Iron Matters: Comparative Impact of Beta-Adrenergic Stimulation and Iron Chelation on Cardiac Iron Metabolism and Mitochondrial Function

Figures

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  Figure 1. Isoproterenol depletes intracellular iron in H9c2 cells. Analysis of intracellular iron levels from the cells challenged with isoproterenol and/or deferoxamine. Data are expressed as mean ± SEM; (*** p < 0.001 vs. control).

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  Figure 2. Isoproterenol modifies iron metabolism-related molecules in H9c2 cells. Analysis of mRNA (a,c,e,f) and protein levels (b,d,g,h) of IRP1 (Aco1) (a,b), IRP2 (Ireb2) (c,d), Fth1 (e), Ftl1 (f) and FERRITIN (g). Representative Western blot image of all the analyzed proteins (h). Western blot original images can be found in File S1. Data were normalized by the Gapdh mRNA or by ACTIN protein levels and expressed as mean ± SEM; (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. ISO; && p < 0.01 vs. DEF).

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  Figure 3. Isoproterenol modifies molecules involved in intracellular iron uptake in H9c2 cells. Analysis of mRNA (a,c) and protein (b,d,e) levels of TFRC (a,b) and DMT1 (c,d). Representative Western blot image of all the analyzed proteins (e). Western blot original images can be found in File S1. Data were normalized by the Gapdh mRNA or ACTIN protein levels and expressed as mean ± SEM; (** p < 0.01 and *** p < 0.001 vs. control).

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  Figure 4. Isoproterenol modifies molecules involved in intracellular iron release in H9c2 cells. Analysis of mRNA (a,c) and protein levels (b,d,e) of FPN (a,b) and HEPCIDIN (c,d). Representative Western blot image of all the analyzed proteins (e). Western blot original images can be found in File S1. Data were normalized by the Gapdh mRNA or ACTIN protein levels and expressed as mean ± SEM; (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control; ### p < 0.001 vs. ISO and &&& p < 0.001 vs. DEF).

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  Figure 5. Isoproterenol modifies the expression of molecules involved in mitochondrial iron uptake and release in H9c2 cells. Analysis of Ftmt, Mfrn1, Mfrn2, Abcb7 and Abcb8 mRNA (a,c,e,g,i) and protein levels (b,d,f,h,j). Representative Western blot image of all the analyzed proteins (k). Western blot original images can be found in File S1. Data were normalized by the Gapdh mRNA or by ACTIN protein levels and expressed as mean ± SEM; (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control; # p < 0.05 and ## p < 0.01 vs. ISO).

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  Figure 6. Isoproterenol downregulates mitofusin 1 and 2 expressions in H9c2 cells. Analysis of Mfn1 and Mfn2 mRNA (a,c) and protein levels (b,d). Representative Western blot image of all the analyzed proteins (e). Western blot original images can be found in File S1. Data were normalized by the Gapdh mRNA or ACTIN protein levels and expressed as mean ± SEM; (* p < 0.05 and *** p < 0.001 vs. control; # p < 0.05 vs. ISO).

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  Figure 7. Isoproterenol impairs mitochondrial function. Quantification of different mitochondrial parameters using Seahorse Analyzer tests. The % maximal respiration (OCR) (a), the % proton leak (OCR) (b), the % spare respiratory capacity (OCR) (c) and the % ATP-production coupled respiration (OCR) (d) are shown. The data are expressed as mean ± SEM; (* p < 0.05, ** p < 0.0 and *** p < 0.001 vs. control).

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  Figure 8. Isoproterenol impairs mitochondrial function and bioenergetic state. Quantification of different mitochondrial parameters using Seahorse Analyzer tests. The ATP production rate (mitoATP and glycoATP) (a), the % oxygen consumption rate (OCR) (b), the % proton efflux rate (PER) (c), the % extracellular acidification rate (ECAR) (d) and % compensatory glycolysis PER (e) are shown. ATP production was expressed by pmol/min/1000 cells and the rest of the data are expressed as mean ± SEM; (* p< 0.05, ** p < 0.0 and *** p < 0.001 vs. control; ## p < 0.01 and ### p < 0.001 vs. ISO; && p < 0.01 vs. DEF).