Duchenne muscular dystrophy: disease mechanism and therapeutic strategies

Duchenne muscular dystrophy (DMD) is a severe, progressive, and ultimately fatal disease of skeletal muscle wasting, respiratory insufficiency, and cardiomyopathy. The identification of the dystrophin gene as central to DMD pathogenesis has led to the understanding of the muscle membrane and the proteins involved in membrane stability as the focal point of the disease. The lessons learned from decades of research in human genetics, biochemistry, and physiology have culminated in establishing the myriad functionalities of dystrophin in striated muscle biology. Here, we review the pathophysiological basis of DMD and discuss recent progress toward the development of therapeutic strategies for DMD that are currently close to or are in human clinical trials. The first section of the review focuses on DMD and the mechanisms contributing to membrane instability, inflammation, and fibrosis. The second section discusses therapeutic strategies currently used to treat DMD. This includes a focus on outlining the strengths and limitations of approaches directed at correcting the genetic defect through dystrophin gene replacement, modification, repair, and/or a range of dystrophin-independent approaches. The final section highlights the different therapeutic strategies for DMD currently in clinical trials

Roteiros para aula prática de laboratório: Afinal, para que serve o coração?

Produção do Projeto Cientísta Mirim: 1. Bactérias a nosso favor? 2. Afinal, para que serve o coração? 3. Por que somos tão diferentes de outros animais e plantas? 4. Por que a malária ainda precisa ser estudada? 5. Remédio ou veneno? Por que é tão importante investigar a morte celular? Todos os produtos veja no link: Material de aprendizagem e prática laboratorial do Projeto Cientista Mirim sobre o coração por meio de um passeio através do sistema cardiovascular; conhecer a anatomia cardíaca com a atividade de dissecação do coração; infarto e valvulopatias.FiocruzCiências e BiologiaPrograma de Pós-graduação em Biociências e Biotecnologia - Cientista MirimPrograma de Pós-Graduação em Formação Científica, Educacional e Tecnológic

Dysregulation of Calcium Handling in Duchenne Muscular Dystrophy-Associated Dilated Cardiomyopathy: Mechanisms and Experimental Therapeutic Strategies

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease resulting in the loss of dystrophin, a key cytoskeletal protein in the dystrophin-glycoprotein complex. Dystrophin connects the extracellular matrix with the cytoskeleton and stabilizes the sarcolemma. Cardiomyopathy is prominent in adolescents and young adults with DMD, manifesting as dilated cardiomyopathy (DCM) in the later stages of disease. Sarcolemmal instability, leading to calcium mishandling and overload in the cardiac myocyte, is a key mechanistic contributor to muscle cell death, fibrosis, and diminished cardiac contractile function in DMD patients. Current therapies for DMD cardiomyopathy can slow disease progression, but they do not directly target aberrant calcium handling and calcium overload. Experimental therapeutic targets that address calcium mishandling and overload include membrane stabilization, inhibition of stretch-activated channels, ryanodine receptor stabilization, and augmentation of calcium cycling via modulation of the Serca2a/phospholamban (PLN) complex or cytosolic calcium buffering. This paper addresses what is known about the mechanistic basis of calcium mishandling in DCM, with a focus on DMD cardiomyopathy. Additionally, we discuss currently utilized therapies for DMD cardiomyopathy, and review experimental therapeutic strategies targeting the calcium handling defects in DCM and DMD cardiomyopathy

Human Heart Explant-Derived Extracellular Vesicles: Characterization and Effects on the In Vitro Recellularization of Decellularized Heart Valves

Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization.</jats:p

Dysregulation of Calcium Handling in Duchenne Muscular Dystrophy-Associated Dilated Cardiomyopathy: Mechanisms and Experimental Therapeutic Strategies

