RESUMEN

Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the ß-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pretreatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability, greater plasma membrane depolarization, and Ca2+ overload, which likely caused prolonged action potentials, delayed after depolarizations, and triggered activity. All these cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac-stress-induced arrhythmogenicity.

Asunto(s)

Cardiomiopatías , Conexina 43 , Ratones , Animales , Conexina 43/genética , Conexina 43/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Arritmias Cardíacas/metabolismo , Uniones Comunicantes , Canales Iónicos/metabolismo , Isoproterenol

RESUMEN

Dilated cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), an inherited degenerative disease of the cardiac and skeletal muscle caused by absence of the protein dystrophin. We showed one hallmark of DMD cardiomyopathy is the dysregulation of cardiac gap junction channel protein connexin-43 (Cx43). Proper Cx43 localization and function at the cardiac intercalated disc (ID) is regulated by post-translational phosphorylation of Cx43-carboxy-terminus residues S325/S328/S330 (pS-Cx43). Concurrently, Cx43 traffics along microtubules (MTs) for targeted delivery to the ID. In DMD hearts, absence of dystrophin results in a hyperdensified and disorganized MT cytoskeleton, yet the link with pS-Cx43 remains unaddressed. To gain insight into the relationship between MTs and pS-Cx43, DMD mice (mdx) and pS-Cx43-deficient (mdxS3A) mice were treated with an inhibitor of MT polymerization, colchicine (Colch). Colch treatment protected mdx, not mdxS3A mice, against Cx43 remodeling, improved MT directionality, and enhanced pS-Cx43/tubulin interaction. Likewise, severe arrhythmias were prevented in isoproterenol-stressed mdx, not mdxS3A mice. Furthermore, MT directionality was improved in pS-Cx43-mimicking mdx (mdxS3E). Mdxutr+/- and mdxutr+/-S3A mice, lacking one copy of dystrophin homolog utrophin, displayed enhanced cardiac fibrosis and reduced lifespan compared with mdxutr+/-S3E; and Colch treatment corrected cardiac fibrosis in mdxutr+/- but not mdxutr+/-S3A. Collectively, the data suggest that improved MT directionality reduces Cx43 remodeling and that pS-Cx43 is necessary and sufficient to regulate MT organization, which plays crucial role in correcting cardiac dysfunction in DMD mice. Thus, identification of novel organizational mechanisms acting on pS-Cx43-MT will help develop novel cardioprotective therapies for DMD cardiomyopathy.NEW & NOTEWORTHY We found that colchicine administration to Cx43-phospho-deficient dystrophic mice fails to protect against Cx43 remodeling. Conversely, Cx43-phospho-mimic dystrophic mice display a normalized MT network. We envision a bidirectional regulation whereby correction of the dystrophic MTs leads to correction of Cx43 remodeling, which in turn leads to further correction of the MTs. Our findings suggest a link between phospho-Cx43 and MTs that provides strong foundations for novel therapeutics in DMD cardiomyopathy.

Asunto(s)

Cardiomiopatías , Distrofia Muscular de Duchenne , Ratones , Animales , Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Conexina 43/genética , Conexina 43/metabolismo , Ratones Endogámicos mdx , Modelos Animales de Enfermedad , Arritmias Cardíacas/genética , Arritmias Cardíacas/prevención & control , Cardiomiopatías/genética , Cardiomiopatías/prevención & control , Microtúbulos/metabolismo , Colchicina , Fibrosis

