A heterozygous TBX5 loss-of-function mutation in an iPSC model led to down-regulated glucose uptake and ATP generation, and higher cholesterol levels in cardiac progenitor cells.
A TBX5 loss-of-function mutation disrupts energy and cholesterol metabolism in iPSC-derived cardiomyocytes, providing a mechanistic link to the cardiac malformations observed in Holt-Oram Syndrome.
Abstract Background TBX5 is a transcription factor with important regulatory functions in cardiogenesis, in particular development of septation, conduction system, and cardiomyogenesis. Multiple involvement of TBX5 in combination with large number of mutations described and the phenotypic patients´variability makes it difficult to establish a clear genotype-phenotype correlation. Purpose To define the impact of a specific TBX5 mutation, we developed a patient-specific iPSC model for Holt-Oram Syndrome based on a well-described phenotype with known genotype and detailed characterisation of the underlying TBX5 mutation. The heterozygous TBX5 loss-of-function mutation leading to haploinsufficiency was investigated in vitro in 2D/3D-model to identify molecular mechanisms involved in impaired cardiac development leading to the patient's congenital heart defects. Methods Patient-specific hiPSCs and isogenic CRISPR/Cas9-corrected iPSCs were differentiated using a cardiac differentiation protocol (2D). Transcriptome profiles (bulk RNAseq) of beating cardiac progenitor cells (CPCs)/early cardiomyocytes (CMs) were analysed (D8/D10/D14) and CM sarcomeres were assessed in detail (D30). Single-cell transcriptomes of the isogenic CPCs (D8) were analysed (sc-RNAseq). The transcriptomes were subjected to downstream bioinformatic analysis, including unsupervised clustering, differential expression testing, and pathway analysis. To prove identified dysregulations, human heart organoids (hHOs) (3D) were generated from the isogenic lines and analysed using bulk RNAseq (D8/10/D15). Glucose uptake, ATP generation, and cholesterol levels were measured during in vitro differentiation (D8/D10/D14) to further validate the identified metabolic dysregulations. Results Bulk and sc-RNAseq (2D) revealed dysregulations in energy metabolism, mitochondrial metabolic pathways relevant to glycolysis and tricarboxylic acid cycle, and alterations in cholesterol metabolism in CPCs/early CMs, which were confirmed in hHOs. This 3D-model identified cell types other than CMs, which are important for septation, being also affected. Glucose uptake and ATP generation were down-regulated in the mutant CPCs/CMs (2D), while cholesterol levels were higher, consistent with bulk and sc-RNAseq data, possibly explaining the differences in CM sarcomeres. Conclusion The investigated loss-of-function TBX5 mutation led to massive dysregulation of energy metabolism and mitochondrial metabolic pathways as well as alterations in cholesterol metabolism in CPCs/early CMs. The observed differences in sarcomere structure are to be seen as consequence of the disturbed metabolism and can be attributed to the TBX5 mutation. We hypothesize that the TBX5 loss-of-function mutation disturbs processes essential for cell physiology, resulting in cell fate perturbation and thus affecting temporal and spatial progression of cardiogenesis, in this case leading to cardiac malformations such as multiple ventricular septal defects.
Dressen et al. (Sat,) conducted a other in Holt-Oram Syndrome. Heterozygous TBX5 loss-of-function mutation (in vitro model) vs. Isogenic CRISPR/Cas9-corrected iPSCs was evaluated on Transcriptome profiles, glucose uptake, ATP generation, and cholesterol levels. A heterozygous TBX5 loss-of-function mutation in an iPSC model led to down-regulated glucose uptake and ATP generation, and higher cholesterol levels in cardiac progenitor cells.