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease resulting in the loss of dystrophin, a key cytoskeletal protein in the dystrophin-glycoprotein complex. Dystrophin connects the extracellular matrix with the cytoskeleton and stabilizes the sarcolemma. Cardiomyopathy is prominent in adolescents and young adults with DMD, manifesting as dilated cardiomyopathy (DCM) in the later stages of disease. Sarcolemmal instability, leading to calcium mishandling and overload in the cardiac myocyte, is a key mechanistic contributor to muscle cell death, fibrosis, and diminished cardiac contractile function in DMD patients. Current therapies for DMD cardiomyopathy can slow disease progression, but they do not directly target aberrant calcium handling and calcium overload. Experimental therapeutic targets that address calcium mishandling and overload include membrane stabilization, inhibition of stretch-activated channels, ryanodine receptor stabilization, and augmentation of calcium cycling via modulation of the Serca2a/phospholamban (PLN) complex or cytosolic calcium buffering. This paper addresses what is known about the mechanistic basis of calcium mishandling in DCM, with a focus on DMD cardiomyopathy. Additionally, we discuss currently utilized therapies for DMD cardiomyopathy, and review experimental therapeutic strategies targeting the calcium handling defects in DCM and DMD cardiomyopathy.</jats:p

Investigations of an inducible intact dystrophin gene excision system in cardiac and skeletal muscle in vivo

AbstractWe sought here to induce the excision of a large intragenic segment within the intact dystrophin gene locus, with the ultimate goal to elucidate dystrophin protein function and stability in striated muscles in vivo. To this end, we implemented an inducible-gene excision methodology using a floxed allele approach, demarcated by dystrophin exons 2–79, in complementation with a cardiac and skeletal muscle directed gene deletion system for spatial–temporal control of dystrophin gene excision in vivo. Main findings of this study include evidence of significant intact dystrophin gene excision, ranging from ~ 25% in heart muscle to ~ 30–35% in skeletal muscles in vivo. Results show that despite evidence of significant dystrophin gene excision, no significant decrease in dystrophin protein content was evident by Western blot analysis, at three months post excision in skeletal muscles or by 6 months post gene excision in heart muscle. Challenges of in vivo dystrophin gene excision revealed acute deleterious effects of tamoxifen on striated muscles, including a transient down regulation in dystrophin gene transcription in the absence of dystrophin gene excision. In addition, technical limitations of incomplete dystrophin gene excision became apparent that, in turn, tempered interpretation. Collectively, these findings are in keeping with earlier studies suggesting the dystrophin protein to be long-lived in striated muscles in vivo; however, more rigorous quantitative analysis of dystrophin stability in vivo will require future works in which more complete gene excision can be demonstrated, and without significant off-target effects of the gene deletion experimental platform per se.</jats:p

Cardiac Sarcomere Signaling in Health and Disease

The cardiac sarcomere is a triumph of biological evolution wherein myriad contractile and regulatory proteins assemble into a quasi-crystalline lattice to serve as the central point upon which cardiac muscle contraction occurs. This review focuses on the many signaling components and mechanisms of regulation that impact cardiac sarcomere function. We highlight the roles of the thick and thin filament, both as necessary structural and regulatory building blocks of the sarcomere as well as targets of functionally impactful modifications. Currently, a new focus emerging in the field is inter-myofilament signaling, and we discuss here the important mediators of this mechanism, including myosin-binding protein C and titin. As the understanding of sarcomere signaling advances, so do the methods with which it is studied. This is reviewed here through discussion of recent live muscle systems in which the sarcomere can be studied under intact, physiologically relevant conditions

Systemic Infusion of Expanded CD133+ Cells and Expanded CD133+ Cell-Derived EVs for the Treatment of Ischemic Cardiomyopathy in a Rat Model of AMI

Myocardial infarction is a leading cause of death among all cardiovascular diseases. Cell therapies using a cell population enriched with endothelial progenitor cells (EPCs), expanded CD133+ cells, have promise as a therapeutic option for the treatment of ischemic areas after infarction. Recently, secreted membrane vesicles, including exosomes and microvesicles, have been recognized as new therapeutic candidates with important roles in intercellular and tissue communication. Expanded CD133+ cells have the ability to produce extracellular vesicles (EVs); however, their effect in the context of the heart is unknown. In the present study, we evaluated the effectiveness of the systemic application of expanded CD133+ cells and expanded CD133+ cell-derived EVs for the treatment of ischemic cardiomyopathy in a rat model of acute myocardial infarction (AMI) and examined the hypothesis that the EVs, because of their critical role in transferring regenerative signals from stem cells to the injured tissues, might elicit an equal or better therapeutic response than the expanded CD133+ cells. We demonstrate that the systemic application of expanded CD133+ cells and EVs has similar effects in infarcted rats. Few animals per group showed improvements in several heart and kidney parameters analyzed, but not significant differences were observed when comparing the groups. The systemic route may not be effective to treat ischemic cardiomyopathy; nonetheless, it may be a beneficial therapy to treat the side effects of AMI such as kidney damage