RESUMEN

The dental pulp has irreplaceable roles in maintaining healthy teeth and its regeneration is a primary aim of regenerative endodontics. This study aimed to replicate the characteristics of dental pulp tissue by using cranial neural crest (CNC)-like cells (CNCLCs); these cells were generated by modifying several steps of a previously established method for deriving NC-like cells from induced pluripotent stem cells (iPSCs). CNC is the anterior region of the neural crest in vertebrate embryos, which contains the primordium of dental pulp cells or odontoblasts. The produced CNCLCs showed approximately 2.5-12,000-fold upregulations of major CNC marker genes. Furthermore, the CNCLCs exhibited remarkable odontoblastic differentiation ability, especially when treated with a combination of the fibroblast growth factors (FGFs) FGF4 and FGF9. The FGFs induced odontoblast marker genes by 1.7-5.0-fold, as compared to bone morphogenetic protein 4 (BMP4) treatment. In a mouse subcutaneous implant model, the CNCLCs briefly fated with FGF4 + FGF9 replicated dental pulp tissue characteristics, such as harboring odontoblast-like cells, a dentin-like layer, and vast neovascularization, induced by the angiogenic self-assembling peptide hydrogel (SAPH), SLan. SLan acts as a versatile biocompatible scaffold in the canal space. This study demonstrated a successful collaboration between regenerative medicine and SAPH technology.

RESUMEN

Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts. Mitochondrial proteins with a twofold change at time points up to 120 days relative to 12 days were subjected to ingenuity pathway analysis (IPA). The highest upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and branched chain amino acid (BCAA) degradation. The top upstream regulators predicted to be activated were peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1-α), the insulin receptor (IR), and the retinoblastoma protein (Rb1) transcriptional repressor. IPA and immunoblotting showed upregulation of the mitochondrial LonP1 protease-a regulator of mitochondrial proteostasis, energetics, and metabolism. LonP1 knockdown increased FAO in neonatal rat ventricular cardiomyocytes (nRVMs). Our results support the notion that LonP1 upregulation negatively regulates FAO in cardiomyocytes to calibrate the flux between glucose and fatty acid oxidation. We discuss potential mechanisms by which IR, Rb1, and LonP1 regulate the metabolic shift from glycolysis to OXPHOS and FAO. These newly identified factors and pathways may help in optimizing the maturation of iPSC-CMs.

Asunto(s)

Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/metabolismo , Biogénesis de Organelos , Proteoma , Proteómica , Animales , Línea Celular , Linaje de la Célula , Metabolismo Energético , Humanos , Ratones , Mitocondrias Cardíacas/genética , Proteínas Mitocondriales/genética , Ratas , Factores de Tiempo

RESUMEN

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder that affects males. However, 8% of female carriers are symptomatic and underrepresented in research due to the lack of animal models. We generated a symptomatic mouse model of DMD carriers via injection of mdx (murine DMD) embryonic stem cells (ESCs) into wild-type (WT) blastocysts (mdx/WT chimera). mdx/WT chimeras developed cardiomyopathic features and dystrophic skeletal muscle phenotypes including elevated mononuclear invasion, central nucleation, fibrosis and declined forelimb grip strength. The disease was accompanied by connexin-43 (Cx43) aberrantly enhanced in both cardiac and skeletal muscles and remodeled in the heart. Genetic reduction of Cx43-copy number in mdx/WT-Cx43(+/-) chimeras protected them from both cardiac and skeletal muscle fiber damage. In dystrophic skeletal muscle, Cx43 expression was not seen in the fibers but in adjacent F4/80+ mononuclear cells. Ethidium Bromide uptake in purified F4/80+/CD11b+ mdx macrophages revealed functional activity of Cx43, which was inhibited by administration of Gap19 peptide mimetic, a Cx43 hemichannel-specific inhibitor. Thus, we suggest that Cx43 reduction in symptomatic DMD carrier mice leads to prevention of Cx43 remodeling in the heart and prevention of aberrant Cx43 hemichannel activity in the skeletal muscle macrophages neighboring Cx43 non-expressing fibers.

