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1.
Circ Res ; 134(5): 529-546, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38348657

RESUMO

BACKGROUND: Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. METHODS: We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). RESULTS: From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. CONCLUSIONS: Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.


Assuntos
Células Endoteliais , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Adulto , Humanos , Animais , Camundongos , Hibridização in Situ Fluorescente , Artérias , Encéfalo , Veias
2.
Circulation ; 150(15): 1199-1210, 2024 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-39155863

RESUMO

BACKGROUND: Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant heterozygous variants in CALM1, CALM2, or CALM3, which each encode the identical CaM (calmodulin) protein. We hypothesized that antisense oligonucleotide (ASO)-mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, whereas the other 2 calmodulin genes would preserve CaM level and function. METHODS: We tested this hypothesis using human induced pluripotent stem cell-derived cardiomyocyte and mouse models of CALM1 pathogenic variants. RESULTS: Human CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1 knockout or CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization duration of CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced bidirectional ventricular tachycardia in Calm1N98S/+ mice without a deleterious effect on cardiac electrical or contractile function. CONCLUSIONS: These results provide proof of concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.


Assuntos
Calmodulina , Miócitos Cardíacos , Oligonucleotídeos Antissenso , Animais , Calmodulina/genética , Calmodulina/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Humanos , Miócitos Cardíacos/metabolismo , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/genética , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/terapia , Síndrome do QT Longo/fisiopatologia , Modelos Animais de Doenças , Potenciais de Ação/efeitos dos fármacos , Camundongos Knockout , Terapia Genética/métodos
3.
Circ Res ; 131(11): e152-e168, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36263775

RESUMO

BACKGROUND: The pioneer transcription factor (TF) GATA4 (GATA Binding Protein 4) is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression. METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays. RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 (NK2 homeobox 5) and ETS1 (ETS Proto-Oncogene 1) motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity. CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.


Assuntos
Endocárdio , Fator de Transcrição GATA4 , Proteína Proto-Oncogênica c-ets-1 , Animais , Camundongos , Cromatina/metabolismo , Endocárdio/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Miócitos Cardíacos/metabolismo , Células NIH 3T3 , Proteína Proto-Oncogênica c-ets-1/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33361330

RESUMO

The paucity of knowledge about cardiomyocyte maturation is a major bottleneck in cardiac regenerative medicine. In development, cardiomyocyte maturation is characterized by orchestrated structural, transcriptional, and functional specializations that occur mainly at the perinatal stage. Sarcomeres are the key cytoskeletal structures that regulate the ultrastructural maturation of other organelles, but whether sarcomeres modulate the signal transduction pathways that are essential for cardiomyocyte maturation remains unclear. To address this question, here we generated mice with cardiomyocyte-specific, mosaic, and hypomorphic mutations of α-actinin-2 (Actn2) to study the cell-autonomous roles of sarcomeres in postnatal cardiomyocyte maturation. Actn2 mutation resulted in defective structural maturation of transverse-tubules and mitochondria. In addition, Actn2 mutation triggered transcriptional dysregulation, including abnormal expression of key sarcomeric and mitochondrial genes, and profound impairment of the normal progression of maturational gene expression. Mechanistically, the transcriptional changes in Actn2 mutant cardiomyocytes strongly correlated with those in cardiomyocytes deleted of serum response factor (SRF), a critical transcription factor that regulates cardiomyocyte maturation. Actn2 mutation increased the monomeric form of cardiac α-actin, which interacted with the SRF cofactor MRTFA and perturbed its nuclear localization. Overexpression of a dominant-negative MRTFA mutant was sufficient to recapitulate the morphological and transcriptional defects in Actn2 and Srf mutant cardiomyocytes. Together, these data indicate that Actn2-based sarcomere organization regulates structural and transcriptional maturation of cardiomyocytes through MRTF-SRF signaling.


