RESUMO
Cardiomyocyte maturation is crucial for generating adult cardiomyocytes and the application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, regulation at the cis-regulatory element level and its role in heart disease remain unclear. Alpha-actinin 2 (ACTN2) levels increase during CM maturation. In this study, we investigated a clinically relevant, conserved ACTN2 enhancer's effects on CM maturation using hPSC and mouse models. Heterozygous ACTN2 enhancer deletion led to abnormal CM morphology, reduced function and mitochondrial respiration. Transcriptomic analyses in vitro and in vivo showed disrupted CM maturation and upregulated anabolic mammalian target for rapamycin (mTOR) signaling, promoting senescence and hindering maturation. As confirmation, ACTN2 enhancer deletion induced heat shock protein 90A expression, a chaperone mediating mTOR activation. Conversely, targeting the ACTN2 enhancer via enhancer CRISPR activation (enCRISPRa) promoted hPSC-CM maturation. Our studies reveal the transcriptional enhancer's role in cardiac maturation and disease, offering insights into potentially fine-tuning gene expression to modulate cardiomyocyte physiology.
Assuntos
Actinina , Diferenciação Celular , Elementos Facilitadores Genéticos , Miócitos Cardíacos , Miócitos Cardíacos/metabolismo , Humanos , Elementos Facilitadores Genéticos/genética , Animais , Actinina/genética , Actinina/metabolismo , Diferenciação Celular/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Transdução de Sinais/genética , Camundongos , Transcrição Gênica , Regulação da Expressão Gênica no Desenvolvimento , Linhagem Celular , FenótipoRESUMO
This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.
RESUMO
Blood vessels serve as intermediate conduits for the extension of sympathetic axons towards target tissues, while also acting as crucial targets for their homeostatic processes encompassing the regulation of temperature, blood pressure, and oxygen availability. How sympathetic axons innervate not only blood vessels but also a wide array of target tissues is not clear. Here we show that in embryonic skin, after the establishment of co-branching between sensory nerves and blood vessels, sympathetic axons invade the skin alongside these sensory nerves and extend their branches towards these blood vessels covered by vascular smooth muscle cells (VSMCs). Our mosaic labeling technique for sympathetic axons shows that collateral branching predominantly mediates the innervation of VSMC-covered blood vessels by sympathetic axons. The expression of nerve growth factor (NGF), previously known to induce collateral axon branching in culture, can be detected in the vascular smooth muscle cell (VSMC)-covered blood vessels, as well as sensory nerves. Indeed, VSMC-specific Ngf knockout leads to a significant decrease of collateral branching of sympathetic axons innervating VSMC-covered blood vessels. These data suggest that VSMC-derived NGF serves as an inductive signal for collateral branching of sympathetic axons innervating blood vessels in the embryonic skin.
Assuntos
Músculo Liso Vascular , Fator de Crescimento Neural , Pele , Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/inervação , Fator de Crescimento Neural/metabolismo , Camundongos , Pele/inervação , Pele/irrigação sanguínea , Pele/metabolismo , Miócitos de Músculo Liso/metabolismo , Axônios/metabolismo , Axônios/fisiologia , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/inervação , Vasos Sanguíneos/metabolismo , Sistema Nervoso Simpático/embriologia , Sistema Nervoso Simpático/fisiologia , Sistema Nervoso Simpático/metabolismo , Camundongos KnockoutRESUMO
Neuropeptide Y (NPY) is best known for its effects in the brain as an orexigenic and anxiolytic agent and in reducing energy expenditure. NPY is also co-expressed with norepinephrine (NE) in sympathetic neurons. Although NPY is generally considered to modulate noradrenergic responses, its specific roles in autonomic physiology remain under-appreciated. Here, we show that sympathetic-derived NPY is essential for metabolic and cardiovascular regulation in mice. NPY and NE are co-expressed in 90% of prevertebral sympathetic neurons and only 43% of paravertebral neurons. NPY-expressing neurons primarily innervate blood vessels in peripheral organs. Sympathetic-specific NPY deletion elicits pronounced metabolic and cardiovascular defects in mice, including reductions in insulin secretion, glucose tolerance, cold tolerance, and pupil size and elevated heart rate, while notably, however, basal blood pressure was unchanged. These findings provide insight into target tissue-specific functions of NPY derived from sympathetic neurons and imply its potential involvement in metabolic and cardiovascular diseases.
