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BACKGROUND: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. METHODS: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia-reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. RESULTS: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin's mechanisms of action. CONCLUSIONS: A single dose of agrin is capable of reducing ischemia-reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.
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Agrina/farmacología , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Recuperación de la Función/efectos de los fármacos , Animales , Humanos , Ratones , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Proteínas Recombinantes/farmacología , PorcinosRESUMEN
Following myonecrosis, muscle satellite cells proliferate, differentiate and fuse, creating new myofibers. The Runx1 transcription factor is not expressed in naïve developing muscle or in adult muscle tissue. However, it is highly expressed in muscles exposed to myopathic damage yet, the role of Runx1 in muscle regeneration is completely unknown. Our study of Runx1 function in the muscle's response to myonecrosis reveals that this transcription factor is activated and cooperates with the MyoD and AP-1/c-Jun transcription factors to drive the transcription program of muscle regeneration. Mice lacking dystrophin and muscle Runx1 (mdx-/Runx1f/f), exhibit impaired muscle regeneration leading to age-dependent muscle waste, gradual decrease in motor capabilities and a shortened lifespan. Runx1-deficient primary myoblasts are arrested at cell cycle G1 and consequently differentiate. Such premature differentiation disrupts the myoblasts' normal proliferation/differentiation balance, reduces the number and size of regenerating myofibers and impairs muscle regeneration. Our combined Runx1-dependent gene expression, ChIP-seq, ATAC-seq and histone H3K4me1/H3K27ac modification analyses revealed a subset of Runx1-regulated genes that are co-occupied by MyoD and c-Jun in mdx-/Runx1f/f muscle. The data provide unique insights into the transcriptional program driving muscle regeneration and implicate Runx1 as an important participant in the pathology of muscle wasting diseases.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Músculo Esquelético/fisiología , Mioblastos/fisiología , Regeneración , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Secuencia de Consenso , Femenino , Expresión Génica , Regulación de la Expresión Génica , Genes jun , Masculino , Ratones Endogámicos mdx , Proteína MioD/metabolismoAsunto(s)
Senescencia Celular , Fibroblastos/metabolismo , Lesiones Cardíacas/metabolismo , Pericardio/metabolismo , Regeneración , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Pez Cebra/metabolismo , Agrina/farmacología , Animales , Senescencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Lesiones Cardíacas/genética , Lesiones Cardíacas/patología , Lesiones Cardíacas/fisiopatología , Cinética , Pericardio/efectos de los fármacos , Pericardio/patología , Regeneración/efectos de los fármacos , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
A fundamental aspect of skeletal myogenesis involves extensive rounds of cell fusion, in which individual myoblasts are incorporated into growing muscle fibers. Here we demonstrate that N-WASp, a ubiquitous nucleation-promoting factor of branched microfilament arrays, is an essential contributor to skeletal muscle-cell fusion in developing mouse embryos. Analysis both in vivo and in primary satellite-cell cultures, shows that disruption of N-WASp function does not interfere with the program of skeletal myogenic differentiation, and does not affect myoblast motility, morphogenesis and attachment capacity. N-WASp-deficient myoblasts, however, fail to fuse. Furthermore, our analysis suggests that myoblast fusion requires N-WASp activity in both partners of a fusing myoblast pair. These findings reveal a specific role for N-WASp during mammalian myogenesis. WASp-family elements appear therefore to act as universal mediators of the myogenic cell-cell fusion mechanism underlying formation of functional muscle fibers, in both vertebrate and invertebrate species.
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Actinas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Músculos/citología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Diferenciación Celular , Fusión Celular , Células Cultivadas , Cruzamientos Genéticos , Drosophila , Heterocigoto , Ratones , Ratones Endogámicos ICR , Modelos Biológicos , Desarrollo de Músculos , Músculos/embriología , Factores de TiempoRESUMEN
Senescence plays a key role in various physiological and pathological processes. We reported that injury-induced transient senescence correlates with heart regeneration, yet the multi-omics profile and molecular underpinnings of regenerative senescence remain obscure. Using proteomics and single-cell RNA sequencing, here we report the regenerative senescence multi-omic signature in the adult mouse heart and establish its role in neonatal heart regeneration and agrin-mediated cardiac repair in adult mice. We identified early growth response protein 1 (Egr1) as a regulator of regenerative senescence in both models. In the neonatal heart, Egr1 facilitates angiogenesis and cardiomyocyte proliferation. In adult hearts, agrin-induced senescence and repair require Egr1, activated by the integrin-FAK-ERK-Akt1 axis in cardiac fibroblasts. We also identified cathepsins as injury-induced senescence-associated secretory phenotype components that promote extracellular matrix degradation and potentially assist in reducing fibrosis. Altogether, we uncovered the molecular signature and functional benefits of regenerative senescence during heart regeneration, with Egr1 orchestrating the process.
