Figure 6.

Detailed characterization of mitochondrial function in response to indole-3-propionic acid (IPA) in HL-1 cardiomyocytes. (A) Schematic representation of respiratory chain including substrates for detailed analysis of respiratory chain complexes. TMPD: N,N,N′,N′-Tetrametyhl-p-phenylenediamine. (B) Measurement of oxygen consumption rate (OCR) over time in response to substrates of respiratory chain complexes injected sequentially. Rot, rotenone; AA, antimycin A. (C) Mitochondrial stress testing in permeabilized HL-1 cardiomyocytes including palmitoyl-L-carnitine in response to 1 mM IPA (24 h). (D) Mitochondrial stress testing in permeabilized HL-1 cardiomyocytes including octanoyl-L-carnitine in response to 1 mM IPA (24 h). (E) Addition of exogenous cytochrome c to permeabilized HL-cardiomyocytes after treatment with IPA (24 h). Measurement of OCR over time. Sap, saponin; suc, succinate; rot, rotenone; cyt c, cytochrome c. (B–E) Data were shown as mean ± SEM with N = 5/group by 2-way ANOVA with Dunnett post-hoc test. (F) Mitochondrial membrane potential was measured by TMRE staining in HL-1 cardiomyocytes after treatment with IPA (24 h). 20 μM FCCP for 10 min was used as positive control. RFU: relative fluorescence units. Data were shown as mean ± SD with N = 4–5/group by 1-way ANOVA with post-hoc test. (G) Measurement of ROS production and hydroxyl radicals by H2DCFDA assay in HL-1 cardiomyocytes. HL-1 cardiomyocytes were treated with 1 mM IPA for 24 h and 30 min., 1 mM iron(II) sulfate for 30 min was used as positive control. Data were shown as mean ± SD with N = 4/group by 1-way ANOVA with post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.