RESUMO
The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.
Assuntos
Ciclo Celular , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Linhagem CelularRESUMO
The immature heart is known to be resistant to ischemia-reperfusion (I/R) injury; however, key proteins engaged in phospho-dependent signaling pathways crucial to cell survival are not yet defined. Our goal was to determine the postnatal changes in myocardial tolerance to I/R, including baseline expression of key proteins governing I/R tolerance and their phosphorylation during I/R. Hearts from male C57Bl/6 mice (neonates, 2, 4, 8, and 12 wk of age, n = 6/group) were assayed for survival signaling/effectors [Akt, p38MAPK, glycogen synthase kinase-3ß (GSK-3ß), heat shock protein 27 (HSP27), connexin-43, hypoxia-inducible factor-1α (HIF-1α), and caveolin-3] and regulators of apoptosis (Bax and Bcl-2) and autophagy (LC3B, Parkin, and Beclin1). The effect of I/R on ventricular function was measured in isolated perfused hearts from immature (4 wk) and adult (12 wk) mice. The neonatal myocardium exhibits a large pool of inactive Akt; high phospho-activation of p38MAPK, HSP27 and connexin-43; phospho-inhibition of GSK-3ß; and high expression of caveolin-3, HIF-1α, LC3B, Beclin1, Bax, and Bcl-2. Immature hearts sustained less dysfunction and infarction following I/R than adults. Emergence of I/R intolerance in adult vs. immature hearts was associated with complex proteomic changes: decreased expression of Akt, Bax, and Bcl-2; increased GSK-3ß, connexin-43, HIF-1α, LC3B, and Bax:Bcl-2; enhanced postischemic HIF-1α, caveolin-3, Bax, and Bcl-2; and greater postischemic GSK-3ß and HSP27 phosphorylation. Neonatal myocardial stress resistance reflects high expression of prosurvival and autophagy proteins and apoptotic regulators. Notably, there is high phosphorylation of GSK-3ß, p38MAPK, and HSP27 and low phosphorylation of Akt (high Akt "reserve"). Subsequent maturation-related reductions in I/R tolerance are associated with reductions in Akt, Bcl-2, LC3B, and Beclin1, despite increased expression and reduced phospho-inhibition of GSK-3ß.
Assuntos
Sobrevivência Celular/fisiologia , Isquemia Miocárdica/metabolismo , Transdução de Sinais/fisiologia , Envelhecimento , Animais , Animais Recém-Nascidos , Regulação da Expressão Gênica , Coração/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Aumento de PesoRESUMO
AIMS: After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell-cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimize cardiac repair and regeneration. METHODS AND RESULTS: We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryoinjured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a, and miR-30e to human stem cell-derived cardiomyocytes and cryoinjured EHM constructs. CONCLUSION: Here, we describe the first characterization of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative miRs, which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation.
Assuntos
Proliferação de Células , Vesículas Extracelulares/transplante , MicroRNAs/metabolismo , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/metabolismo , Pericárdio/transplante , Regeneração , Animais , Animais Recém-Nascidos , Linhagem Celular , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Contração Miocárdica , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Comunicação Parácrina , Pericárdio/metabolismo , Ratos , Recuperação de Função Fisiológica , Fatores de TempoRESUMO
BACKGROUND: Under certain conditions, the physiological repair of connective tissues might fail to restore the original structure and function. Optimized engineered connective tissues (ECTs) with biophysical properties adapted to the target tissue could be used as a substitution therapy. This study aimed to investigate the effect of ECT enforcement by a complex of multiwall carbon nanotubes with chitosan (C-MWCNT) to meet in vivo demands. MATERIALS AND METHODS: ECTs were constructed from human foreskin fibroblasts (HFF-1) in collagen type I and enriched with the three different percentages 0.025, 0.05 and 0.1% of C-MWCNT. Characterization of the physical properties was performed by biomechanical studies using unidirectional strain. RESULTS: Supplementation with 0.025% C-MWCNT moderately increased the tissue stiffness, reflected by Young's modulus, compared to tissues without C-MWCNT. Supplementation of ECTs with 0.1% C-MWCNT reduced tissue contraction and increased the elasticity and the extensibility, reflected by the yield point and ultimate strain, respectively. Consequently, the ECTs with 0.1% C-MWCNT showed a higher resilience and toughness as control tissues. Fluorescence tissue imaging demonstrated the longitudinal alignment of all cells independent of the condition. CONCLUSION: Supplementation with C-MWCNT can enhance the biophysical properties of ECTs, which could be advantageous for applications in connective tissue repair.
Assuntos
Quitosana/farmacologia , Tecido Conjuntivo/fisiologia , Nanotubos de Carbono/química , Engenharia Tecidual , Animais , Fenômenos Biomecânicos , Bovinos , Linhagem Celular , Quitosana/química , Módulo de Elasticidade , Fibroblastos/efeitos dos fármacos , HumanosRESUMO
This protocol describes a robust method for the generation of engineered human myocardium (EHM) from pluripotent stem cells (PSCs) in a multi-well plate under defined, serum-free conditions. By parallel culture of up to 48 EHM in one plate, contractile heart muscle can be obtained to serve numerous applications, including drug screening and disease modelling. This protocol has been successfully applied to human embryonic stem (HES) cell- and induced PSC-derived cardiomyocytes, subtype-specific, i.e., atrial and ventricular, and commercially available cardiomyocyte preparations. For complete details on the use and execution of this protocol, please refer to Tiburcy et al. (2017).