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Proliferación Celular , Senescencia Celular , Proteína 1 de la Respuesta de Crecimiento Precoz , Miocitos Cardíacos , Regeneración , Animales , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regeneración/fisiología , Senescencia Celular/fisiología , Miocitos Cardíacos/metabolismo , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Transducción de Señal , Fibroblastos/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Cultivadas , Animales Recién Nacidos , Modelos Animales de Enfermedad , Fenotipo Secretor Asociado a la Senescencia , Proteómica , Análisis de la Célula Individual , Masculino , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones Noqueados , Quinasa 1 de Adhesión FocalRESUMEN
Cardiomyocyte proliferation and dedifferentiation have fueled the field of regenerative cardiology in recent years, whereas the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterized by the restoration of function lost during dedifferentiation. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: transient dedifferentiation and redifferentiation. Here we survey the temporal transcriptomic and proteomic landscape of dedifferentiation-redifferentiation in adult mouse hearts and reveal that well-characterized dedifferentiation features largely return to normal, although elements of residual dedifferentiation remain, even after the contractile function is restored. These hearts appear rejuvenated and show robust resistance to ischemic injury, even 5 months after redifferentiation initiation. Cardiomyocyte redifferentiation is driven by negative feedback signaling and requires LATS1/2 Hippo pathway activity. Our data reveal the importance of cardiomyocyte redifferentiation in functional restoration during regeneration but also protection against future insult, in what could lead to a potential prophylactic treatment against ischemic heart disease for at-risk patients.
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In mammals, the physiological activation of the glucocorticoid receptor (GR) by glucocorticoids (GCs) promotes the maturation of cardiomyocytes during late gestation, but the effect on postnatal cardiac growth and regenerative plasticity is unclear. Here we demonstrate that the GC-GR axis restrains cardiomyocyte proliferation during postnatal development. Cardiomyocyte-specific GR ablation in conditional knockout (cKO) mice delayed the postnatal cardiomyocyte cell cycle exit, hypertrophic growth and cytoarchitectural maturation. GR-cKO hearts showed increased expression of genes involved in glucose catabolism and reduced expression of genes promoting fatty acid oxidation and mitochondrial respiration. Accordingly, oxygen consumption in GR-cKO cardiomyocytes was less dependent on fatty acid oxidation, and glycolysis inhibition reverted GR-cKO effects on cardiomyocyte proliferation. GR ablation or transient pharmacological inhibition after myocardial infarction in juvenile and/or adult mice facilitated cardiomyocyte survival, cell cycle re-entry and division, leading to cardiac muscle regeneration along with reduced scar formation. Thus, GR restrains heart regeneration and may represent a therapeutic target.
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Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Fibras Musculares Esqueléticas/citología , Mioblastos/citología , Actinas/metabolismo , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Fusión Celular , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2-Neuregulin and Hippo-YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial-mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Transición Epitelial-Mesenquimal , Insuficiencia Cardíaca/metabolismo , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Receptor ErbB-2/metabolismo , Regeneración , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/patología , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibrosis , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Mecanotransducción Celular , Ratones Transgénicos , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/patología , Fosforilación , Receptor ErbB-2/genética , Proteínas Señalizadoras YAPRESUMEN
The adult mammalian heart regenerates poorly after injury and, as a result, ischemic heart diseases are among the leading causes of death worldwide. The recovery of the injured heart is dependent on orchestrated repair processes including inflammation, fibrosis, cardiomyocyte survival, proliferation, and contraction properties that could be modulated in patients. In this work we designed an automated high-throughput screening system for small molecules that induce cardiomyocyte proliferation in vitro and identified the small molecule Chicago Sky Blue 6B (CSB). Following induced myocardial infarction, CSB treatment reduced scar size and improved heart function of adult mice. Mechanistically, we show that although initially identified using in vitro screening for cardiomyocyte proliferation, in the adult mouse CSB promotes heart repair through (i) inhibition of CaMKII signaling, which improves cardiomyocyte contractility; and (ii) inhibition of neutrophil and macrophage activation, which attenuates the acute inflammatory response, thereby contributing to reduced scarring. In summary, we identified CSB as a potential therapeutic agent that enhances cardiac repair and function by suppressing postinjury detrimental processes, with no evidence for cardiomyocyte renewal.
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Corazón/efectos de los fármacos , Infarto del Miocardio/metabolismo , Miocitos Cardíacos , Azul de Tripano/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cicatriz/metabolismo , Femenino , Ratones , Ratones Endogámicos ICR , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismoRESUMEN
Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered anti-proliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.
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Corazón/anatomía & histología , Corazón/fisiología , Miocitos Cardíacos/citología , Regeneración/efectos de los fármacos , Vitamina D/farmacología , Pez Cebra/anatomía & histología , Pez Cebra/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Corazón/efectos de los fármacos , Mitógenos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos , Transducción de Señal/efectos de los fármacos , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismoRESUMEN
In response to muscle damage the muscle adult stem cells are activated and differentiate into myoblasts that regenerate the damaged tissue. We have recently showed that following myopathic damage the level of the Runx1 transcription factor (TF) is elevated and that during muscle regeneration this TF regulates the balance between myoblast proliferation and differentiation (Umansky et al.). We employed Runx1-dependent gene expression, Chromatin Immunoprecipitation sequencing (ChIP-seq), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and histone H3K4me1/H3K27ac modification analyses to identify a subset of Runx1-regulated genes that are co-occupied by the TFs MyoD and c-Jun and are involved in muscle regeneration (Umansky et al.). The data is available at the GEO database under the superseries accession number GSE56131.