Fig. 2.

Identification of ivermectin and nifuroxazide as mitochondrial ATP modulators under hypoxia by phenotypic screens. (a) Assay workflow to identify small molecules that maintain mitochondrial ATP levels under hypoxia with cardiac anti-hypertrophic effects. (b) Stacked histogram of the primary screen. Thirty-four primary hits (orange) were identified by the hit criteria (vehicle average + 3 SD = 0.931) indicated by dashed line and other parameters. (c) Scatter plot of results of mito-ATeam assays (protection against hypoxia- or oligomycin A [OA]-mediated mitochondrial ATP decrease). Each data point represents the average of three independent experiments, normalized to initial values (OA) or negative and positive controls (hypoxia) (initial value was set to 1). Grey dashed lines indicate the thresholds (> 80% for x axis and < 0.9 for y axis). Blue dashed line indicates OA response in vehicle group. Twenty-six compounds shown in black were excluded based on the counter-assays (cell viability and mitochondrial membrane potential) (Fig. S2a). Out of eight selected compounds, ivermectin and nifuroxazide (shown in orange) exhibited protective effects on mitochondrial ATP under hypoxia, but not in response to OA, without activating caspase-3/7 (Fig. S2b). Source data are available online for this figure (Table S1). (d and e) Representative images of fluorescence resonance energy transfer (FRET) signal in mito-ATeam stable HL-1 cardiomyocytes treated with ivermectin (d, upper panel) or nifuroxazide (e, upper panel) for 24 h under hypoxia (1% O₂), followed by reoxygenation (21% O₂). Scale bars, 20 μm. Quantified FRET ratios of mito-ATeam in HL-1 cardiomyocytes treated with ivermectin (0, 1, 3, 10 μM, d, lower panel) or nifuroxazide (0, 1, 3, 10 μM, e, lower panel) under hypoxia/reoxygenation are shown. Data are presented as means ± SD (n = 3 biologically independent samples).