Assuntos
Técnicas de Cultura de Células/métodos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Engenharia Tecidual/métodos , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Pyruvate dehydrogenase (PDH) complex, a multienzyme complex at the nexus of glycolytic and Krebs cycles, provides acetyl-CoA to the Krebs cycle and NADH to complex I thus supporting a critical role in mitochondrial energy production and cellular survival. PDH activity is regulated by pyruvate dehydrogenase phosphatases (PDP1, PDP2), pyruvate dehydrogenase kinases (PDK 1-4), and mitochondrial pyruvate carriers (MPC1, MPC2). As NADH-dependent oxidative phosphorylation is diminished in systolic heart failure, we tested whether the left ventricular myocardium (LV) from end-stage systolic adult heart failure patients (n = 26) exhibits altered expression of PDH complex subunits, PDK, MPC, PDP, and PDH complex activity, compared to LV from nonfailing donor hearts (n = 21). Compared to nonfailing LV, PDH activity and relative expression levels of E2, E3bp, E1α, and E1ß subunits were greater in LV failure. PDK4, MPC1, and MPC2 expressions were decreased in failing LV, whereas PDP1, PDP2, PDK1, and PDK2 expressions did not differ between nonfailing and failing LV. In order to examine PDK4 further, donor human LV cardiomyocytes were induced in culture to hypertrophy with 0.1 µM angiotensin II and treated with PDK inhibitors (0.2 mM dichloroacetate, or 5 mM pyruvate) or activators (0.6 mM NADH plus 50 µM acetyl CoA). In isolated hypertrophic cardiomyocytes in vitro, PDK activators and inhibitors increased and decreased PDK4, respectively. In conclusion, in end-stage failing hearts, greater expression of PDH proteins and decreased expression of PDK4, MPC1, and MPC2 were evident with higher rates of PDH activity. These adaptations support sustained capacity for PDH to facilitate glucose metabolism in the face of other failing bioenergetic pathways.
Assuntos
Insuficiência Cardíaca Sistólica/tratamento farmacológico , Complexo Piruvato Desidrogenase/uso terapêutico , Animais , Insuficiência Cardíaca Sistólica/patologia , Humanos , Pessoa de Meia-Idade , Complexo Piruvato Desidrogenase/farmacologia , RatosRESUMO
BACKGROUND: The combination of cardiomyocyte (CM) and vascular cell (VC) fetal reprogramming upon stress culminates in end-stage heart failure (HF) by mechanisms that are not fully understood. Previous studies suggest KLF15 as a key regulator of CM hypertrophy. OBJECTIVES: This study aimed to characterize the impact of KLF15-dependent cardiac transcriptional networks leading to HF progression, amenable to therapeutic intervention in the adult heart. METHODS: Transcriptomic bioinformatics, phenotyping of Klf15 knockout mice, Wnt-signaling-modulated hearts, and pressure overload and myocardial ischemia models were applied. Human KLF15 knockout embryonic stem cells and engineered human myocardium, and human samples were used to validate the relevance of the identified mechanisms. RESULTS: The authors identified a sequential, postnatal transcriptional repression mediated by KLF15 of pathways implicated in pathological tissue remodeling, including distinct Wnt-pathways that control CM fetal reprogramming and VC remodeling. The authors further uncovered a vascular program induced by a cellular crosstalk initiated by CM, characterized by a reduction of KLF15 and a concomitant activation of Wnt-dependent transcriptional signaling. Within this program, a so-far uncharacterized cardiac player, SHISA3, primarily expressed in VCs in fetal hearts and pathological remodeling was identified. Importantly, the KLF15 and Wnt codependent SHISA3 regulation was demonstrated to be conserved in mouse and human models. CONCLUSIONS: The authors unraveled a network interplay defined by KLF15-Wnt dynamics controlling CM and VC homeostasis in the postnatal heart and demonstrated its potential as a cardiac-specific therapeutic target in HF. Within this network, they identified SHISA3 as a novel, evolutionarily conserved VC marker involved in pathological remodeling in HF.
Assuntos
Insuficiência Cardíaca/metabolismo , Fatores de Transcrição Kruppel-Like/deficiência , Proteínas de Membrana/biossíntese , Regulação para Cima/fisiologia , Remodelação Ventricular/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Genome editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (DMD). Most of these mutations are clustered in "hotspots." There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, we screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting. In proof-of-concept studies, we performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. We conclude that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.
