RESUMO
BACKGROUND: Modeling of human arrhythmias with induced pluripotent stem cell-derived cardiomyocytes has focused on single-cell phenotypes. However, arrhythmias are the emergent properties of cells assembled into tissues, and the impact of inherited arrhythmia mutations on tissue-level properties of human heart tissue has not been reported. METHODS: Here, we report an optogenetically based, human engineered tissue model of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia caused by mutation of the cardiac ryanodine channel and triggered by exercise. We developed a human induced pluripotent stem cell-derived cardiomyocyte-based platform to study the tissue-level properties of engineered human myocardium. We investigated pathogenic mechanisms in CPVT by combining this novel platform with genome editing. RESULTS: In our model, CPVT tissues were vulnerable to developing reentrant rhythms when stimulated by rapid pacing and catecholamine, recapitulating hallmark features of the disease. These conditions elevated diastolic Ca2+ levels and increased temporal and spatial dispersion of Ca2+ wave speed, creating a vulnerable arrhythmia substrate. Using Cas9 genome editing, we pinpointed a single catecholamine-driven phosphorylation event, ryanodine receptor-serine 2814 phosphorylation by Ca2+/calmodulin-dependent protein kinase II, that is required to unmask the arrhythmic potential of CPVT tissues. CONCLUSIONS: Our study illuminates the molecular and cellular pathogenesis of CPVT and reveals a critical role of calmodulin-dependent protein kinase II-dependent reentry in the tissue-scale mechanism of this disease. We anticipate that this approach will be useful for modeling other inherited and acquired cardiac arrhythmias.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Taquicardia Ventricular/patologia , Taquicardia Ventricular/fisiopatologia , Engenharia Tecidual/métodos , Potenciais de Ação/fisiologia , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , Optogenética/métodosRESUMO
Biomedical research has relied on animal studies and conventional cell cultures for decades. Recently, microphysiological systems (MPS), also known as organs-on-chips, that recapitulate the structure and function of native tissues in vitro, have emerged as a promising alternative. However, current MPS typically lack integrated sensors and their fabrication requires multi-step lithographic processes. Here, we introduce a facile route for fabricating a new class of instrumented cardiac microphysiological devices via multimaterial three-dimensional (3D) printing. Specifically, we designed six functional inks, based on piezo-resistive, high-conductance, and biocompatible soft materials that enable integration of soft strain gauge sensors within micro-architectures that guide the self-assembly of physio-mimetic laminar cardiac tissues. We validated that these embedded sensors provide non-invasive, electronic readouts of tissue contractile stresses inside cell incubator environments. We further applied these devices to study drug responses, as well as the contractile development of human stem cell-derived laminar cardiac tissues over four weeks.
Assuntos
Miocárdio/citologia , Impressão Tridimensional/instrumentação , Análise Serial de Tecidos/instrumentaçãoRESUMO
Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (â¼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (â¼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease.
Assuntos
Forma Celular/fisiologia , Matriz Extracelular/fisiologia , Contração Muscular/fisiologia , Miócitos Cardíacos/fisiologia , Actinas/fisiologia , Animais , Elasticidade , Hidrogéis , Miócitos Cardíacos/citologia , Ratos , Ratos Sprague-DawleyRESUMO
The Murphy Roths Large (MRL) mouse, a strain capable of regenerating right ventricular myocardium, has a high postmyocardial infarction (post-MI) survival rate compared with C57BL/6J (C57) mice. The biological processes responsible for this survival advantage are unknown. To assess the effect of genetic background, the LG/J strain, which harbours 75% of the MRL composite genome, was included in the study. The MRL survival advantage versus C57 mice (92 versus 68%, P < 0.05) occurred primarily in the first 5 days; LG/J survival was intermediate (P = n.s.). Microarray data analysis revealed an attenuation of apoptotic (P < 0.05) and stress response transcripts in MRL hearts compared with C57 hearts post-MI. Supporting the microarray results, there were fewer TUNEL-positive cells 1 day post-MI in MRL infarcts compared with C57 infarcts (P = 0.001) and fewer CD45-positive cells in the MRL infarct border zone 2 days post-MI (P < 0.01); the LG/J results were intermediate (P = n.s.). The MRL hearts had smaller infarct scars and attenuated ventricular dilatation 30 days post-MI compared with C57 hearts (P < 0.05). We conclude that the early post-MI survival advantage of MRL mice over the C57 strain is mediated at least in part by reductions in apoptosis and inflammatory infiltration, and that these reductions may influence chronic remodelling. The intermediate survival, apoptosis and inflammation profile of LG/J mice suggests that this high tolerance for MI in the MRL mouse could be derived from its shared genetic background with the LG/J mouse.
