RESUMEN
RATIONALE: Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr+/- mice after a single systemic delivery of recombinant adeno-associated virus. OBJECTIVE: To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. METHODS AND RESULTS: Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat-associated 9 from Staphylococcus aureus) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr+/- neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. CONCLUSIONS: This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.
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Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Cardiomiopatías/terapia , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Distrofina/genética , Edición Génica/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Contracción Miocárdica , Músculos Papilares/metabolismo , Animales , Proteínas Asociadas a CRISPR/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Exones , Fibrosis , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Vectores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Mutación , Músculos Papilares/patología , Músculos Papilares/fisiopatología , Fenotipo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Recuperación de la Función , Utrofina/genéticaAsunto(s)
COVID-19/inmunología , Proteína Inhibidora del Complemento C1/metabolismo , Complemento C1s/antagonistas & inhibidores , SARS-CoV-2/inmunología , Trombosis/inmunología , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/inmunología , COVID-19/sangre , COVID-19/virología , Activación de Complemento/efectos de los fármacos , Activación de Complemento/inmunología , Complemento C1s/metabolismo , Inactivadores del Complemento/farmacología , Inactivadores del Complemento/uso terapéutico , Humanos , Inmunidad Innata/efectos de los fármacos , SARS-CoV-2/metabolismo , Trombosis/sangre , Trombosis/prevención & control , Tratamiento Farmacológico de COVID-19RESUMEN
Regulatory single-nucleotide polymorphisms (rSNPs) alter gene expression. Common approaches for identifying rSNPs focus on sequence variants in conserved regions; however, it is unknown what fraction of rSNPs is undetectable using this approach. We present a systematic analysis of gene expression variation at the single-nucleotide level in the Saccharomyces cerevisiae GAL1-10 regulatory region. We exhaustively mutated nearly every base and measured the expression of each variant with a sensitive dual reporter assay. We observed an expression change for 7% (43/582) of the bases in this region, most of which (35/43, 81%) reside in conserved positions. The most dramatic changes were caused by variants that produced AUGs upstream of the translation start (uAUGs), and we sought to understand the consequences and molecular mechanisms underlying this class of mutations. A genome-wide analysis showed that genes with uAUGs display significantly lower mRNA and protein levels than genes without uAUGs. To determine the generality of this mechanism, we introduced uAUGs into S. cerevisiae genes and observed significantly reduced expression in 17/21 instances (p < 0.01), suggesting that uAUGs are functional in a wide variety of sequence contexts. Quantification of mRNA and protein levels for uAUG mutants showed that uAUGs affect both transcription and translation. Expression of uAUG mutants under the upf1Δ strain demonstrated that uAUGs stimulate the nonsense-mediated decay pathway. Our results suggest that uAUGs are potent and widespread regulators of gene expression that act by attenuating both protein and RNA levels.
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Polimorfismo de Nucleótido Simple , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Regiones no Traducidas 5' , Secuencia de Bases , Secuencia Conservada , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Peripheral artery disease (PAD) is a major health burden in the world. Stem cell-based therapy has emerged as an attractive treatment option in regenerative medicine. In this study, we sought to test the hypothesis that stem cell-based therapy can ameliorate ischemia induced limb injury. METHODS: We isolated mesenchymal stem cells derived from human placentas (PMSCs) and intramuscularly transplanted them into injured hind limbs. Treatment with PMSCs reduced acute muscle fibers apoptosis induced by ischemia. RESULTS: PMSC treatment significantly enhanced regeneration of the injured hind limb by reducing fibrosis and enhancing running capacity when the animals were subjected to treadmill training. Mechanistically, injected PMSCs can modulate acute inflammatory responses by reducing neutrophil and macrophage infiltration following limb ischemia. ELISA assays further confirmed that PMSC treatment can also reduce pro-inflammatory cytokines, TNF-α and IL-6, and enhance anti-inflammatory cytokine, IL-10 at the injury sites. CONCLUSION: Taken together, our results demonstrated that PMSCs can be a potential effective therapy for treatment of PAD via immunomodulation.