Asunto(s)

Conexina 43/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatología , Animales , Cardiomiopatías/metabolismo , Conexina 43/genética , Modelos Animales de Enfermedad , Distrofina/genética , Femenino , Corazón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/metabolismo , Fuerza Muscular , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo

RESUMEN

The goal of this investigation was to compare the effects of chronic (4 wk) transverse aortic constriction (TAC) in Sprague-Dawley rats and C57BL/6J mice. TAC, after 1 day, induced similar left ventricular (LV) pressure gradients in both rats (n = 7) and mice (n = 7) (113 ± 5.4 vs. 103 ± 11.5 mmHg), and after 4 wk, the percent increase in LV hypertrophy, as reflected by LV/tibial length (51% vs 49%), was similar in rats (n = 12) and mice (n = 12). After 4 wk of TAC, LV systolic and diastolic function were preserved in TAC rats. In contrast, in TAC mice, LV ejection fraction decreased by 31% compared with sham, along with increases in LV end-diastolic pressure (153%) and LV systolic wall stress (86%). Angiogenesis, as reflected by Ki67 staining of capillaries, increased more in rats (n = 6) than in mice (n = 6; 10 ± 2 vs. 6 ± 1 Ki67-positive cells/field). Myocardial blood flow fell by 55% and coronary reserve by 28% in mice with TAC (n = 4), but they were preserved in rats (n = 4). Myogenesis, as reflected by c-kit-positive myocytes staining positively for troponin I, is another mechanism that can confer protection after TAC. However, the c-kit-positive cells in rats with TAC were all negative for troponin I, indicating the absence of myogenesis. Thus, rats showed relative tolerance to severe pressure overload compared with mice, with mechanisms involving angiogenesis but not myogenesis.

Asunto(s)

Hipertrofia Ventricular Izquierda/fisiopatología , Disfunción Ventricular Izquierda/fisiopatología , Función Ventricular Izquierda , Presión Ventricular , Remodelación Ventricular , Animales , Aorta/fisiopatología , Aorta/cirugía , Capilares/metabolismo , Capilares/fisiopatología , Circulación Coronaria , Modelos Animales de Enfermedad , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/metabolismo , Antígeno Ki-67/metabolismo , Ligadura , Ratones Endogámicos C57BL , Desarrollo de Músculos , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas Sprague-Dawley , Transducción de Señal , Especificidad de la Especie , Factores de Tiempo , Troponina I/metabolismo , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/metabolismo

RESUMEN

Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD); however, a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable alanines (mdxS3A). The mdxS3E, but not mdxS3A, mice were resistant to Cx43 remodeling, with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NADPH oxidase 2 (NOX2)/ROS production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality, and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation, and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43 remodeling and suggest that targeting Cx43 may be a therapeutic strategy for preventing heart dysfunction and arrhythmias in DMD patients.

Asunto(s)

Señalización del Calcio , Cardiomiopatías/metabolismo , Conexina 43/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Cardiomiopatías/genética , Cardiomiopatías/patología , Conexina 43/genética , Humanos , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Microtúbulos/genética , Microtúbulos/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Miocardio/patología , Miocitos Cardíacos/patología , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo

RESUMEN

Patients with Duchenne muscular dystrophy (DMD) commonly present with severe ventricular arrhythmias that contribute to heart failure. Arrhythmias and lethality are also consistently observed in adult Dmdmdx mice, a mouse model of DMD, after acute ß-adrenergic stimulation. These pathological features were previously linked to aberrant expression and remodeling of the cardiac gap junction protein connexin43 (Cx43). Here, we report that remodeled Cx43 protein forms Cx43 hemichannels in the lateral membrane of Dmdmdx cardiomyocytes and that the ß-adrenergic agonist isoproterenol (Iso) aberrantly activates these hemichannels. Block of Cx43 hemichannels or a reduction in Cx43 levels (using Dmdmdx Cx43+/- mice) prevents the abnormal increase in membrane permeability, plasma membrane depolarization, and Iso-evoked electrical activity in these cells. Additionally, Iso treatment promotes nitric oxide (NO) production and S-nitrosylation of Cx43 hemichannels in Dmdmdx heart. Importantly, inhibition of NO production prevents arrhythmias evoked by Iso. We found that NO directly activates Cx43 hemichannels by S-nitrosylation of cysteine at position 271. Our results demonstrate that opening of remodeled and S-nitrosylated Cx43 hemichannels plays a key role in the development of arrhythmias in DMD mice and that these channels may serve as therapeutic targets to prevent fatal arrhythmias in patients with DMD .