Assuntos
Apoptose/genética , Inflamação/genética , Infarto do Miocárdio/genética , Remodelação Ventricular/genética , Animais , Dilatação/métodos , Coração/fisiologia , Marcação In Situ das Extremidades Cortadas/métodos , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Taxa de SobrevidaRESUMO
The dynamic changes in estrogen levels throughout aging and during the menstrual cycle influence wound healing. Elevated estrogen levels during the pre-ovulation phase accelerate tissue repair, whereas reduced estrogen levels in post-menopausal women lead to slow healing. Although previous reports have shown that estrogen may potentiate healing by triggering the estrogen receptor (ER)-ß signaling pathway, its binding to ER-α has been associated with severe collateral effects and has therefore limited its use as a therapeutic agent. To this end, soy phytoestrogens, which preferentially bind to the ER-ß, are currently being explored as a safer therapeutic alternative to estrogen. However, the development and evaluation of phytoestrogen-based materials as local ER-ß modulators remains largely unexplored. Here, we engineered biomimetic and estrogenic nanofiber wound dressings built from soy protein isolate (SPI) and hyaluronic acid (HA) using immersion rotary jet spinning. These engineered scaffolds were shown to successfully recapitulate the native dermal architecture, while delivering an ER-ß-triggering phytoestrogen (genistein). When tested in ovariectomized mouse and ex vivo human skin tissues, HA/SPI scaffolds outperformed controls (no treatment or HA only scaffolds) towards promoting cutaneous tissue repair. These improved healing outcomes were prevented when the ER-ß pathway was genetically or chemically inhibited. Our findings suggest that estrogenic fibrous scaffolds facilitate skin repair by ER-ß activation.
Assuntos
Biomimética , Receptor beta de Estrogênio , Animais , Receptor alfa de Estrogênio , Humanos , Camundongos , Fitoestrógenos , Pele , CicatrizaçãoRESUMO
Engineering bioscaffolds for improved cutaneous tissue regeneration remains a healthcare challenge because of the increasing number of patients suffering from acute and chronic wounds. To help address this problem, we propose to utilize alfalfa, an ancient medicinal plant that contains antibacterial/oxygenating chlorophylls and bioactive phytoestrogens, as a building block for regenerative wound dressings. Alfalfa carries genistein, which is a major phytoestrogen known to accelerate skin repair. The scaffolds presented herein were built from composite alfalfa and polycaprolactone (PCL) nanofibers with hydrophilic surface and mechanical stiffness that recapitulate the physiological microenvironments of skin. This composite scaffold was engineered to have aligned nanofibrous architecture to accelerate directional cell migration. As a result, alfalfa-based composite nanofibers were found to enhance the cellular proliferation of dermal fibroblasts and epidermal keratinocytes in vitro. Finally, these nanofibers exhibited reproducible regenerative functionality by promoting re-epithelialization and granulation tissue formation in both mouse and human skin, without requiring additional proteins, growth factors, or cells. Overall, these findings demonstrate the potential of alfalfa-based nanofibers as a regenerative platform toward accelerating cutaneous tissue repair.
Assuntos
Derme , Queratinócitos , Medicago sativa/química , Nanocompostos , Nanofibras , Cicatrização/efeitos dos fármacos , Linhagem Celular , Derme/lesões , Derme/metabolismo , Derme/patologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Nanocompostos/química , Nanocompostos/uso terapêutico , Nanofibras/química , Nanofibras/uso terapêutico , Poliésteres/químicaRESUMO
Recent reports suggest the utility of extracellular matrix (ECM) molecules as raw components in scaffolding of engineered materials. However, rapid and tunable manufacturing of ECM molecules into fibrous structures remains poorly developed. Here we report on an immersion rotary jet-spinning (iRJS) method to show high-throughput manufacturing (up to â¼1 g/min) of hyaluronic acid (HA) and other ECM fiber scaffolds using different spinning conditions and postprocessing modifications. This system allowed control over a variety of scaffold material properties, which enabled the fabrication of highly porous (70-95%) and water-absorbent (swelling ratio â¼2000-6000%) HA scaffolds with soft-tissue mimetic mechanical properties (â¼0.5-1.5 kPa). Tuning these scaffolds' properties enabled the identification of porosity (â¼95%) as a key facilitator for rapid and in-depth cellular ingress in vitro. We then demonstrated that porous HA scaffolds accelerated granulation tissue formation, neovascularization, and reepithelialization in vivo, altogether potentiating faster wound closure and tissue repair. Collectively, this scalable and versatile manufacturing approach enabled the fabrication of tunable ECM-mimetic nanofiber scaffolds that may provide an ideal first building block for the design of all-in-one healing materials.
