IGF-1 boosts mitochondrial function by a Ca2+ uptake-dependent mechanism in cultured human and rat cardiomyocytes

Figures

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  FIGURE 1. IGF-1 increases mitochondrial Ca2+ uptake and reduces the PDH phosphorylation in NRVMs and hES-CMs. (A) Rhod2 and Mitotracker Green fluorescence pattern. Scale bar: 20 µm. (B) Representative confocal images of Rhod2 fluorescence were recorded for 100 s in permeabilized NRVMs treated with IGF-1 for 24 h and exposed to a pulse of 2 μM free Ca2+ (n = 3–6). Scale bar: 20 µm. (C) Rhod2 fluorescence kinetic obtained from B, including ruthenium red (RuRed, 5 μM) treated conditions. (D) Area under the curve and maximal fluorescence (amplitude) registered in C for each condition. (E) Representative Western blot and quantification of phosphorylated PDH in serine 293 in NRVMs treated with IGF-1 for 24 h (n = 6). *p < 0.05 vs. control **p < 0.01 vs. control. ***p < 0.001 vs. control; #p < 0.05 vs. non-RuRed treated condition. (F) TMRE+ and Mitycam fluorescence pattern. Scale bar: 10 µm. (G) Representative images of Mitycam fluorescence were recorded for 550 s in hES-CMs treated with IGF-1 for 24 h and exposed to an electric field stimulation (2 Hz) and isoproterenol (Iso, 10 µM). (n = 3—6). Scale bar: 10 µm. (H) Mitycam fluorescence kinetic. (I) Area under the curve and maximum fluorescence…

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  FIGURE 2. Effect of IGF-1 on MCU complex elements expression and ∆Ψmt in hES-CMs. (A) Scheme of MCU complex components (MCU, MCUB, SMDT1, MICU1, MICU2, MCUR1) inserted in the inner mitochondrial membrane (IMM) and a representation of the ∆Ψmt between the intermembrane space (IMS) and the mitochondrial matrix (MM); (B) Relative mRNA and (C) protein levels of MCU in hES-CMs stimulated with IGF-1 for 1–48 h (n = 6—8); (D) Relative mRNA levels of MCU complex regulatory subunits (MCUB, SMDT1, MICU1, MICU2, MCUR1) in hES-CMs stimulated with IGF-1 for 1–48 h (n = 6—8). The dotted line represents the mRNA levels of the untreated (control) condition; (E) Relative levels of basal TMRE+ in cells treated with IGF-1 for 24 h *p < 0.05 vs. control. Scale bar: 10 µm.

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  FIGURE 3. IGF-1 increases mitochondrial respiration by a mechanism dependent on mitochondrial Ca2+ uptake. (A) Representative plot of OCR over time. (B) Quantification of the relative changes in OCR (basal respiration, ATP-linked respiration, maximal respiration, non-mitochondrial respiration, spare capacity, and proton leak) in hES-CMs stimulated with IGF-1 (10 nM for 24 h) or vehicle. (C) Quantification of the relative changes in OCR in hES-CMs stimulated with ru360 (10 µM) or vehicle respiration (n = 3).