| Autor: |
Nager, Mireia, Bhujabal, Zambarlal, Bowitz Larsen, Kenneth B, Kalstad, Trine B, Myrmel, Truls, Birgisdottir, Asa B |
| Zdroj: |
Circulation (Ovid); November 2022, Vol. 146 Issue: Supplement 1 pA14720-A14720, 1p |
| Abstrakt: |
Introduction:Engineered heart tissue (EHT) consisting of beating cardiomyocytes differentiated from human inducible pluripotent stem cells (hiPSCs) has the potential to recapitulate molecular and functional features of the human heart. Cardiac homeostasis is dependent on lysosomal removal of damaged mitochondria through selective autophagy (mitophagy). The level of mitophagy and cardioprotective impact during ischemia and reperfusion remains elusive and demands further investigation.Objective:To assess mitophagy during ischemia-reperfusion simulation in human EHTs.Methods and results:A hiPSC line was genetically modified using CRISPR/Cas9 knock-in to insert a fluorescent tag (mCherry or EGFP) on Translocase of the Outer Mitochondrial Membrane 20(TOMM20)gene resulting in hiPSCs with fluorescent mitochondria. The hiPSCs were differentiated to cardiomyocytes that were embedded in a fibrin hydrogel between two flexible silicone posts to generate EHTs. The EHTs (21 days or older) were subjected to 90 min of ischemia simulation followed by 120 min of simulated reperfusion with and without the lysosomal inhibitor Bafilomycin A1 (BafA1) to measure flux. Time-matched control EHTs remained in normal conditions (with and without BafA1). The EHTs were fixed and sections were immunostained with the lysosomal marker LAMP1A and analyzed by confocal microscopy. Co-localization of fluorescent mitochondria and stained lysosomes was observed after 120 min of reperfusion. To further assess this co-localization we used proximity ligation assay (PLA), an antibody-based assay allowing verification of close proximity (within 40 nm) of two proteins resulting in a single fluorescent signal (PLA dot). We performed the assay using anti-TOMM20 and anti-LAMP1A antibodies to localize mitochondrial content within lysosomes. Notably, there was a significant increase in the number of PLA dots in response to 120 min of reperfusion in the presence of BafA1 (3.41±0.56 fold vs. control, p<0.01; n=4), indicative of induced mitophagy flux.Conclusion:We observed a marked increase in lysosomal degradation of mitochondria in EHTs during reperfusion. The functional impact on EHTs during ischemia and reperfusion needs further research. |
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