Assuntos
Materiais Biomiméticos/farmacologia , Ácido Hialurônico/química , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Materiais Biomiméticos/química , Matriz Extracelular/química , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/farmacologia , Humanos , Ácido Hialurônico/farmacologia , Nanofibras/química , Porosidade , Regeneração/efeitos dos fármacos , Cicatrização/efeitos dos fármacosRESUMO
Historically, soy protein and extracts have been used extensively in foods due to their high protein and mineral content. More recently, soy protein has received attention for a variety of its potential health benefits, including enhanced skin regeneration. It has been reported that soy protein possesses bioactive molecules similar to extracellular matrix (ECM) proteins and estrogen. In wound healing, oral and topical soy has been heralded as a safe and cost-effective alternative to animal protein and endogenous estrogen. However, engineering soy protein-based fibrous dressings, while recapitulating ECM microenvironment and maintaining a moist environment, remains a challenge. Here, the development of an entirely plant-based nanofibrous dressing comprised of cellulose acetate (CA) and soy protein hydrolysate (SPH) using rotary jet spinning is described. The spun nanofibers successfully mimic physicochemical properties of the native skin ECM and exhibit a high water retaining capability. In vitro, CA/SPH nanofibers promote fibroblast proliferation, migration, infiltration, and integrin ß1 expression. In vivo, CA/SPH scaffolds accelerate re-epithelialization and epidermal thinning as well as reduce scar formation and collagen anisotropy in a similar fashion to other fibrous scaffolds, but without the use of animal proteins or synthetic polymers. These results affirm the potential of CA/SPH nanofibers as a novel wound dressing.
Assuntos
Bandagens , Materiais Biomiméticos/química , Celulose/química , Matriz Extracelular/química , Nanofibras/química , Pele , Proteínas de Soja/química , Alicerces Teciduais/química , Cicatrização , Ferimentos e Lesões/terapia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Organ-on-chip platforms aim to improve preclinical models for organ-level responses to novel drug compounds. Heart-on-a-chip assays in particular require tissue engineering techniques that rely on labor-intensive photolithographic fabrication or resolution-limited 3D printing of micropatterned substrates, which limits turnover and flexibility of prototyping. We present a rapid and automated method for large scale on-demand micropatterning of gelatin hydrogels for organ-on-chip applications using a novel biocompatible laser-etching approach. Fast and automated micropatterning is achieved via photosensitization of gelatin using riboflavin-5'phosphate followed by UV laser-mediated photoablation of the gel surface in user-defined patterns only limited by the resolution of the 15 µm wide laser focal point. Using this photopatterning approach, we generated microscale surface groove and pillar structures with feature dimensions on the order of 10-30 µm. The standard deviation of feature height was 0.3 µm, demonstrating robustness and reproducibility. Importantly, the UV-patterning process is non-destructive and does not alter gelatin micromechanical properties. Furthermore, as a quality control step, UV-patterned heart chip substrates were seeded with rat or human cardiac myocytes, and we verified that the resulting cardiac tissues achieved structural organization, contractile function, and long-term viability comparable to manually patterned gelatin substrates. Start-to-finish, UV-patterning shortened the time required to design and manufacture micropatterned gelatin substrates for heart-on-chip applications by up to 60% compared to traditional lithography-based approaches, providing an important technological advance enroute to automated and continuous manufacturing of organ-on-chips.
Assuntos
Hidrogéis/química , Análise Serial de Tecidos/instrumentação , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Animais , Automação , Células Cultivadas , Gelatina/química , Humanos , Miócitos Cardíacos/citologia , Impressão Tridimensional , RatosRESUMO
Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 µl (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle), and ejection fractions and contractile work 50-250 times smaller and 104-108 times smaller than the corresponding values for rodent and human ventricles, respectively. We also measured tissue coverage and alignment, calcium-transient propagation and pressure-volume loops in the presence or absence of test compounds. Moreover, we describe an instrumented bioreactor with ventricular-assist capabilities, and provide a proof-of-concept disease model of structural arrhythmia. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings.