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Tratamiento Basado en Trasplante de Células y Tejidos , Trasplante de Células Madre Mesenquimatosas , Enfermedad Arterial Periférica/terapia , Daño por Reperfusión/terapia , Animales , Extremidades/lesiones , Femenino , Humanos , Inmunomodulación , Ratones , Enfermedad Arterial Periférica/inmunología , Enfermedad Arterial Periférica/patología , Placenta/citología , Placenta/inmunología , Embarazo , Daño por Reperfusión/inmunología , Daño por Reperfusión/patologíaRESUMEN
BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) and adiposity measures are independently associated with the development of diabetes in African American (AA) adults. However, studies have not examined the combined interaction between RAAS and adiposity measures in relation to diabetes risk in AA adults. OBJECTIVE: We examined the longitudinal association of combined RAAS and adiposity measures with incident diabetes among AAs in the Jackson Heart Study. METHODS: AA adults were assessed at baseline (2000-2004) and over 12 years of follow-up. RAAS, anthropometric (waist circumference [WC], body mass index [BMI]) and adipokine (adiponectin, leptin, leptin: adiponectin ratio [LAR]) measures were collected at baseline. Aldosterone, WC, and LAR were chosen as the best predictor variables. The final model, adjusting for age, sex, education, occupation, systolic blood pressure, smoking, physical activity and RAAS altering medications, incorporated these variables and their interactions (WC*Aldosterone + LAR*Aldosterone) to explore their impact on incident diabetes. RESULTS: Among 3,220 participants without diabetes at baseline, there were 554 incident cases over a median follow-up of 7.5 years. Aldosterone, WC, and LAR were positively associated with incident diabetes (all p < 0.05). A significant interaction was found between WC and aldosterone with a greater association among individuals with lower WC. This interaction was significant in participants with prediabetes but not in those with normoglycemia. No significant interaction was found between log-LAR and aldosterone with risk of incident diabetes. CONCLUSION: Higher aldosterone in participants is associated with greater risk of diabetes, particularly among individuals with prediabetes and lower WC.
RESUMEN
TRIM family proteins play integral roles in the innate immune response to virus infection. MG53 (TRIM72) is essential for cell membrane repair and is believed to be a muscle-specific TRIM protein. Here we show human macrophages express MG53, and MG53 protein expression is reduced following virus infection. Knockdown of MG53 in macrophages leads to increases in type I interferon (IFN) upon infection. MG53 knockout mice infected with influenza virus show comparable influenza virus titres to wild type mice, but display increased morbidity accompanied by more accumulation of CD45+ cells and elevation of IFNß in the lung. We find that MG53 knockdown results in activation of NFκB signalling, which is linked to an increase in intracellular calcium oscillation mediated by ryanodine receptor (RyR). MG53 inhibits IFNß induction in an RyR-dependent manner. This study establishes MG53 as a new target for control of virus-induced morbidity and tissue injury.
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Gripe Humana/inmunología , Interferón beta/metabolismo , Proteínas de la Membrana/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Animales , Señalización del Calcio/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Inmunidad Innata , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Interferón beta/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , ARN Interferente Pequeño , Transducción de Señal/inmunología , Proteínas de Motivos Tripartitos/genéticaRESUMEN
Background The aortic valve of the heart experiences constant mechanical stress under physiological conditions. Maladaptive valve injury responses contribute to the development of valvular heart disease. Here, we test the hypothesis that MG 53 (mitsugumin 53), an essential cell membrane repair protein, can protect valvular cells from injury and fibrocalcific remodeling processes associated with valvular heart disease. Methods and Results We found that MG 53 is expressed in pig and human patient aortic valves and observed aortic valve disease in aged Mg53-/- mice. Aortic valves of Mg53-/- mice showed compromised cell membrane integrity. In vitro studies demonstrated that recombinant human MG 53 protein protects primary valve interstitial cells from mechanical injury and that, in addition to mediating membrane repair, recombinant human MG 53 can enter valve interstitial cells and suppress transforming growth factor-ß-dependent activation of fibrocalcific signaling. Conclusions Together, our data characterize valve interstitial cell membrane repair as a novel mechanism of protection against valvular remodeling and assess potential in vivo roles of MG 53 in preventing valvular heart disease.