Asunto(s)

Arritmias Cardíacas/patología , Conexina 43/metabolismo , Distrofia Muscular de Duchenne/complicaciones , Miocitos Cardíacos/patología , Agonistas Adrenérgicos beta/toxicidad , Animales , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/patología , Permeabilidad de la Membrana Celular , Conexina 43/genética , Cisteína/metabolismo , Modelos Animales de Enfermedad , Distrofina/genética , Potenciales Evocados/efectos de los fármacos , Humanos , Isoproterenol/toxicidad , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Distrofia Muscular de Duchenne/genética , Mutación , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/metabolismo , Oocitos , Técnicas de Placa-Clamp , Xenopus laevis

RESUMEN

Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here, we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent on the autophagy-related 7 (Atg) conjugation system and LC3. This form of mitophagy played an essential role in protecting the heart against ischemia and was mediated by a protein complex consisting of unc-51 like kinase 1 (Ulk1), Rab9, receptor-interacting serine/thronine protein kinase 1 (Rip1), and dynamin-related protein 1 (Drp1). This complex allowed the recruitment of trans-Golgi membranes associated with Rab9 to damaged mitochondria through S179 phosphorylation of Rab9 by Ulk1 and S616 phosphorylation of Drp1 by Rip1. Knockin of Rab9 (S179A) abolished mitophagy and exacerbated the injury in response to myocardial ischemia, without affecting conventional autophagy. Mitophagy mediated through the Ulk1/Rab9/Rip1/Drp1 pathway protected the heart against ischemia by maintaining healthy mitochondria.

Asunto(s)

Mitocondrias Cardíacas , Mitofagia/genética , Isquemia Miocárdica , Miocardio , Transducción de Señal/genética , Proteínas de Unión al GTP rab , Animales , Autofagosomas/metabolismo , Autofagosomas/patología , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Isquemia Miocárdica/prevención & control , Miocardio/metabolismo , Miocardio/patología , Fosforilación/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo

RESUMEN

Duchenne muscular dystrophy (DMD) associated cardiomyopathy remains incurable. Connexin 43 (Cx43) is upregulated and remodeled in the hearts of mdx mice, a mouse model of DMD. Hearts from Wild Type, mdx, and mdx:Cx43(+/-) mice were studied before (4-6 months) and after (10-15 months) the onset of cardiomyopathy to assess the impact of decreasing Cx43 levels on cardiac pathology in dystrophic mice. Increased connexin 43 protein levels in mdx hearts were not observed in mdx:Cx43(+/-) hearts. Cx43 remodeling in mdx hearts was attenuated in mdx:Cx43(+/-) hearts. At time-point 4-6 months, isolated cardiomyocytes from mdx hearts displayed enhanced ethidium bromide uptake, augmented intracellular calcium signals and increased production of reactive oxygen species. These pathological features were improved in mdx:Cx43(+/-) cardiomyocytes. Isoproterenol-challenged mdx:Cx43(+/-) mice did not show arrhythmias or acute lethality observed in mdx mice. Likewise, isoproterenol-challenged mdx:Cx43(+/-) isolated hearts were also protected from arrhythmogenesis. At time-point 10-15 months, mdx:Cx43(+/-) mice showed decreased cardiac fibrosis and improved ventricular function, relative to mdx mice. These results suggest that normalization of connexin 43 protein levels in mdx mice reduces overall cardiac pathology.

