Bulk RNA-sequencing from SVBP or TTL gene knock-out and WT engineered heart tissues
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE233852 | GEO | 2024/08/27
REPOSITORIES: GEO
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Project description:Rationale Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and is associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with findings that microtubule detyrosination (dTyr-MTs) is markedly elevated in heart failure. Acute reduction of dTyr-MTs by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, presenting a new perspective for HCM treatment. Objective In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model (Mybpc3-knock-in; KI), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents, in 3-week-old KI mice, and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show: i) TTL for 6 weeks dose-dependently reduced dTyr-MTs and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes. ii) TTL for 12 weeks reduced the abundance of dTyr-MTs in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in KI mice. iii) TTL for 10 days normalized cell area in HCM hiPSC-cardiomyocytes. iv) TTL overexpression activates transcription of several tubulin isoforms, and omics GO analysis revealed enrichment of components of the cytoskeleton, sarcomere Z-disc, mitochondria, and intercalated disc in KI mice. v) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.
2024-08-28 | PXD054236 | Pride
Project description:Desmosomes are specialized cell-cell junctions critically important in the heart, where their disruption promotes arrhythmogenic cardiomyopathy (ACM). Individuals carrying truncating variants in the desmosomal gene desmoplakin (DSP) often show a prominent inflammatory component in the absence of known antigenic stimuli, termed “hot phase” arrythmia. To assess the role of innate immune activation in DSP-cardiomyopathy, we generated engineered heart tissues (EHTs) from two patients with heterozygous DSP truncation variants (DSPtv, p.E1597X and p.R1951X). We also generated EHTs with homozygous DSP deletion (DSP-/-) for modeling. DSP-/- EHTs revealed impaired force production and reduced conduction velocity compared to isogenic controls, features consistent with ACM. RNA sequencing and protein characterization demonstrated a dramatic increase in innate immune activation in DSP-/- EHTs. Similarly, patient-derived DSPtv EHTs show enhanced sensitivity to inflammatory stimuli seen as reduced contractility and greater cytokine release. Inhibition of NFκB signaling improved baseline force production and mechanical response to strain in DSPtv EHTs, indicating that anti-inflammatory modulation may improve mechanical function in DSP deficient hearts. Furthermore, genomic correction of p.R1951X using adenine base editing reduced inflammatory biomarkers in cultured EHTs. Taken together, these data identify that DSPtv promote innate immune activation, creating an arrhythmogenic substrate in the concealed phase of ACM.
2024-07-03 | GSE248187 | GEO
Project description:Tubulin detyrosination-tyrosination cycle regulates the stability of microtubules. Thus far described on α-tubulins, the tyrosination level is maintained by a single tubulin-tyrosine ligase (TTL). However, the precise dynamics and tubulin isoforms which undergo (de)tyrosination in neurons are unknown. Here, we exploit the substrate promiscuity of the TTL to introduce an O-propargyl-L-tyrosine in cancer cell lines and neurons. Mass spectrometry-based chemical proteomics in neuroblastoma cells using the O-propargyl-L-tyrosine probe revealed previously discussed tyrosination of TUBA4A, MAPRE1, and other non-tubulin proteins. This finding was further corroborated in differentiating neurons. We present the method for tubulin tyrosination profiling in living cells. Our results show that detyrosination-tyrosination is not restricted to α-tubulins with coded C-terminal tyrosine and is thus involved in fine-tuning of the tubulin and non-tubulin proteins during neuronal differentiation.
2022-10-20 | PXD037402 | Pride
Project description:Reducing microtubule detyrosination has been shown to inprove contractility in heart failure cardiomyocytes. To evaluate the feasibility of targeting the detyrosinated microtubule network for treatment of cardiomyopathy in an in-vivo context we overexpressed tubulin tyrosine ligase (TTL) by AAV9 delivery in a mouse model of HCM (Mybpc3-/-) and an empty vector as control in HCM and WT mice.
2024-08-27 | GSE233851 | GEO
Project description:Low-LET radiation can cause cardiovascular dysfunctions at high-dose rates. For example, photons used in thoracic radiotherapy are known to cause acute cardiac tissue damage with elevated serum cardiac Troponin I level and long-term cardiac complications when delivered as fractionated exposures at high-dose rates. However, the effects of continuous low-dose rate radiation exposure on the heart, which simulate the space radiation environment, have not been well-studied. In this study, we aim to model low-LET space radiation-induced cardiovascular dysfunction using human induced pluripotent stem cell (iPSC)-derived engineered heart tissues (EHTs) exposed to protracted γ-ray irradiation. The investigation of pathophysiological changes in this model may provide insights and guide the development of countermeasures. As a proof-of-principle for the application of this model in drug development, we also tested the protective effect of a mitochondrial-specific antioxidant, MitoTempo, on irradiated EHTs.