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Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/metabolismo , Calcinosis/metabolismo , Proteínas de Motivos Tripartitos/biosíntesis , Remodelación Ventricular , Animales , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/fisiopatología , Biomarcadores/metabolismo , Western Blotting , Calcinosis/diagnóstico , Calcinosis/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Ecocardiografía , Humanos , Inmunohistoquímica , Masculino , Ratones , Transducción de Señal , Estrés Mecánico , PorcinosRESUMEN
The cornea plays an important role in transmitting light and providing protection to the eye, but is susceptible to injury and infection. Standard treatments for corneal wounds include topical lubricants, antibiotics, bandage contact lens, and surgery. However, these measures are often ineffective. Here we show that MG53, a protein with an essential role in cell membrane repair, contributes to the corneal injury-repair process. Native MG53 is present in the corneal epithelia, tear film, and aqueous humor, suggesting its potential function in corneal homeostasis. Knockout of MG53 in mice causes impaired healing and regenerative capacity following injury. Exogenous recombinant human MG53 (rhMG53) protein protects the corneal epithelia against mechanical injury and enhances healing by promoting migration of corneal fibroblasts. Using in vivo alkaline-induced injury to the rat cornea, we show that rhMG53 promotes re-epithelialization and reduces post-injury fibrosis and vascularization. Finally, we show that rhMG53 modulates TGF-ß-mediated fibrotic remodeling associated with corneal injury. Overall, our data support the bi-functional role of MG53 in facilitating corneal healing and maintaining corneal transparency by reducing fibrosis and vascularization associated with corneal injuries.
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Córnea/metabolismo , Lesiones de la Cornea/genética , Proteínas de la Membrana/genética , Cicatrización de Heridas/genética , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Córnea/efectos de los fármacos , Córnea/patología , Lesiones de la Cornea/metabolismo , Lesiones de la Cornea/fisiopatología , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis , Humanos , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Ratas , Proteínas Recombinantes/farmacología , Regeneración/efectos de los fármacos , Regeneración/genética , Roedores/genética , Roedores/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body.
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Proteínas de la Membrana/fisiología , Cicatrización de Heridas , Animales , Calcio/metabolismo , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Proteínas de la Membrana/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/metabolismo , Regeneración/genética , Biología de SistemasRESUMEN
Autophagy, a form of lysosomal degradation capable of eliminating dysfunctional proteins and organelles, is a cellular process associated with homeostasis. Autophagy functions in cell survival by breaking down proteins and organelles and recycling them to meet metabolic demands. However, aberrant up regulation of autophagy can function as an alternative to apoptosis. The duality of autophagy, and its regulation over cell survival/death, intimately links it with human disease. Non-coding RNAs regulate mRNA levels and elicit diverse effects on mammalian protein expression. The most studied non-coding RNAs to-date are microRNAs (miRNA). MicroRNAs function in post-transcriptional regulation, causing profound changes in protein levels, and affect many biological processes and diseases. The role and regulation of autophagy, whether it is beneficial or harmful, is a controversial topic in cardiovascular disease. A number of recent studies have identified miRNAs that target autophagy-related proteins and influence the development, progression, or treatment of cardiovascular disease. Understanding the mechanisms by which these miRNAs work can provide promising insight and potential progress towards the development of therapeutic treatments in cardiovascular disease.
