RESUMEN
BACKGROUND: Discovering determinants of cardiomyocyte maturity is critical for deeply understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration, but elucidation of endogenous developmental regulators of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. MBNL1 (muscleblind-like 1) regulates fibroblast, thymocyte, and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. METHODS: MBNL1 gain- and loss-of-function mouse models were studied at several developmental time points and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro assays to determine the mechanisms through which MBNL1 exerts its effects. RESULTS: MBNL1 is coexpressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deficiency was insufficient to promote regeneration in the adult heart because of cell cycle checkpoint activation. CONCLUSIONS: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Targeting loss of cardiomyocyte maturity and downregulation of cell cycle inhibitors through MBNL1 deletion was not sufficient to promote adult regeneration.
Asunto(s)
Diferenciación Celular , Miocitos Cardíacos , Proteínas de Unión al ARN , Regeneración , Animales , Miocitos Cardíacos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratones , Proliferación Celular , Transducción de Señal , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Proteínas de Unión al ADNRESUMEN
Long intergenic non-coding RNAs (lincRNAs) have been implicated in gene regulation, but their requirement for development needs empirical interrogation. We computationally identified nine murine lincRNAs that have developmentally regulated transcriptional and epigenomic profiles specific to early heart differentiation. Six of the nine lincRNAs had in vivo expression patterns supporting a potential function in heart development, including a transcript downstream of the cardiac transcription factor Hand2, which we named Handlr (Hand2-associated lincRNA), Rubie and Atcayos We genetically ablated these six lincRNAs in mouse, which suggested genomic regulatory roles for four of the cohort. However, none of the lincRNA deletions led to severe cardiac phenotypes. Thus, we stressed the hearts of adult Handlr and Atcayos mutant mice by transverse aortic banding and found that absence of these lincRNAs did not affect cardiac hypertrophy or left ventricular function post-stress. Our results support roles for lincRNA transcripts and/or transcription in the regulation of topologically associated genes. However, the individual importance of developmentally specific lincRNAs is yet to be established. Their status as either gene-like entities or epigenetic components of the nucleus should be further considered.
Asunto(s)
Diferenciación Celular , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Miocardio/metabolismo , ARN Largo no Codificante/biosíntesis , Animales , Eliminación de Gen , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Ratones , Ratones Mutantes , ARN Largo no Codificante/genéticaRESUMEN
RATIONALE: Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart's ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart. OBJECTIVE: To investigate the role of collagen organization in cardiac fibroblast fate determination. METHODS AND RESULTS: Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFß (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here. CONCLUSIONS: Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.
Asunto(s)
Diferenciación Celular , Colágeno/química , Infarto del Miocardio/metabolismo , Miofibroblastos/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Colágeno/metabolismo , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/citología , Fibras de Estrés/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Mechanical stress from cardiomyocyte contraction causes misfolded sarcomeric protein replacement. Sarcomeric maintenance utilizes localized pools of mRNAs and translation machinery, yet the importance of localized translation remains unclear. In this issue of the JCI, Haddad et al. identify the Z-line as a critical site for localized translation of sarcomeric proteins, mediated by ribosomal protein SA (RPSA). RPSA localized ribosomes at Z-lines and was trafficked via microtubules. Cardiomyocyte-specific loss of RPSA in mice resulted in mislocalized protein translation and caused structural dilation from myocyte atrophy. These findings demonstrate the necessity of RPSA-dependent spatially localized translation for sarcomere maintenance and cardiac structure and function.
Asunto(s)
Miocitos Cardíacos , Biosíntesis de Proteínas , Proteínas Ribosómicas , Sarcómeros , Sarcómeros/metabolismo , Sarcómeros/patología , Animales , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ribosomas/metabolismo , Ribosomas/genética , Humanos , Microtúbulos/metabolismoRESUMEN
Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns.
Asunto(s)
Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/metabolismo , Cromosomas de los Mamíferos/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Cricetinae , Drosophila , Ratones , Mutación , Motivos de Nucleótidos , Unión Proteica , Relación Estructura-Actividad , CohesinasRESUMEN
To investigate the role of immune cells in lung regeneration, we used a unilateral pneumonectomy model that promotes the formation of new alveoli in the remaining lobes. Immunofluorescence and single-cell RNA sequencing found CD115+ and CCR2+ monocytes and M2-like macrophages accumulating in the lung during the peak of type 2 alveolar epithelial stem cell (AEC2) proliferation. Genetic loss of function in mice and adoptive transfer studies revealed that bone marrow-derived macrophages (BMDMs) traffic to the lung through a CCL2-CCR2 chemokine axis and are required for optimal lung regeneration, along with Il4ra-expressing leukocytes. Our data suggest that these cells modulate AEC2 proliferation and differentiation. Finally, we provide evidence that group 2 innate lymphoid cells are a source of IL-13, which promotes lung regeneration. Together, our data highlight the potential for immunomodulatory therapies to stimulate alveologenesis in adults.