Assuntos
Actinina/genética , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Actinina/metabolismo , Animais , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Regulação da Expressão Gênica/genética , Camundongos , Mitocôndrias/metabolismo , Morfogênese , Mutação , Miócitos Cardíacos/patologia , Sarcômeros/patologia , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
5.
Circ Res ; 126(3): 377-394, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31999538

RESUMO

The heart is lined by a single layer of mesothelial cells called the epicardium that provides important cellular contributions for embryonic heart formation. The epicardium harbors a population of progenitor cells that undergo epithelial-to-mesenchymal transition displaying characteristic conversion of planar epithelial cells into multipolar and invasive mesenchymal cells before differentiating into nonmyocyte cardiac lineages, such as vascular smooth muscle cells, pericytes, and fibroblasts. The epicardium is also a source of paracrine cues that are essential for fetal cardiac growth, coronary vessel patterning, and regenerative heart repair. Although the epicardium becomes dormant after birth, cardiac injury reactivates developmental gene programs that stimulate epithelial-to-mesenchymal transition; however, it is not clear how the epicardium contributes to disease progression or repair in the adult. In this review, we will summarize the molecular mechanisms that control epicardium-derived progenitor cell migration, and the functional contributions of the epicardium to heart formation and cardiomyopathy. Future perspectives will be presented to highlight emerging therapeutic strategies aimed at harnessing the regenerative potential of the fetal epicardium for cardiac repair.


Assuntos
Cardiopatias/etiologia , Pericárdio/crescimento & desenvolvimento , Regeneração , Animais , Humanos , Miocárdio/citologia , Miocárdio/metabolismo , Comunicação Parácrina , Pericárdio/citologia , Pericárdio/metabolismo , Pericárdio/fisiologia
6.
Proc Natl Acad Sci U S A ; 116(2): 556-565, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30584088

RESUMO

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.


Assuntos
Autofagossomos/metabolismo , Doença de Depósito de Glicogênio Tipo IIb/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Miócitos Cardíacos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Técnicas de Inativação de Genes , Doença de Depósito de Glicogênio Tipo IIb/genética , Doença de Depósito de Glicogênio Tipo IIb/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Lisossomos/genética , Lisossomos/patologia , Miócitos Cardíacos/patologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo
7.
J Mol Cell Cardiol ; 132: 1-12, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31042488

RESUMO

Heart failure is the leading cause of morbidity and mortality worldwide. Several lines of evidence suggest that physical activity and exercise can pre-condition the heart to improve the response to acute cardiac injury such as myocardial infarction or ischemia/reperfusion injury, preventing the progression to heart failure. It is becoming more apparent that cardioprotection is a concerted effort between multiple cell types and converging signaling pathways. However, the molecular mechanisms of cardioprotection are not completely understood. What is clear is that the mechanisms underlying this protection involve acute activation of transcriptional activators and their corresponding gene expression programs. Here, we review the known stress-dependent transcriptional programs that are activated in cardiomyocytes and cardiac fibroblasts to preserve function in the adult heart after injury. Focus is given to prominent transcriptional pathways such as mechanical stress or reactive oxygen species (ROS)-dependent activation of myocardin-related transcription factors (MRTFs) and transforming growth factor beta (TGFß), and gene expression that positively regulates protective PI3K/Akt signaling. Together, these pathways modulate both beneficial and pathological responses to cardiac injury in a cell-specific manner.


Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica , Insuficiência Cardíaca/prevenção & controle , Infarto do Miocárdio/prevenção & controle , Miócitos Cardíacos/metabolismo , Transcrição Gênica , Animais , Fibroblastos/citologia , Insuficiência Cardíaca/metabolismo , Humanos , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Transdução de Sinais
8.
Circulation ; 138(17): 1864-1878, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-29716942

RESUMO

BACKGROUND: Hypertrophic cardiomyocyte growth and dysfunction accompany various forms of heart disease. The mechanisms responsible for transcriptional changes that affect cardiac physiology and the transition to heart failure are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling cardiomyocyte force transmission and propagation of electrical activity. The ID is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. METHODS: Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor (MRTF)-A and MRTF-B specifically in adult cardiomyocytes to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. RESULTS: We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure. Although mice lacking MRTFs in adult cardiomyocytes display normal cardiac physiology at baseline, pressure overload leads to rapid heart failure characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and cardiomyocyte adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by single-molecule localization microscopy may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. CONCLUSIONS: Our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates cross-talk between the actin and microtubule cytoskeleton and maintains ID integrity and cardiomyocyte homeostasis in heart disease.