Asunto(s)

Calcio/metabolismo , Cardiomiopatías/metabolismo , Conexina 43/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animales , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones Transgénicos , Distrofia Muscular de Duchenne/patología , Miocardio/patología , Miocitos Cardíacos/patología

RESUMEN

Sarcolipin (SLN) is an inhibitor of the sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) and is abnormally elevated in the muscle of Duchenne muscular dystrophy (DMD) patients and animal models. Here we show that reducing SLN levels ameliorates dystrophic pathology in the severe dystrophin/utrophin double mutant (mdx:utr -/-) mouse model of DMD. Germline inactivation of one allele of the SLN gene normalizes SLN expression, restores SERCA function, mitigates skeletal muscle and cardiac pathology, improves muscle regeneration, and extends the lifespan. To translate our findings into a therapeutic strategy, we knock down SLN expression in 1-month old mdx:utr -/- mice via adeno-associated virus (AAV) 9-mediated RNA interference. The AAV treatment markedly reduces SLN expression, attenuates muscle pathology and improves diaphragm, skeletal muscle and cardiac function. Taken together, our findings suggest that SLN reduction is a promising therapeutic approach for DMD.

Asunto(s)

Cardiomiopatías/fisiopatología , Regulación de la Expresión Génica/genética , Silenciador del Gen , Terapia Genética , Proteínas Musculares/genética , Distrofia Muscular de Duchenne/fisiopatología , Distrofia Muscular de Duchenne/terapia , Proteolípidos/genética , Animales , Cardiomiopatías/genética , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Proteínas Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Proteolípidos/metabolismo , Interferencia de ARN , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Utrofina/genética , Utrofina/metabolismo

RESUMEN

The process of human cardiac development can be faithfully recapitulated in a culture dish with human pluripotent stem cells, where the impact of environmental stressors can be evaluated. The consequences of ionizing radiation exposure on human cardiac differentiation are largely unknown. In this study, human-induced pluripotent stem cell cultures (hiPSCs) were subjected to an external beam of 3.7 MeV α-particles at low mean absorbed doses of 0.5, 3, and 10 cGy. Subsequently, the hiPSCs were differentiated into beating cardiac myocytes (hiPSC-CMs). Pluripotent and cardiac markers and morphology did not reveal differences between the irradiated and nonirradiated groups. While cell number was not affected during CM differentiation, cell number of differentiated CMs was severely reduced by ionizing radiation in a dose-responsive manner. ß-adrenergic stimulation causes calcium (Ca2+) overload and oxidative stress. Although no significant increase in Ca2+ transient amplitude was observed in any group after treatment with 1 µmol/L isoproterenol, the incidence of spontaneous Ca2+ waves/releases was more frequent in hiPSC-CMs of the irradiated groups, indicating arrhythmogenic activities at the single cell level. Increased transcript expression of mitochondrial biomarkers (LONP1, TFAM) and mtDNA-encoded genes (MT-CYB, MT-RNR1) was detected upon differentiation of hiPSC-CMs suggesting increased organelle biogenesis. Exposure of hiPSC-CM cultures to 10 cGy significantly upregulated MT-CYB and MT-RNR1 expression, which may reflect an adaptive response to ionizing radiation. Our results indicate that important aspects of differentiation of hiPSCs into cardiac myocytes may be affected by low fluences of densely ionizing radiations such as α-particles.

Asunto(s)

Diferenciación Celular/efectos de la radiación , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/efectos de la radiación , Proliferación Celular/efectos de la radiación , Células Cultivadas , ADN Mitocondrial/metabolismo , ADN Mitocondrial/efectos de la radiación , Humanos , Células Madre Pluripotentes Inducidas , Contracción Miocárdica/efectos de la radiación , Radiación Ionizante , Estrés Fisiológico/efectos de la radiación

RESUMEN

Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFß, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.