Systemic Infusion of Expanded CD133⁺ Cells and Expanded CD133⁺ Cell-Derived EVs for the Treatment of Ischemic Cardiomyopathy in a Rat Model of AMI

Myocardial infarction is a leading cause of death among all cardiovascular diseases. Cell therapies using a cell population enriched with endothelial progenitor cells (EPCs), expanded CD133+ cells, have promise as a therapeutic option for the treatment of ischemic areas after infarction. Recently, secreted membrane vesicles, including exosomes and microvesicles, have been recognized as new therapeutic candidates with important roles in intercellular and tissue communication. Expanded CD133+ cells have the ability to produce extracellular vesicles (EVs); however, their effect in the context of the heart is unknown. In the present study, we evaluated the effectiveness of the systemic application of expanded CD133+ cells and expanded CD133+ cell-derived EVs for the treatment of ischemic cardiomyopathy in a rat model of acute myocardial infarction (AMI) and examined the hypothesis that the EVs, because of their critical role in transferring regenerative signals from stem cells to the injured tissues, might elicit an equal or better therapeutic response than the expanded CD133+ cells. We demonstrate that the systemic application of expanded CD133+ cells and EVs has similar effects in infarcted rats. Few animals per group showed improvements in several heart and kidney parameters analyzed, but not significant differences were observed when comparing the groups. The systemic route may not be effective to treat ischemic cardiomyopathy; nonetheless, it may be a beneficial therapy to treat the side effects of AMI such as kidney damage.</jats:p

Human heart explant-derived extracellular vesicles: characterization and effects on the in vitro recellularization of decellularized heart valves

Submitted by Manoel Barata ([email protected]) on 2019-04-30T15:45:04Z No. of bitstreams: 1 ijms-20-01279.pdf: 4316554 bytes, checksum: 85ff9eae04abc5318258f7c318223836 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-05-20T14:02:52Z (GMT) No. of bitstreams: 1 ijms-20-01279.pdf: 4316554 bytes, checksum: 85ff9eae04abc5318258f7c318223836 (MD5)Made available in DSpace on 2019-05-20T14:02:52Z (GMT). No. of bitstreams: 1 ijms-20-01279.pdf: 4316554 bytes, checksum: 85ff9eae04abc5318258f7c318223836 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Curitiba, PR, Brasil.Universidade Tecnológica Federal do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Extracellular vesicles (EVs) are particles released from different cell types and represent key components of paracrine secretion. Accumulating evidence supports the beneficial effects of EVs for tissue regeneration. In this study, discarded human heart tissues were used to isolate human heart-derived extracellular vesicles (hH-EVs). We used nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to physically characterize hH-EVs and mass spectrometry (MS) to profile the protein content in these particles. The MS analysis identified a total of 1248 proteins. Gene ontology (GO) enrichment analysis in hH-EVs revealed the proteins involved in processes, such as the regulation of cell death and response to wounding. The potential of hH-EVs to induce proliferation, adhesion, angiogenesis and wound healing was investigated in vitro. Our findings demonstrate that hH-EVs have the potential to induce proliferation and angiogenesis in endothelial cells, improve wound healing and reduce mesenchymal stem-cell adhesion. Last, we showed that hH-EVs were able to significantly promote mesenchymal stem-cell recellularization of decellularized porcine heart valve leaflets. Altogether our data confirmed that hH-EVs modulate cellular processes, shedding light on the potential of these particles for tissue regeneration and for scaffold recellularization