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Autofagia/genética , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/patología , MicroARNs/genética , Animales , Autofagia/fisiología , Enfermedades Cardiovasculares/fisiopatología , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/patología , Cardiomiopatías Diabéticas/fisiopatología , Humanos , MicroARNs/metabolismo , Modelos Cardiovasculares , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Remodelación Ventricular/genética , Remodelación Ventricular/fisiologíaRESUMEN
BACKGROUND: Timely follow-up after hospitalization for heart failure (HF) is recommended. However, follow-up is suboptimal, especially in lower socioeconomic groups. Patient-centered solutions for facilitating follow-up post-HF hospitalization have not been extensively evaluated. METHODS AND RESULTS: Face-to-face surveys were conducted between 2015 and 2016 among 83 racially diverse adult patients (61% African American, 34% Caucasian, and 5% Other) hospitalized for HF at a university hospital centered in a low-income area of Columbus, Ohio. Patient perceptions of methods to facilitate follow-up post-HF hospitalization and likelihood of using interventions were investigated using a Likert scale: 1=very much to 5=not at all. Results were analyzed by Wilcoxon signed-rank test with Bonferroni correction. The response rate was 82%. The annual household income was <$35 000 for 49% of patients. An appointment near the patient's home was the most desired intervention (77%), followed by reminder message (73%), transportation to appointment (63%), and elimination of copayment (59%). Interventions most likely to be used if provided were similarly ranked: reminder message (48%), appointment near home (46%), elimination of copay (46%), and transportation to appointment (39%). There were significant differences (P=0.001) in high-ranking interventions related to location (appointment near home, transportation, home appointment) and reminder for visit compared with low-ranking interventions related to time (weekend appointment, appointment after 5 pm) and telemedicine. CONCLUSIONS: Among this cohort of racially diverse low-income patients hospitalized with HF, an appointment near the patient's home and a reminder message were the most desired interventions to facilitate follow-up. Further study of similar populations nationwide is warranted.
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Citas y Horarios , Insuficiencia Cardíaca/terapia , Hospitalización/estadística & datos numéricos , Cooperación del Paciente , Sistemas Recordatorios/instrumentación , Anciano , Femenino , Estudios de Seguimiento , Humanos , Masculino , Estudios Retrospectivos , Encuestas y CuestionariosRESUMEN
Metabolic syndrome is a cluster of risk factors, such as obesity, insulin resistance, and hyperlipidemia that increases the individual's likelihood of developing cardiovascular diseases. Patients inflicted with metabolic disorders also suffer from tissue repair defect. Mitsugumin 53 (MG53) is a protein essential to cellular membrane repair. It facilitates the nucleation of intracellular vesicles to sites of membrane disruption to create repair patches, contributing to the regenerative capacity of skeletal and cardiac muscle tissues upon injury. Since individuals suffering from metabolic syndrome possess tissue regeneration deficiency and MG53 plays a crucial role in restoring membrane integrity, we studied MG53 activity in mice models exhibiting metabolic disorders induced by a 6 month high-fat diet (HFD) feeding. Western blotting showed that MG53 expression is not altered within the skeletal and cardiac muscles of mice with metabolic syndrome. Rather, we found that MG53 levels in blood circulation were actually reduced. This data directly contradicts findings presented by Song et. al that indict MG53 as a causative factor for metabolic syndrome (Nature 494, 375-379). The diminished MG53 serum level observed may contribute to the inadequate tissue repair aptitude exhibited by diabetic patients. Furthermore, immunohistochemical analyses reveal that skeletal muscle fibers of mice with metabolic disorders experience localization of subcellular MG53 around mitochondria. This clustering may represent an adaptive response to oxidative stress resulting from HFD feeding and may implicate MG53 as a guardian to protect damaged mitochondria. Therapeutic approaches that elevate MG53 expression in serum circulation may be a novel method to treat the degenerative tissue repair function of diabetic patients.
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Proteínas Portadoras/sangre , Dieta Alta en Grasa/efectos adversos , Síndrome Metabólico/metabolismo , Mitocondrias/metabolismo , Animales , Proteínas Portadoras/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Proteínas de la Membrana , Síndrome Metabólico/inducido químicamente , Ratones , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Estrés OxidativoRESUMEN
We report a complex case of multivessel CAD in a patient with a porcelain aorta and high-grade left subclavian artery stenosis. Utilizing a staged left subclavian artery stent placement with a next-day plan for a four-vessel, on-pump CABG and ascending aortic replacement, this case highlights an organized approach to diagnosing and dealing with a heavily calcified aorta while describing a stepwise algorithm to deal with aortic calcifications prior to initiating cardiac surgery.