Asunto(s)

Fibrosis Endomiocárdica/genética , Hematopoyesis/genética , Proteínas Inhibidoras de la Diferenciación/deficiencia , Enfermedades Vasculares/genética , Animales , Apoptosis/genética , Células de la Médula Ósea/metabolismo , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Fibrosis Endomiocárdica/metabolismo , Fibrosis Endomiocárdica/patología , Células Endoteliales/metabolismo , Endotelio/metabolismo , Regulación de la Expresión Génica , Genotipo , Proteínas Inhibidoras de la Diferenciación/genética , Ratones , Ratones Noqueados , Trombospondina 1/metabolismo , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patología

RESUMEN

Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

Asunto(s)

Envejecimiento/patología , Quimera/metabolismo , Células Madre Embrionarias/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/patología , Miocardio/patología , Animales , Calcio/metabolismo , Conexina 43/metabolismo , Distrofina/metabolismo , Femenino , Pruebas de Función Cardíaca , Humanos , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Miocitos Cardíacos/metabolismo , Regeneración

RESUMEN

In both human chronic lymphocytic leukemia (CLL) and the New Zealand Black (NZB) murine model of CLL, decreased levels of microRNAs miR-15a/16 play an important role in the disease. Here we investigate the effects of this microRNA on early steps of B cell development and the capacity of miR-15a-deficient hematopoietic stem cells (HSC) and B1 progenitor cells (B1P) to reproduce CLL-like phenotype both in vitro and in vivo. Our results demonstrate that both miR-15a deficient HSC and B1P cells are capable of repopulating irradiated recipients and produce higher numbers of B1 cells than sources with normal miR-15a/16 levels. Furthermore, induced pluripotent stem (iPS) cells derived for the first time from NZB mice, provided insights into the B cell differentiation roadblock inherent in this strain. In addition, exogenously delivered miR-15a into the NZB derived B cell line provided valuable clues into novel targets such as Mmp10 and Mt2. Our data supports the hypothesis that miR-15a/16 deficient stem cells and B1Ps experience a maturation blockage, which contributes to B1 cells bias in development. This work will help understand the role of miR-15a in early events of CLL and points to B1P cells as potential cells of origin for this incurable disease.

Asunto(s)

Leucemia Linfocítica Crónica de Células B/metabolismo , MicroARNs/metabolismo , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Separación Celular , Modelos Animales de Enfermedad , Leucemia Linfocítica Crónica de Células B/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Células Madre Neoplásicas/metabolismo , Células Madre/metabolismo

RESUMEN

The Inhibitor of DNA Binding (Id) proteins play a crucial role in regulating hematopoiesis and are known to interact with E proteins and the bHLH family of transcription factors. Current efforts seek to elucidate the individual roles of Id members in regulating hematopoietic development and specification. However, the nature of their functional redundancies remains elusive since ablation of multiple Id genes is embryonically lethal. We developed a model to test this compensation in the adult. We report that global Id3 ablation with Tie2Cre-mediated conditional ablation of Id1 in both hematopoietic and endothelial cells (Id cDKO) extends viability to 1 year but leads to multi-lineage hematopoietic defects including the emergence of anemia associated with defective erythroid development, a novel phenotype unreported in prior single Id knockout studies. We observe decreased cell counts in the bone marrow and splenomegaly to dimensions beyond what is seen in single Id knockout models. Transcriptional dysregulation of hematopoietic regulators observed in bone marrow cells is also magnified in the spleen. E47 protein levels were elevated in Id cDKO bone marrow cell isolates, but decreased in the erythroid lineage. Chromatin immunoprecipitation (ChIP) studies reveal increased occupancy of E47 and GATA1 at the promoter regions of ß-globin and E2A. Bone marrow transplantation studies highlight the importance of intrinsic Id signals in maintaining hematopoietic homeostasis while revealing a strong extrinsic influence in the development of anemia. Together, these findings demonstrate that loss of Id compensation leads to dysregulation of the hematopoietic transcriptional network and multiple defects in erythropoietic development in adult mice.