Assuntos
Insuficiência Cardíaca/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Idoso , Animais , Animais Recém-Nascidos , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Miócitos Cardíacos/ultraestrutura , Células NIH 3T3 , Imagem Individual de Molécula , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Função Ventricular Esquerda , Remodelação Ventricular
9.
Development ; 142(1): 21-30, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25516967

RESUMO

An important pool of cardiovascular progenitor cells arises from the epicardium, a single layer of mesothelium lining the heart. Epicardium-derived progenitor cell (EPDC) formation requires epithelial-to-mesenchymal transition (EMT) and the subsequent migration of these cells into the sub-epicardial space. Although some of the physiological signals that promote EMT are understood, the functional mediators of EPDC motility and differentiation are not known. Here, we identify a novel regulatory mechanism of EPDC mobilization. Myocardin-related transcription factor (MRTF)-A and MRTF-B (MKL1 and MKL2, respectively) are enriched in the perinuclear space of epicardial cells during development. Transforming growth factor (TGF)-ß signaling and disassembly of cell contacts leads to nuclear accumulation of MRTFs and the activation of the motile gene expression program. Conditional ablation of Mrtfa and Mrtfb specifically in the epicardium disrupts cell migration and leads to sub-epicardial hemorrhage, partially stemming from the depletion of coronary pericytes. Using lineage-tracing analyses, we demonstrate that sub-epicardial pericytes arise from EPDCs in a process that requires the MRTF-dependent motile gene expression program. These findings provide novel mechanisms linking EPDC motility and differentiation, shed light on the transcriptional control of coronary microvascular maturation and suggest novel therapeutic strategies to manipulate epicardium-derived progenitor cells for cardiac repair.


Assuntos
Movimento Celular , Vasos Coronários/crescimento & desenvolvimento , Pericárdio/citologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Pericárdio/metabolismo , Pericárdio/ultraestrutura , Pericitos/citologia , Pericitos/efeitos dos fármacos , Fator de Resposta Sérica/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/farmacologia
10.
Nat Biomed Eng ; 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39237710

RESUMO

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) lack nanoscale structures essential for efficient excitation-contraction coupling. Such nanostructures, known as dyads, are frequently disrupted in heart failure. Here we show that the reduced expression of cardiomyopathy-associated 5 (CMYA5), a master protein that establishes dyads, contributes to dyad disorganization in heart failure and to impaired dyad assembly in hiPSC-CMs, and that a miniaturized form of CMYA5 suitable for delivery via an adeno-associated virus substantially improved dyad architecture and normalized cardiac function under pressure overload. In hiPSC-CMs, the miniaturized form of CMYA5 increased contractile forces, improved Ca2+ handling and enhanced the alignment of sarcomere Z-lines with ryanodine receptor 2, a protein that mediates the sarcoplasmic release of stored Ca2+. Our findings clarify the mechanisms responsible for impaired dyad structure in diseased cardiomyocytes, and suggest strategies for promoting dyad assembly and stability in heart disease and during the derivation of hiPSC-CMs.

11.
bioRxiv ; 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38464269

RESUMO

In the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.

12.
Nat Commun ; 15(1): 5929, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39009604

RESUMO

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol, and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines, the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs, bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks, which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible, scalable, and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications, and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Organoides , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Organoides/citologia , Técnicas de Cultura de Células/métodos , Reprodutibilidade dos Testes , Células Cultivadas , Criopreservação/métodos
13.
Stem Cell Reports ; 18(9): 1811-1826, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37595583

RESUMO

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that causes life-threatening arrhythmias and myocardial dysfunction. Pathogenic variants in Plakophilin-2 (PKP2), a desmosome component within specialized cardiac cell junctions, cause the majority of ACM cases. However, the molecular mechanisms by which PKP2 variants induce disease phenotypes remain unclear. Here we built bioengineered platforms using genetically modified human induced pluripotent stem cell-derived cardiomyocytes to model the early spatiotemporal process of cardiomyocyte junction assembly in vitro. Heterozygosity for truncating variant PKP2R413X reduced Wnt/ß-catenin signaling, impaired myofibrillogenesis, delayed mechanical coupling, and reduced calcium wave velocity in engineered tissues. These abnormalities were ameliorated by SB216763, which activated Wnt/ß-catenin signaling, improved cytoskeletal organization, restored cell junction integrity in cell pairs, and improved calcium wave velocity in engineered tissues. Together, these findings highlight the therapeutic potential of modulating Wnt/ß-catenin signaling in a human model of ACM.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , beta Catenina/genética , Sinalização do Cálcio , Junções Intercelulares , Miócitos Cardíacos , Placofilinas/genética
14.
bioRxiv ; 2023 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-37131696

RESUMO

Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa , and conversely, increased the expression of ventricular identity genes including Myl2 . Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.