2023-09-05 | GSE242108 | GEO
Project description:Chronic stimulation of cardiac a1A-adrenergic receptors (a1A-ARs) improves symptoms in multiple preclinical models of heart failure. However, the translational significance remains unclear. This study tested the effects of chronic a1A-AR stimulation on human engineered heart tissue (EHT). EHTs were created from thin slices of decellularized pig myocardium seeded with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and fibroblasts. Using a paired experimental design, EHTs were cultured for 3 wk., mechanically tested, cultured again for 2 wk. with a1A-AR agonist A61603 (10 nM) or vehicle control, and retested after drug washout for 24-hr. Separate control experiments determined the effects of EHT age (3-5 wk.) or repeat mechanical testing. We found that chronic A61603 treatment caused a 25% increase of length-dependent activation (LDA) of contraction compared to vehicle treatment (n=7/group, P=0.035). EHT force was not increased after chronic A61603 treatment. However, after vehicle treatment, EHT force was increased 35% relative to baseline testing (n=7/group, P=0.022), suggesting EHT maturation. Control experiments showed increased EHT force resulted from repeat mechanical testing, not from EHT aging. RNA-seq analysis confirmed the a1A-AR is expressed in human EHTs and found chronic A61603 treatment affected gene expression in biological pathways which are known to be activated by a1A-ARs, including the MAP kinase signaling pathway. In conclusion, chronic stimulation of the a1A-AR, a promising preclinical target for treating heart failure, gave mixed results in human EHTs. Chronic a1A-AR stimulation had a positive effect (increased LDA) but potentially, a negative effect (lower EHT force). The translational significance of these findings requires further study.
2023-01-01 | GSE216439 | GEO
Project description:Tridimensional cardiac differentiation from hiPSCs has been largely described in the literature. However, the exact impact that 3D culture has throughout the entire process of cardiac differentiation remains poorly defined. We developed a robust and efficient 3D platform for cardiomyocyte differentiation from hiPSCs, based on the temporal modulation of WNT signalling using small molecules. 3D aggregates of hiPSCs were generated by forced aggregation in microwells and subsequently differentiated. In order to determine the differences in gene expression profile due to 3D culture throughout the different stages of cardiac differentiation, we compared transcriptional changes between cells in 3D aggregates and standard 2D monolayer cardiac differentiation. Analysis of these data suggests a faster commitment of hiPSCs toward the cardiac lineage and also higher degree of cardiomyocyte functional maturation after 20 days of culture in the 3D aggregates when compared with the 2D monolayer.
2019-06-24 | GSE116574 | GEO
Project description:The purpose of this experiment was to compared the transcriptome of hepatocyte-like cells (HLCs) generated in vitro and adult primary human hepatocytes (PHHs). HLCs were differentiated from either hESCs or hIPSCs using previously established protocols (Hannah et al 2013; Segeritz et al 2018). Undifferentiated hIPSCs were used as control to confirm differentiation status. PHHs were commercially sourced as well as freshly isolated from donors.
2022-08-11 | E-MTAB-10634 | biostudies-arrayexpress
Project description:We generated hiPSCs from patients fibloblast with retinitis pigmentosa (RP) using retrovirus and Sendai virus vectors, which we differentiated into hiPSC derived retinal pigment epithelium using two different methods (SDIA and SFEB methods). We investigated whether these hiPSC-RPE colonies, which were differentiated from various cell lines and methods, showed similar gene expression patterns to those of native RPE. We classified hiPSC-RPE, hiPSCs, and fibroblasts from RP patients, hRPE (commercially available human fetal RPE, Lonza) , ARPE19 (a human RPE cell line), and other human tissues from 54,675 probe sets using microarray data.
2014-11-03 | E-GEOD-50738 | biostudies-arrayexpress
Project description:Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart and high concentrations of RA have been shown to steer differentiation towards atrial CMs, whereas lower concentrations of RA promote a more ventricular-like CM profile. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of retinoic acid (RA) during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts. Different concentrations of RA (Control group without RA, LRA group with 0.05 µM and HRA group with 0.1 µM) were administered during third to sixth days of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. The maturation and growth of EHTs were induced in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. The function of CMs and EHTs was analyzed under different conditions. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPRC and immunofluorescence staining analysis. RNA sequencing was conducted on EHTs to examine how RA affects both the function and structure of EHT. Results: In the HRA group, hiPSC-CMs exhibited the first onset of beating and showed the highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are the marker genes for left ventricular CMs, was also the highest in the HRA group. The transcription factor MEF2C associated with RA and ventricular development genes, NPPA and MYH7, were highly expressed in the HRA group, while GATA4 was less expressed in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was more functionally similar to the left ventricle. The expression of TBX5 and NKX2.5 was found to be the highest expression in the HRA group of EHT, while expression of TBX20 and ISL1 was found to be the highest expression in control group. When the electrical stimulation frequency of EHT in the HRA group was raised, it correspondingly increased its contractile force. The heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paved the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine.
2023-10-25 | GSE245954 | GEO