Asunto(s)

Anemia , Eritropoyesis/genética , Eliminación de Gen , Células Madre Hematopoyéticas/metabolismo , Proteína 1 Inhibidora de la Diferenciación/deficiencia , Proteínas Inhibidoras de la Diferenciación/deficiencia , Anemia/genética , Anemia/metabolismo , Anemia/patología , Animales , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/metabolismo , Ratones , Ratones Noqueados , Factor de Transcripción 3/genética , Factor de Transcripción 3/metabolismo

RESUMEN

The neuronal nitric-oxide synthase (nNOS) splice variant nNOSµ is essential for skeletal muscle function. Its localization is dependent on dystrophin, which stabilizes the dystrophin glycoprotein complex (DGC) at the sarcolemma of skeletal muscle fibers. In Duchenne muscular dystrophy (DMD) dystrophin is absent and sarcolemmal nNOS is lost. This leads to functional ischemia due to a decrease in contraction-induced vasodilation. In cardiomyocytes, nNOSµ is believed to be the predominant NOS isoform. However, the association of nNOS with the DGC in the heart is unclear. Here, we report nNOS localization at the intercalated discs (IDs) of cardiomyocytes, where utrophin is highly expressed. In mdx, mdx:utr, nNOSµ knock-out (KO), and mdx:nNOSµ KO mice, we observed a gradual reduction of nNOS at IDs and disrupted ID morphology, compared to wild-type. In mdx:nNOSµ KO mice, but not in mdx or nNOSµ KO mice, we also observed an early development of cardiac fibrosis. These findings suggest that nNOS localization in the heart may not depend exclusively on the presence of dystrophin. Additionally, the ß1 subunit of soluble guanylyl cyclase (sGC), responsible for the production of cGMP through nitric oxide (NO) signaling, was also detected at the IDs. Together, our results suggest a new role of nNOS at the IDs for the cGMP-dependent NO pathway and the maintenance of ID morphology.

Asunto(s)

Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Utrofina/metabolismo , Animales , Guanilato Ciclasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo I/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Sarcolema/enzimología , Guanilil Ciclasa Soluble , Utrofina/genética

RESUMEN

Duchenne muscular dystrophy (DMD) is caused by an X-linked mutation that leads to the absence of dystrophin, resulting in life-threatening arrhythmogenesis and associated heart failure. We targeted the gap junction protein connexin43 (Cx43) responsible for maintaining cardiac conduction. In mild mdx and severe mdx:utr mouse models of DMD, and human DMD tissues, Cx43 was found to be pathologically mislocalized to lateral sides of cardiomyocytes. In addition, overall Cx43 protein levels were markedly increased in mouse and human DMD heart tissues examined. Electrocardiography on isoproterenol challenged mice showed that both models developed arrhythmias and died within 24 hours, while wild-type mice were free of pathology. Administering peptide mimetics to inhibit lateralized Cx43 function prior to challenge protected mdx mice from arrhythmogenesis and death, while mdx:utr mice displayed markedly improved ECG scores. These findings suggest that Cx43 lateralization contributes significantly to DMD arrhythmogenesis and that selective inhibition may provide substantial benefit.

Asunto(s)

Arritmias Cardíacas/tratamiento farmacológico , Conexina 43/antagonistas & inhibidores , Distrofia Muscular Animal/complicaciones , Distrofia Muscular de Duchenne/complicaciones , Adolescente , Animales , Arritmias Cardíacas/patología , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Isoproterenol , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/patología , Péptidos/farmacología , Péptidos/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética

RESUMEN

According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

Asunto(s)

Núcleo Celular/metabolismo , ADN Mitocondrial/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Animales , Transporte Biológico/fisiología , Diferenciación Celular/fisiología , Línea Celular , Cromosomas/genética , ADN Mitocondrial/genética , Células Madre Embrionarias/metabolismo , Dosificación de Gen/genética , Ratones , Ratones Endogámicos C57BL