15.
Nat Cardiovasc Res ; 2(10): 881-898, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38344303

RESUMO

Understanding how the atrial and ventricular heart chambers maintain distinct identities is a prerequisite for treating chamber-specific diseases. Here, we selectively knocked out (KO) the transcription factor Tbx5 in the atrial working myocardium to evaluate its requirement for atrial identity. Atrial Tbx5 inactivation downregulated atrial cardiomyocyte (aCM) selective gene expression. Using concurrent single nucleus transcriptome and open chromatin profiling, genomic accessibility differences were identified between control and Tbx5 KO aCMs, revealing that 69% of the control-enriched ATAC regions were bound by TBX5. Genes associated with these regions were downregulated in KO aCMs, suggesting they function as TBX5-dependent enhancers. Comparing enhancer chromatin looping using H3K27ac HiChIP identified 510 chromatin loops sensitive to TBX5 dosage, and 74.8% of control-enriched loops contained anchors in control-enriched ATAC regions. Together, these data demonstrate TBX5 maintains the atrial gene expression program by binding to and preserving the tissue-specific chromatin architecture of atrial enhancers.

16.
Nat Commun ; 12(1): 4155, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230480

RESUMO

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Pericárdio/citologia , Pericárdio/metabolismo , Animais , Quimiocinas , Vasos Coronários/metabolismo , Embrião de Mamíferos , Transição Epitelial-Mesenquimal , Expressão Gênica , Coração , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso , Proteínas Nucleares , Pericárdio/embriologia , Fator de Resposta Sérica , Transdução de Sinais , Transativadores , Fatores de Transcrição/metabolismo , Transcriptoma
17.
Nat Rev Cardiol ; 16(9): 519-537, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31028357

RESUMO

Arrhythmogenic cardiomyopathy is a genetic disorder characterized by the risk of life-threatening arrhythmias, myocardial dysfunction and fibrofatty replacement of myocardial tissue. Mutations in genes that encode components of desmosomes, the adhesive junctions that connect cardiomyocytes, are the predominant cause of arrhythmogenic cardiomyopathy and can be identified in about half of patients with the condition. However, the molecular mechanisms leading to myocardial destruction, remodelling and arrhythmic predisposition remain poorly understood. Through the development of animal, induced pluripotent stem cell and other models of disease, advances in our understanding of the pathogenic mechanisms of arrhythmogenic cardiomyopathy over the past decade have brought several signalling pathways into focus. These pathways include canonical and non-canonical WNT signalling, the Hippo-Yes-associated protein (YAP) pathway and transforming growth factor-ß signalling. These studies have begun to identify potential therapeutic targets whose modulation has shown promise in preclinical models. In this Review, we summarize and discuss the reported molecular mechanisms underlying the pathogenesis of arrhythmogenic cardiomyopathy.


Assuntos
Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Miócitos Cardíacos/patologia , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipogenia/genética , Animais , Apoptose , Arritmias Cardíacas/patologia , Cardiomiopatias/patologia , Linhagem Celular , Modelos Animais de Doenças , Fibrose , Via de Sinalização Hippo , Humanos , Inflamação/metabolismo , MicroRNAs/metabolismo , Mutação , Miócitos Cardíacos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP
19.
Dev Cell ; 42(6): 600-615.e4, 2017 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-28950101

RESUMO

Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.


Assuntos
Endorreduplicação , Pericárdio/fisiologia , Regeneração , Animais , Fenômenos Biomecânicos , Movimento Celular , Células Gigantes/patologia , Hipertrofia , Camundongos Endogâmicos C57BL , Mitose , Poliploidia , Peixe-Zebra
20.
J Vis Exp ; (109)2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-27023710

RESUMO

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.


Assuntos
Movimento Celular/fisiologia , Pericárdio/citologia , Pericárdio/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
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