Isoprenaline modified the lipidomic profile and reduced β-oxidation in HL-1 cardiomyocytes: In vitro model of takotsubo syndrome

Figures

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  FIGURE 1. Increasing doses of isoprenaline impair mitochondrial functions. (A) Representative graph of the oxygen consumption rate (OCR) of HL-1 cardiomyocytes exposed to different levels of isoprenaline showing the calculation of mitochondrial functions after acute ISO exposure. NR, non-mitochondrial respiration, ATP, ATP production, PL, proton leak, MR, maximal respiration, (B) Quantification of ATP production calculated from (A), one-way ANOVA: p = 5.83 × 10–5. (C) Quantification of maximal respiration from (A), one-way ANOVA: p = 0.0009. (D) Quantification of proton leak from (A), one-way ANOVA: p = 2.79 × 10–7. (A–D) n = 3–6 biological replicates per group, one technical replicate, (E) Representative graph of the oxygen consumption rate (OCR) of HL-1 cardiomyocytes exposed to different levels of isoprenaline showing the calculation of mitochondrial functions after 24 h of exposure to ISO. (F) Quantification of ATP production from (E), one-way ANOVA: p = 0.0041. (G) Quantification of maximal respiration from (E), one-way ANOVA: p = 0.0006. (H) Quantification of proton leak calculated from (E), one-way ANOVA: p = 0.028. (E–H) n = 6 biological replicates per group, one technical…

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  FIGURE 2. Reduce utilization of medium-length fatty acids and increase lipid accumulation after high-level and long-term exposure to isoprenaline. (A) Representative graph of the oxygen consumption rate (OCR) of HL-1 cardiomyocytes before and after 40 μM etomoxir inhibition. (B) Quantification of fatty acid oxidation calculated from (A). Statistical significance was assessed using two-way ANOVA (effect of concentrations: p = 4.53 × 10–7, time: p = 0.5643, interaction: p = 1.76 × 10–6) followed by Sidakholm multiple comparisons. (A,B) n = 12 biological replicates per group, one technical replicate, (C) Intracellular lipid accumulation in HL-1 cardiomyocytes expose to 0 mM, 0.5 mM, 1.0 mM, and 2.5 mM levels of isoprenaline for 2 h and 24 h (effect of concentrations: p = 7.03 × 10–5, time: p = 0.0043, interaction: p = 2.13 × 10–7). n = 6 replicates per group, 2 technical replicates.

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  FIGURE 3. Altered gene and protein expression of lipid transporters in HL-1 cardiomyocytes exposed to isoprenaline. (A–C) qPCR analysis of HL-1 cardiomyocytes exposed to 0.0 mM and 2.5 mM levels of ISO for 2 h (green boxes) and 24 h (red boxes). Statistical significance was assessed using a two-tailed t-test. N = 6 replicates per group. All values shown for the PCR analyses are relative to the housekeeping gene TBP and GUSB. n = 6 replicates per group, 3 technical replicates, (D–F) Western blot analyses of HL-1 cardiomyocytes exposed to 0.0 mM and 2.5 mM levels of ISO for 2 h (green boxes) and 24 h (red boxes). Statistical significance was assessed using a two-tailed t-test. All values shown for the PCR analyses are relative to the loading control GAPDH. n = 6 replicates per group, 2 technical replicates.

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  FIGURE 4. Changes in the lipidomics profile induced by high-level and long-term exposure to isoprenaline. (A,B) Principal Component Analyses showed that prolonged exposure of HL-1 cardiomyocytes to 2.5 mM of isoprenaline promotes a distinct shift of the lipidomic profile compared to 2 h of exposure. (C) Clustering result of statistically significant molecules of lipids in the HL-1 cardiomyocytes exposed to 2.5 mM of isoprenaline for 24 h. n = 5–9 biological replicates per group, one technical replicate.