Assuntos
Edição de Genes , Genoma Humano , Distrofia Muscular de Duchenne/genética , Mutação/genética , Miocárdio/patologia , Engenharia Tecidual/métodos , Sequência de Bases , Distrofina/genética , Éxons/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction and is associated with life-threating complications in the acute phase. The underlying disease mechanism in TTS is still unknown. A genetic basis has been suggested to be involved in the pathogenesis. OBJECTIVES: The aims of the study were to establish an in vitro induced pluripotent stem cell (iPSC) model of TTS, to test the hypothesis of altered ß-adrenergic signaling in TTS iPSC-cardiomyocytes (CMs), and to explore whether genetic susceptibility underlies the pathophysiology of TTS. METHODS: Somatic cells of patients with TTS and control subjects were reprogrammed to iPSCs and differentiated into CMs. Three-month-old CMs were subjected to catecholamine stimulation to simulate neurohumoral overstimulation. We investigated ß-adrenergic signaling and TTS cardiomyocyte function. RESULTS: Enhanced ß-adrenergic signaling in TTS-iPSC-CMs under catecholamine-induced stress increased expression of the cardiac stress marker NR4A1; cyclic adenosine monophosphate levels; and cyclic adenosine monophosphate-dependent protein kinase A-mediated hyperphosphorylation of RYR2-S2808, PLN-S16, TNI-S23/24, and Cav1.2-S1928, and leads to a reduced calcium time to transient 50% decay. These cellular catecholamine-dependent responses were mainly mediated by ß1-adrenoceptor signaling in TTS. Engineered heart muscles from TTS-iPSC-CMs showed an impaired force of contraction and a higher sensitivity to isoprenaline-stimulated inotropy compared with control subjects. In addition, altered electrical activity and increased lipid accumulation were detected in catecholamine-treated TTS-iPSC-CMs, and were confirmed by differentially expressed lipid transporters CD36 and CPT1C. Furthermore, we uncovered genetic variants in different key regulators of cardiac function. CONCLUSIONS: Enhanced ß-adrenergic signaling and higher sensitivity to catecholamine-induced toxicity were identified as mechanisms associated with the TTS phenotype. (International Takotsubo Registry [InterTAK Registry] [InterTAK]; NCT01947621).
Assuntos
Catecolaminas/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Cardiomiopatia de Takotsubo/metabolismo , Adulto , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais , Cardiomiopatia de Takotsubo/patologiaRESUMO
Recreating the beating heart in the laboratory continues to be a formidable bioengineering challenge. The fundamental feature of the heart is its pumping action, requiring considerable mechanical forces to compress a blood filled chamber with a defined in- and outlet. Ventricular output crucially depends on venous loading of the ventricles (preload) and on the force generated by the preloaded ventricles to overcome arterial blood pressure (afterload). The rate of contraction is controlled by the spontaneously active sinus node and transmission of its electrical impulses into the ventricles. The underlying principles for these physiological processes are described by the Frank-Starling mechanism and Bowditch phenomenon. It is essential to consider these principles in the design and evaluation of tissue engineered myocardium. This review focuses on current strategies to evoke mechanical loading in hydrogel-based heart muscle engineering.
Assuntos
Ventrículos do Coração , Modelos Cardiovasculares , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Engenharia Tecidual/métodos , Função Ventricular , Animais , Fenômenos Biomecânicos , Fenômenos Biofísicos , Ventrículos do Coração/citologia , HumanosRESUMO
BACKGROUND: Our previous randomized controlled trial demonstrated cardiorespiratory protection by remote ischemic preconditioning (RIPC) in children before cardiac surgery. However, the impact of RIPC on myocardial prosurvival intracellular signaling remains unknown in cyanosis. RIPC may augment phosphorylated protein signaling in myocardium and circulating leukocytes during tetralogy of Fallot (ToF) repair. METHODS AND RESULTS: Children (n=40) undergoing ToF repair were double-blind randomized to RIPC (n=11 boys, 9 girls) or control (sham RIPC: n=9 boys, 11 girls). Blood samples were taken before, immediately after, and 24 hours after cardiopulmonary bypass. Resected right ventricular outflow tract muscle and leukocytes were processed for protein expression and mitochondrial respiration. There was no difference in age (7.1 ± 3.4 versus 7.1 ± 3.4 months), weight (7.7 ± 1.8 versus 7.5 ± 1.9 kg), or bypass or aortic cross-clamp times between the groups (control versus RIPC, mean±SD). No differences were seen between the groups for an increase in the ratio of phosphorylated to total protein for protein kinase B, p38 mitogen activated protein kinase, signal transducer and activator of transcription 3, glycogen synthase kinase 3ß, heat shock protein 27, Connexin43, or markers associated with promotion of necrosis (serum cardiac troponin I), apoptosis (Bax, Bcl-2), and autophagy (Parkin, Beclin-1, LC3B). A high proportion of total proteins were in phosphorylated form in control and RIPC myocardium. In leukocytes, mitochondrial respiration and assessed protein levels did not differ between groups. CONCLUSIONS: In patients with cyanotic heart disease, a high proportion of proteins are in phosphorylated form. RIPC does not further enhance phosphorylated protein signaling in myocardium or circulating leukocytes in children undergoing ToF repair. CLINICAL TRIAL REGISTRATION: URL: (http://www.anzctr.org.au/trial_view.aspx?id=335613. Unique identifier: Australian New Zealand Clinical Trials Registry number ACTRN12610000496011.