Assuntos
Ansiolíticos , Neuropeptídeo Y , Animais , Camundongos , Pressão Sanguínea , Norepinefrina , Homeostase , GlucoseRESUMO
Neuropeptide Y (NPY) is best known for its effects in the brain as an orexigenic and anxiolytic agent and in reducing energy expenditure. NPY is also co-expressed with Norepinephrine (NE) in sympathetic neurons. Although NPY is generally considered to modulate noradrenergic responses, its specific roles in autonomic physiology remain under-appreciated. Here, we show that sympathetic-derived NPY is essential for metabolic and cardiovascular regulation in mice. NPY and NE are co-expressed in 90% of prevertebral sympathetic neurons and only 43% of paravertebral neurons. NPY-expressing neurons primarily innervate blood vessels in peripheral organs. Sympathetic-specific deletion of NPY elicits pronounced metabolic and cardiovascular defects in mice, including reductions in insulin secretion, glucose tolerance, cold tolerance, pupil size, and an elevation in heart rate, while notably, however, basal blood pressure was unchanged. These findings provide new knowledge about target tissue-specific functions of NPY derived from sympathetic neurons and imply its potential involvement in metabolic and cardiovascular diseases.
RESUMO
Cardiomyopathies remain one of the leading causes of morbidity and mortality worldwide. Environmental risk factors and genetic predisposition account for most cardiomyopathy cases. As with all complex diseases, there are significant challenges in the interpretation of the molecular mechanisms underlying cardiomyopathy-associated genetic variants. Given the technical improvements and reduced costs of DNA sequence technologies, an increasing number of patients are now undergoing genetic testing, resulting in a continuously expanding list of novel mutations. However, many patients carry noncoding genetic variants, and although emerging evidence supports their contribution to cardiac disease, their role in cardiomyopathies remains largely understudied. In this review, we summarize published studies reporting on the association of different types of noncoding variants with various types of cardiomyopathies. We focus on variants within transcriptional enhancers, promoters, intronic sites, and untranslated regions that are likely associated with cardiac disease. Given the broad nature of this topic, we provide an overview of studies that are relatively recent and have sufficient evidence to support a significant degree of causality. We believe that more research with additional validation of noncoding genetic variants will provide further mechanistic insights on the development of cardiac disease, and noncoding variants will be increasingly incorporated in future genetic screening tests.
RESUMO
The heart develops in a synchronized sequence of proliferation and differentiation of cardiac progenitor cells (CPCs) from two anatomically distinct pools of cells, the first heart field (FHF) and second heart field (SHF). Congenital heart defects arise upon dysregulation of these processes, many of which are restricted to derivatives of the FHF or SHF. Of the conserved set of signaling pathways that regulate development, the Wnt signaling pathway has long been known for its importance in SHF development. The source of such Wnts has remained elusive, though it has been postulated that these Wnts are secreted from ectodermal or endodermal sources. The central question remains unanswered: Where do these Wnts come from? Here, we show that CPCs autoregulate SHF development via Wnt through genetic manipulation of a key Wnt export protein (Wls), scRNA-seq analysis of CPCs, and use of our precardiac organoid system. Through this, we identify dysregulated developmental trajectories of anterior SHF cell fate, leading to a striking single ventricle phenotype in knockout embryos. We then applied our findings to our precardiac organoid model and found that Wnt2 is sufficient to restore SHF cell fate in our model of disrupted endogenous Wnt signaling. In this study, we provide a basis for SHF cell fate decision-proliferation vs. differentiation-autoregulated by CPCs through Wnt.
