Investigating the role of PITX2 in atrial metabolic function

E. Thiemann (Hamburg)¹, C. Schulz (Hamburg)², T. Christ (Hamburg)³, M. Heine (Hamburg)⁴, J. Heeren (Hamburg)⁴, L. Fabritz (Hamburg)⁵, T. Eschenhagen (Hamburg)³, P. Kirchhof (Hamburg)⁵, F. Cuello (Hamburg)^3
1Universitätsklinikum Hamburg-Eppendorf Institut für Experimentelle Pharmakologie und Toxikologie Hamburg, Deutschland; ²Universitäres Herz- und Gefäßzentrum Hamburg Hamburg, Deutschland; ³Universitätsklinikum Hamburg-Eppendorf Institut für Klinische Pharmakologie und Toxikologie Hamburg, Deutschland; ⁴Universitätsklinikum Hamburg-Eppendorf Institut für Biochemie und Molekulare Zellbiologie Hamburg, Deutschland; ⁵Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Lübeck, Deutschland

Aim: Atrial fibrillation is the most common cardiac arrhythmia and is associated with increased morbidity and mortality. Genome wide association studies revealed several risk regions which are closely related to the development of atrial fibrillation. Single nucleotide polymorphisms in the chromosomal 4q25 region, which is located in vicinity to the PITX2 gene, showed the strongest association with atrial fibrillation. Based on previous studies suggesting a role of PITX2 in cardiac metabolic function, we aimed to further investigate the role of PITX2 in atrial metabolism using human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes and engineered heart tissue (EHT).

Methods: HiPSCs with CRISPR/Cas9-induced PITX2 knockout (PITX2KO) and isogenic control hiPSCs were differentiated into atrial cardiomyocytes and used for generating atrial EHTs. Changes in the expression of genes involved in metabolic pathways were analyzed by RNA sequencing with subsequent bioinformatic analysis using R. To compare the response of ventricular versus atrial tissue to metabolic stress, atrial and ventricular wildtype EHTs were put under acute metabolic stress by increasing media glucose levels or adding palmitic acid. Contractile function was assessed and glucose and fatty acid uptake analyzed via incubation with radioactively labeled metabolic tracers.

Results: Analysis of RNA sequencing data from PITX2KO and isogenic control EHTs revealed that the oxidative phosphorylation pathway was among the most altered KEGG pathways with a high number of differentially expressed genes in PITX2KO. In addition, analysis of the PPAR signaling pathway revealed differential expression of several lipid metabolic genes. PPARy and genes encoding for fatty acid transporters, such as SLC27A6 and CD36, showed changed expression in PITX2KO. Furthermore, PITX2KO also led to differential expression of genes involved in lipoprotein metabolism, such as ANGPTL4, LPL and LIPG. Induction of metabolic stress did not alter the contractile function of ventricular EHTs, whereas atrial EHTs exhibited decreased beating force and irregular beating pattern after palmitic acid treatment. In addition, the uptake of radioactively labeled glucose and oleic acid was increased in palmitic acid-treated atrial EHTs, whereas ventricular EHTs showed increased glucose uptake but decreased oleic acid uptake.

Conclusion: RNA sequencing analysis of PITX2KO atrial EHTs suggests a role of the transcription factor PITX2 in the transcriptional regulation of mitochondrial and lipid metabolic genes. Metabolic stress conditions induce a different response in atrial versus ventricular EHTs with atrial EHTs showing a higher sensitivity to treatment with saturated fatty acids.