myoblasts derived from muscle stem cells (also called satellite cells). Although it is known that inflammation triggers skeletal muscle regeneration, the underlying molecular mechanisms remain incompletely understood. In this study, we show that granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is expressed in developing somites. G-CSFR and G-CSF were expressed in myoblasts of mouse embryos during the midgestational stage but not in mature myocytes. Furthermore, G-CSFR was specifically but transiently expressed in regenerating myocytes present in injured adult mouse skeletal muscle. Neutralization of endogenous G-CSF with a blocking antibody impaired the regeneration process, whereas exogenous G-CSF supported muscle regeneration by promoting the proliferation of regenerating myoblasts. Furthermore, muscle regeneration was markedly impaired in G-CSFR-knockout mice. These findings indicate that G-CSF is crucial for skeletal myocyte development and regeneration and demonstrate the importance of inflammation-mediated induction of muscle regeneration.