Pervasive environmental contaminant acrolein compromises myocardial geometry and function through the induction of cuproptosis

Figures

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  Figure 1 Biometrics and echocardiographic indices of acrolein-challenged mice Mice were exposed to acrolein at 2.5 mg/kg/day (i.p.) for 20 days. (A) Body weight. (B) Heart weight. (C) Liver weight. (D) Septal thickness. (E) LV posterior wall thickness. (F) LV end systolic diameter (LVESD). (G) LV end diastolic diameter (LVEDD). (H) Fractional shortening. (I) Ejection fraction. (J) LV mass. (K) Normalized LV mass (to body weight). (L) Heart rate. Data are presented as the mean ± SEM, n = 8 mice per group, *P < 0.05 between the indicated groups.

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  Figure 2 Cardiomyocyte contractile function and intracellular Ca 2+ properties in acrolein-challenged mice Mice were exposed to acrolein at 2.5 mg/kg/day (i.p.) for 20 days. (A) Resting cell length. (B) Peak shortening (% of resting cell length). (C) Maximal velocity of shortening (+ dL/dt). (D) Maximal velocity of relengthening (–dL/dt). (E) Time-to-peak shortening (TPS). (F) Time-to-90% relengthening (TR90). (G) Resting Fura-2 fluorescence intensity (FFI). (H) Electrically stimulated rise in FFI (ΔFFI). (I) Intracellular Ca2+ clearance rate. Mean ± SEM, n = 35 cells (panel A–F) or 28 cells (panel G–I) from 3 mice per group, * P < 0.05 between the indicated groups.

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  Figure 3 H&E and Masson’s trichrome staining and free radical accumulation in the myocardium of acrolein-challenged mice Mice were exposed to acrolein at 2.5 mg/kg/day (i.p.) for 20 days. (A) Representative micrographs from H&E staining. (B) Pooled cardiomyocyte cross-sectional area. (C) Representative Masson trichrome staining. (D) Pooled interstitial fibrosis analysis. (E) Representative DCF staining (DAPI) top row, DCF) middle row and merged) bottom row). (F) Pooled DCF fluorescence. Data are presented as the mean ± SEM, n = 6 mice per group, *P < 0.05 between the indicated groups.

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  Figure 4 TEM ultrastructure, mitochondrial function, and TUNEL apoptosis in the myocardium of acrolein-challenged mice Mice were exposed to acrolein at 2.5 mg/kg/day (i.p.) for 20 days. (A) Representative TEM ultrastructure for damaged and/or deformed mitochondria. (B) Mitochondrial circular index (short-to-long axis ratio). (C) Mitochondrial area (individual). (D) Mitochondrial function assessed via aconitase activity assay. (E) Representative TUNEL staining depicting apoptosis (DAPI) top row. (TUNEL) middle row. (bottom) merged). (F) Pooled TUNEL staining depicting the percentage of TUNEL-positive apoptotic cells. (G) Level of the mitochondrial protein PGC1α. (H) Level of the mitochondrial protein UCP2. Inset: Representative gel blots showing PGC1α and UCP2 using specific antibodies (GAPDH or α-tubulin as loading controls). Data are presented as the mean ± SEM, n = 30–31 mitochondria from 5 micrographs (panels B,C) or 6 mice (all other panels) per group, *P < 0.05 between the indicated groups.

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  Figure 5 Levels of protein markers for apoptosis, ferroptosis, cuproptosis, and mitochondrial complexes as well as intracellular Cu 2+ in hearts from acrolein-challenged mice Mice were exposed to acrolein at 2.5 mg/kg/day (i.p.) for 20 days. (A) Representative immunoblots depicting the levels of Bax, Bcl2, caspase-3, GPX4, SLC7A11, SLC31A1, ATP7A, DLAT, FDX1, ACO2, NDUFS8 and MTF1 using specific antibodies (GAPDH as a loading control). (B) Level of Bax. (C) Level of Bcl2. (D) Level of Caspase-3. (E) Level of GPX4. (F) Level of SLC7A11. (G) Level of SLC31A1 (CTR1). (H) Level of ATP7A. (I) Level of DLAT. (J) Level of FDX1. (K) Level of ACO2. (L) Level of NDUFS8. (M) Level of MTF1. (N) Intracellular Cu2+ level. Data are presented as the mean ± SEM. n = 6–7 hearts per group. * P < 0.05 between the indicated groups.

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  Figure 6 Levels of MTF2, interaction between acrolein and MTF2, and impact of TTM, mitoTEMPO, or Act D on acrolein -evoked cardiomyocyte contractile defects Adult C57BL/6 mice were injected with acrolein (2.5 mg/kg/day, i.p.) for 20 days in the presence or absence of TTM (5 mg/kg, p.o., every 5 days), mitoTEMPO (10 mg/kg, i.p., every 5 days) or Act D (0.1 mg/kg, i.p., every 5 days) prior to assessment of mechanical function. (A) Level of MTF2 following acrolein challenge (2.5 mg/kg/day, i.p.). (B) Interaction between acrolein and the MTF2 dimer predicted by CB-DOCK2. (C–H) Impact of TTM and actinomycin D on acrolein-induced cardiomyocyte dysfunction) c) Resting cell length. (D) Peak shortening. (E) +dL/dt. (F) –dL/dt. (G) Time-to-peak shortening (TPS). (H) Time-to-90% lengthening (TR90). (I) Chemical structure of acrolein. (J) Schematic diagram illustrating acrolein-induced myocardial responsiveness (including Cu2+ regulatory and Fe-S cluster proteins, mitochondrial integrity, cell death, myocardial remodeling and function). Data are presented as the mean ± SEM, n = 7 mice (in panel A) or 20 cells from 3 mice (in panel C–H) per group. *P < 0.05 between the indicated groups.