Assuntos
Ventrículos do Coração/citologia , Modelos Biológicos , Engenharia Tecidual , Animais , Arritmias Cardíacas/patologia , Desenho Assistido por Computador , Matriz Extracelular/química , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Contração Miocárdica , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Nanofibras/química , Polímeros/química , Ratos , Ratos Sprague-Dawley , Alicerces Teciduais/química , Função VentricularRESUMO
Wounds in the fetus can heal without scarring. Consequently, biomaterials that attempt to recapitulate the biophysical and biochemical properties of fetal skin have emerged as promising pro-regenerative strategies. The extracellular matrix (ECM) protein fibronectin (Fn) in particular is believed to play a crucial role in directing this regenerative phenotype. Accordingly, Fn has been implicated in numerous wound healing studies, yet remains untested in its fibrillar conformation as found in fetal skin. Here, we show that high extensional (â¼1.2 ×105 s-1) and shear (â¼3 ×105 s-1) strain rates in rotary jet spinning (RJS) can drive high throughput Fn fibrillogenesis (â¼10â¯mL/min), thus producing nanofiber scaffolds that are used to effectively enhance wound healing. When tested on a full-thickness wound mouse model, Fn nanofiber dressings not only accelerated wound closure, but also significantly improved tissue restoration, recovering dermal and epidermal structures as well as skin appendages and adipose tissue. Together, these results suggest that bioprotein nanofiber fabrication via RJS could set a new paradigm for enhancing wound healing and may thus find use in a variety of regenerative medicine applications.
Assuntos
Materiais Biocompatíveis , Fibronectinas , Nanofibras , Cicatrização , Administração Cutânea , Animais , Materiais Biocompatíveis/química , Fibronectinas/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanofibras/química , Pele/efeitos dos fármacos , Pele/patologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Cicatrização/efeitos dos fármacosRESUMO
Microphysiological systems and organs-on-chips promise to accelerate biomedical and pharmaceutical research by providing accurate in vitro replicas of human tissue. Aside from addressing the physiological accuracy of the model tissues, there is a pressing need for improving the throughput of these platforms. To do so, scalable data acquisition strategies must be introduced. To this end, we here present an instrumented 24-well plate platform for higher-throughput studies of engineered human stem cell-derived cardiac muscle tissues that recapitulate the laminar structure of the native ventricle. In each well of the platform, an embedded flexible strain gauge provides continuous and non-invasive readout of the contractile stress and beat rate of an engineered cardiac tissue. The sensors are based on micro-cracked titanium-gold thin films, which ensure that the sensors are highly compliant and robust. We demonstrate the value of the platform for toxicology and drug-testing purposes by performing 12 complete dose-response studies of cardiac and cardiotoxic drugs. Additionally, we showcase the ability to couple the cardiac tissues with endothelial barriers. In these studies, which mimic the passage of drugs through the blood vessels to the musculature of the heart, we regulate the temporal onset of cardiac drug responses by modulating endothelial barrier permeability in vitro.
Assuntos
Ensaios de Triagem em Larga Escala/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Modelos Cardiovasculares , Miócitos Cardíacos/citologia , Engenharia Tecidual/instrumentação , Animais , Fármacos Cardiovasculares/farmacologia , Células Cultivadas , Desenho de Equipamento , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Células-Tronco/citologiaRESUMO
In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the ß-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.
Assuntos
Ventrículos do Coração/citologia , Procedimentos Analíticos em Microchip/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Engenharia Tecidual/métodos , Função Ventricular/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Dispositivos Lab-On-A-Chip , Contração Miocárdica/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region-specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin-R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin-R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin-R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309-1336, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica , Agrecanas/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Brevicam/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Imageamento Tridimensional , Espectrometria de Massas , Rede Nervosa/metabolismo , Neuritos/metabolismo , Neuroimagem , Neurônios/metabolismo , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Tubulina (Proteína)/metabolismoRESUMO
The efficacy of cardiac cell therapy depends on the integration of existing and newly formed cardiomyocytes. Here, we developed a minimal in vitro model of this interface by engineering two cell microtissues (µtissues) containing mouse cardiomyocytes, representing spared myocardium after injury, and cardiomyocytes generated from embryonic and induced pluripotent stem cells, to model newly formed cells. We demonstrated that weaker stem cell-derived myocytes coupled with stronger myocytes to support synchronous contraction, but this arrangement required focal adhesion-like structures near the cell-cell junction that degrade force transmission between cells. Moreover, we developed a computational model of µtissue mechanics to demonstrate that a reduction in isometric tension is sufficient to impair force transmission across the cell-cell boundary. Together, our in vitro and in silico results suggest that mechanotransductive mechanisms may contribute to the modest functional benefits observed in cell-therapy studies by regulating the amount of contractile force effectively transmitted at the junction between newly formed and spared myocytes.