Assuntos
Cardiopatias Congênitas , Coração , Humanos , Coração/fisiologia , Diferenciação Celular , Via de Sinalização Wnt , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Satellite glia are the major glial cells in sympathetic ganglia, enveloping neuronal cell bodies. Despite this intimate association, the extent to which sympathetic functions are influenced by satellite glia in vivo remains unclear. Here, we show that satellite glia are critical for metabolism, survival, and activity of sympathetic neurons and modulate autonomic behaviors in mice. Adult ablation of satellite glia results in impaired mTOR signaling, soma atrophy, reduced noradrenergic enzymes, and loss of sympathetic neurons. However, persisting neurons have elevated activity, and satellite glia-ablated mice show increased pupil dilation and heart rate, indicative of enhanced sympathetic tone. Satellite glia-specific deletion of Kir4.1, an inward-rectifying potassium channel, largely recapitulates the cellular defects observed in glia-ablated mice, suggesting that satellite glia act in part via K+-dependent mechanisms. These findings highlight neuron-satellite glia as functional units in regulating sympathetic output, with implications for disorders linked to sympathetic hyper-activity such as cardiovascular disease and hypertension.
Assuntos
Gânglios Simpáticos , Neuroglia , Animais , Sobrevivência Celular , Camundongos , Neuroglia/fisiologia , Neurônios , Transdução de SinaisRESUMO
Embryos devoid of autonomic innervation suffer sudden cardiac death. However, whether autonomic neurons have a role in heart development is poorly understood. To investigate if sympathetic neurons impact cardiomyocyte maturation, we co-cultured phenotypically immature cardiomyocytes derived from human induced pluripotent stem cells with mouse sympathetic ganglion neurons. We found that 1) multiple cardiac structure and ion channel genes related to cardiomyocyte maturation were up-regulated when co-cultured with sympathetic neurons; 2) sarcomere organization and connexin-43 gap junctions increased; 3) calcium imaging showed greater transient amplitudes. However, sarcomere spacing, relaxation time, and level of sarcoplasmic reticulum calcium did not show matured phenotypes. We further found that addition of endothelial and epicardial support cells did not enhance maturation to a greater extent beyond sympathetic neurons, while administration of isoproterenol alone was insufficient to induce changes in gene expression. These results demonstrate that sympathetic neurons have a significant and complex role in regulating cardiomyocyte development.
RESUMO
Neurons can regulate the development, pathogenesis, and regeneration of target organs. However, the role of neurons during heart development and regeneration remains unclear. We genetically inhibited sympathetic innervation in vivo, which resulted in heart enlargement with an increase in cardiomyocyte number. Transcriptomic and protein analysis showed down-regulation of the two clock gene homologs Period1/Period2 (Per1/Per2) accompanied by up-regulation of cell cycle genes. Per1/Per2 deletion increased heart size and cardiomyocyte proliferation, recapitulating sympathetic neurondeficient hearts. Conversely, increasing sympathetic activity by norepinephrine treatment induced Per1/Per2 and suppressed cardiomyocyte proliferation. We further found that the two clock genes negatively regulate myocyte mitosis entry through the Wee1 kinase pathway. Our findings demonstrate a previously unknown link between cardiac neurons and clock genes in regulation of cardiomyocyte proliferation and heart size and provide mechanistic insights for developing neuromodulation strategies for cardiac regen5eration.
RESUMO
The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.
Assuntos
Coração/embriologia , Miocárdio/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Células Cultivadas , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miocárdio/citologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The heart undergoes profound morphological and functional changes as it continues to mature postnatally. However, this phase of cardiac development remains understudied. More recently, cardiac maturation research has attracted a lot of interest due to the need for more mature stem cell-derived cardiomyocytes for disease modeling, drug screening and heart regeneration. Additionally, neonatal heart injury models have been utilized to study heart regeneration, and factors regulating postnatal heart development have been associated with adult cardiac disease. Critical components of cardiac maturation are systemic and local biochemical cues. Specifically, cardiac innervation and the concentration of various metabolic hormones appear to increase perinatally and they have striking effects on cardiomyocytes. Here, we first report some of the key parameters of mature cardiomyocytes and then discuss the specific effects of neurons and hormonal cues on cardiomyocyte maturation. We focus primarily on the structural, electrophysiologic, metabolic, hypertrophic and hyperplastic effects of each factor. This review highlights the significance of underappreciated regulators of cardiac maturation and underscores the need for further research in this exciting field.
