The Citrus Flavanone Naringenin Produces Cardioprotective Effects in Hearts from 1 Year Old Rat, through Activation of mitoBK Channels

Figures

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  FIGURE 1. Post-ischemic functional parameters of RPP (A), CF (B), and dP/dt (C) recorded during the reperfusion time in Langendorff-perfused hearts of 1 year old rats pre-treated with vehicle, NAR 100 mg/kg or DIAZO 40 mg/kg. Two way ANOVA indicated that the curves obtained following NAR-treatment show significant differences (P < 0.01) vs. vehicle. The corresponding data emerging from the morphometric analysis of the tissue injury are also shown; asterisks indicate significant differences (∗P < 0.05) vs. vehicle (D).

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  FIGURE 2. Values of injured areas (expressed as % of the left ventricle area Ai/ALV %) observed in the left ventricles isolated from 1 year old rats treated with vehicle, NAR, NAR+PAX or submitted to pre-conditioning ischemia (IPC) before the protocol of in vivo acute infarct. The asterisks indicate a statistically significant difference from the value observed in the hearts of vehicle treated animals (∗P < 0.05).

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  FIGURE 3. (A) Concentration-dependent changes in mitochondrial membrane potential, induced by the cumulative administration of increasing concentrations of NAR to isolated cardiac 1 year old mitochondria. Two-way ANOVA indicated statistically significant differences between the curve obtained in control condition and those obtained in the presence of PAX (10 μM). (B) Increase of the fluorescence (expressed as a % of that induced by the potassium-ionophore valinomycin) produced by a Tl+-sensitive probe accumulated into the mitochondrial matrix, after the addition of Tl+ ions to a suspension of isolated cardiac 1 year old mitochondria in the presence of NAR (100 μM). The asterisks indicate a statistically significant difference (∗P < 0.05). (C) The reduction of the extra-mitochondrial calcium concentration (in μM), following the administration of isolated cardiac mitochondria from 1 year old rats in the presence of NAR 100 μM, DIAZO 100 μM, or their vehicle. The asterisks indicate a statistically significant difference (∗P < 0.05).

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  FIGURE 4. (A) Percentage of β-galactosidase positive cells observed after treatment with different increasing concentrations of Dox. (B) Representative phase contrast photomicrographs of treated cells (control, Dox 0.05 μM and Dox 0.1 μM, respectively). (C) Real mRNA levels, indicative of an induction of p16 and p21 mRNA, and (D) cell cycle phases analyses. The asterisks indicate a statistically significant difference (∗∗∗p < 0.001, ∗∗p < 0.01 vs. control group).

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  FIGURE 5. Cell viability of senescent H9c2 cells under hypoxic conditions. Percentage of cell viability compared to the control (non-senescent normoxic H9c2 cells) to which a viability of 100% was attributed. The asterisks indicate a statistically significant difference vs. control (∗∗∗p < 0.001); the hashtags indicate statistically significant difference vs. A/R injury vehicle. (###p < 0.001, ##p < 0.01, #p < 0.05).

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  FIGURE 6. Reduction of α and β subunits of BK channel, indicated as maxiKa and slo-β1, respectively, in mitochondrial membranes. (A) Western blot analysis of both α and β subunits in mitochondrial membranes obtained from young and old rat ventricle. Left panel shows a representative example of western blot experiment. Right panel shows a significant reduction of protein expression in “old” ventricle (black bars) respect to “young” ventricle (white bars). (B) Western blot analysis of both α and β subunits in mitochondrial membranes obtained from H9c2 cell line after treatment with Dox (“old” cell) or DMSO alone (“young” cell). Left panel shows a representative example of western blot experiment. Right panel shows a significant reduction of protein expression in “old” H9c2 (black bar) compared to the “young” H9c2 (white bars). Bars represent the mean ± SEM (n = 3 independent experiments). Statistic t-test ∗P < 0.05, ∗∗P < 0.01.