Assuntos
Comunicação Celular , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Simulação por Computador , Adesões Focais/metabolismo , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos BALB C , Modelos Cardiovasculares , Miócitos Cardíacos/transplante , Fenótipo , Cultura Primária de Células , Transplante de Células-Tronco , Estresse Mecânico , Fatores de TempoRESUMO
Inspired by the relatively simple morphological blueprint provided by batoid fish such as stingrays and skates, we created a biohybrid system that enables an artificial animal--a tissue-engineered ray--to swim and phototactically follow a light cue. By patterning dissociated rat cardiomyocytes on an elastomeric body enclosing a microfabricated gold skeleton, we replicated fish morphology at 1/10 scale and captured basic fin deflection patterns of batoid fish. Optogenetics allows for phototactic guidance, steering, and turning maneuvers. Optical stimulation induced sequential muscle activation via serpentine-patterned muscle circuits, leading to coordinated undulatory swimming. The speed and direction of the ray was controlled by modulating light frequency and by independently eliciting right and left fins, allowing the biohybrid machine to maneuver through an obstacle course.
Assuntos
Luz , Robótica , Rajidae/fisiologia , Natação/fisiologia , Engenharia Tecidual , Nadadeiras de Animais/fisiologia , Animais , Fenômenos Biomecânicos , Sinais (Psicologia) , Músculo Esquelético/fisiologia , OptogenéticaRESUMO
Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of heart disease. While the transcriptional regulator friend of Gata 2 (FOG2) is known to be essential for heart morphogenesis and coronary development, its tissue-specific function has not been previously investigated. Additionally, little is known about the role of FOG2 in the adult heart. Here we used spatiotemporally regulated inactivation of Fog2 to delineate its function in both the embryonic and adult mouse heart. Early cardiomyocyte- restricted loss of Fog2 recapitulated the cardiac and coronary defects of the Fog2 germline murine knockouts. Later cardiomyocyte-restricted loss of Fog2 (Fog2MC) did not result in defects in cardiac structure or coronary vessel formation. However, Fog2MC adult mice had severely depressed ventricular function and died at 8-14 weeks. Fog2MC adult hearts displayed a paucity of coronary vessels, associated with myocardial hypoxia, increased cardiomyocyte apoptosis, and cardiac fibrosis. Induced inactivation of Fog2 in the adult mouse heart resulted in similar phenotypes, as did ablation of the FOG2 interaction with the transcription factor GATA4. Loss of the FOG2 or FOG2-GATA4 interaction altered the expression of a panel of angiogenesis-related genes. Collectively, our data indicate that FOG2 regulates adult heart function and coronary angiogenesis.
Assuntos
Envelhecimento/fisiologia , Vasos Coronários/metabolismo , Proteínas de Ligação a DNA/metabolismo , Coração , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Humanos , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Mutação/genética , Neovascularização Fisiológica , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Biglycan, a small leucine-rich proteoglycan, has been shown to interact with extracellular matrix (ECM) collagen and may influence fibrillogenesis. We hypothesized that biglycan contributes to post-myocardial infarction (MI) scar development and that the absence of biglycan would result in altered scar structure and mechanics. Anterior MI was induced in biglycan hemizygous null and wild-type mice by permanent ligation of the left coronary artery. The initial extent of ischemic injury was similar in the two groups, as was the infarct size after 30 days, although there was some tendency toward reduced expansion in the biglycan-null. Electron microscopy revealed that collagen fibrils had a smaller average diameter and a narrower range in the biglycan-null scar, as well as appearing more densely packed. In vivo strain analysis showed that biglycan-null scars were stiffer than the wild-type. Remote LV collagen concentration tended to be reduced in biglycan-null hearts, but the difference was not statistically significant. Null-expression of biglycan may alter collagen fibril ultrastructure, and thereby influence scar mechanics and remodeling.