Figure 1.

BT2 acutely uncouples mitochondria in rat and human cardiomyocytes.A, representative oxygen consumption trace of neonatal rat ventricular myocytes (NRVMs) offered 40 μM BT2, 80 μM BT2, or vehicle control (DMSO) 5 min prior to conducting measurements. Forty micro molars and eighty micro molars BT2 were chosen as concentrations because they are 10-fold above the EC₅₀ (40 μM) or previously used in the literature (80 μM) (84). (n = 10 technical replicates from a single experiment). B, collated oxygen consumption rate parameters for NRVMs offered 40 μM BT2 in the presence or the absence of 1 μM norepinephrine. (n = 4 biological replicates). C, proton leak–linked respiration in NRVMs. Experiments conducted as in (A and B). [BT2] = 5, 10, 20, 40, and 80 μM. (n = 4 biological replicates). D, collated oxygen consumption rate parameters for human iPSC-derived iCell cardiomyocytes offered 40 μM BT2 in the presence or the absence of 1 μM norepinephrine as in (B). (n = 4 biological replicates). E, representative images for iCell cardiomyocytes treated for 20 to 40 min with 80 μM BT2, 10 μM DNP, 1 μM FCCP, or vehicle control. Ten nano molars TMRE and two hundred nano molars MitoTracker Green FM were given 1 h to equilibrate prior to compound addition (further details available in the Experimental procedures section). F, left, average TMRE intensity relative to vehicle control for treatments as in (D) with iCell cardiomyocytes (top) and NRVMs (bottom). Right, circularity as a measure of mitochondrial morphology for treatments as in (D) with iCell cardiomyocytes (top) and NRVMs (bottom). (n = 7–15 technical replicates collated from n = 3 biological replicates for each cell type). G, left, simplified schematic of uniformly labeled ¹³C₆-glucose or ¹³C₆-leucine enriching TCA cycle intermediates. Right, mole percent enrichment (M.P.E.) of TCA cycle intermediates citrate and malate in NRVMs with the ¹³C label provided on either glucose or leucine. (n = 3 biological replicates). Panels (B–D, and G) show data as mean ± SD of individual biological replicates. Panels (A and F) show data as mean ± SD from technical replicates. Statistical analysis was conducted with a pairwise Student’s t test {[(B and C), each BT2 concentration against vehicle control], and (D)} or ANOVA followed by Dunnett’s posthoc multiple comparison tests (E) as appropriate. ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001. BT2, 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid; DMSO, dimethyl sulfoxide; DNP, 2,4-dinitrophenol; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazine; iPSC, induced pluripotent stem cell; TCA, tricarboxylic acid; TMRE, tetramethylrhodamine, ethyl ester.