Assuntos
Hormônios/metabolismo , Miócitos Cardíacos/fisiologia , Neurônios/metabolismo , HumanosRESUMO
Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Receptores Ativados por Proliferador de Peroxissomo/genética , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Transcriptoma , Proteínas de Sinalização YAPRESUMO
PURPOSE OF REVIEW: Heart development is a meticulously coordinated process that involves the specification of two distinct populations of cardiac progenitor cells, namely the first and the second heart field. Disruption of heart field progenitors can result in congenital heart defects. In this review, we aim to describe the signaling pathways and transcription factors that link heart field development and congenital heart disease. RECENT FINDINGS: Single-cell transcriptomics, lineage-tracing mouse models, and stem cell-based in vitro modeling of cardiogenesis have significantly improved the spatiotemporal characterization of cardiac progenitors. Additionally, novel functional genomic studies have now linked more genetic variants with congenital heart disease. Dysregulation of cardiac progenitor cells causes malformations that can be lethal. Ongoing research will continue to shed light on cardiac morphogenesis and help us better understand and treat patients with congenital heart disease.
Assuntos
Cardiopatias Congênitas , Coração , Animais , Humanos , Camundongos , Miocárdio , Transdução de Sinais , Células-TroncoAssuntos
Cardiomiopatia Dilatada/genética , Conectina/genética , Mutação/genética , Adolescente , Adulto , Família , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Heart failure is a major public health problem affecting over 23 million people worldwide. In this study, we present the results of a large scale meta-analysis of heart failure GWAS and replication in a comparable sized cohort to identify one known and two novel loci associated with heart failure. Heart failure sub-phenotyping shows that a new locus in chromosome 1 is associated with left ventricular adverse remodeling and clinical heart failure, in response to different initial cardiac muscle insults. Functional characterization and fine-mapping of that locus reveal a putative causal variant in a cardiac muscle specific regulatory region activated during cardiomyocyte differentiation that binds to the ACTN2 gene, a crucial structural protein inside the cardiac sarcolemma (Hi-C interaction p-value = 0.00002). Genome-editing in human embryonic stem cell-derived cardiomyocytes confirms the influence of the identified regulatory region in the expression of ACTN2. Our findings extend our understanding of biological mechanisms underlying heart failure.
Assuntos
Actinina/genética , Predisposição Genética para Doença/genética , Insuficiência Cardíaca/genética , Sistema ABO de Grupos Sanguíneos/genética , Fibrilação Atrial/genética , Cromossomos Humanos Par 1 , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Insuficiência Cardíaca/patologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Doenças Musculoesqueléticas/genética , Miócitos Cardíacos/citologia , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) - are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.
Assuntos
Coração/embriologia , Mioblastos Cardíacos/citologia , Miocárdio/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Mioblastos Cardíacos/metabolismo , Miocárdio/metabolismo , Organoides/citologia , Organoides/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
The discovery of the first heart field (FHF) and the second heart field (SHF) led us to understand how cardiac lineages and structures arise during development. However, it remains unknown how they are specified. Here, we generate precardiac spheroids with pluripotent stem cells (PSCs) harboring GFP/RFP reporters under the control of FHF/SHF markers, respectively. GFP+ cells and RFP+ cells appear from two distinct areas and develop in a complementary fashion. Transcriptome analysis shows a high degree of similarities with embryonic FHF/SHF cells. Bmp and Wnt are among the most differentially regulated pathways, and gain- and loss-of-function studies reveal that Bmp specifies GFP+ cells and RFP+ cells via the Bmp/Smad pathway and Wnt signaling, respectively. FHF/SHF cells can be isolated without reporters by the surface protein Cxcr4. This study provides novel insights into understanding the specification of two cardiac origins, which can be leveraged for PSC-based modeling of heart field/chamber-specific disease.