Leptin Modifies the Rat Heart Performance Associated with Mitochondrial Dysfunction Independently of Its Prohypertrophic Effects

Figures

• 

  Figure 1. Effect of leptin on the cardiac rate-pressure product. (a) Plot of heart rates (beats per minute, mmHg) of leptin-perfused and control hearts. (b) Plot of heart contraction pressures of leptin-perfused and control hearts. (c) Heart rate-pressure product of leptin-perfused and control hearts; ∗p < 0.05 (n = 4). The concentration of leptin was 3.1 nM in the perfusion solution.

• 

  Figure 2. Calcium transported by mitochondria. (a) Plot of calcium internalization during active oxidative phosphorylation for control (gray) and leptin-treated (black) mitochondria. Arrows indicate the time points when the indicated additions were made. The reaction was stopped by the addition of CCCP to induce a complete release of calcium. (b), (c), and (d) are representative plots of control and leptin-treated mitochondria after 2, 3, and 4 hours of perfusion, respectively. CCCP: carbonyl cyanide-m-chlorophenyl hydrazone; Mit: mitochondria; AUF: arbitrary units of fluorescence.

• 

  Figure 3. Determination of mitochondrial potential. (a) Representative plots of the experimental measurement of potential of control (gray) and leptin-treated (black) mitochondria during active oxidative phosphorylation. The reaction was started by the addition of glutamate/malate (G/M), and the potential was collapsed by the addition of CCCP. (b), (c), and (d) are representative plots of control and leptin-treated mitochondria after 2, 3, and 4 hours of perfusion, respectively. Panel d includes a plot of leptin-treated mitochondria treated with the mPTP inhibitor cyclosporine A (CsA). Arrows indicate the time points when the indicated additions were made. Note that the potential of leptin-treated mitochondria was neither established since the first hour of perfusion nor recovered with CsA.

• 

  Figure 4. Semiquantitative analysis of the membrane potential of isolated mitochondria. Bars represent the median ± SEM of 4 independent determinations with different mitochondrial preparations. The delta AUF was measured after CCCP addition and was considered the membrane potential. ∗p < 0.05 (n = 4).

• 

  Figure 5. Mitochondrial swelling induced by calcium. Panels (a), (b), (c), and (d) are plots of the median ± SEM of 4 independent determinations of mitochondrial swelling of different mitochondrial preparations after 1, 2, 3, and 4 hours of perfusion, respectively. Reactions were started by the addition of CaCl2, and absorbance was recorded each minute for ten minutes. ∗p < 0.05 (n = 4).

• 

  Figure 6. Leptin induces overexpression of the protein c-ACT and increases phosphorylation of STAT3. (a) The c-ACT protein content was measured by Western blotting of the heart homogenate using GAPDH as the loading control. Bars represent the median ± SEM of four independent mitochondrial preparations. (b) The phosphorylated STAT3 protein content was measured by Western blotting of the heart homogenate using total STAT3 as a reference control. (c) The STAT3 protein content was measured by Western blotting of the heart homogenate using GAPDH as the loading control. The values indicate the mean ± SEM, n = 4; ∗p < 0.05 versus the control group.

• 

  Figure 7. Quantification of oxidized lipids in heart homogenates and isolated mitochondria. Levels of oxidized lipids were measured by TBARS methodology in (a) homogenates after 1, 2, 4, or 4 hours of perfusion with/without leptin. Bars represent the median ± SEM of four independent preparations; ∗p < 0.05 vs the control group. (b) Isolated mitochondria. (c) Values of TBARS from homogenates and mitochondria were analyzed by Pearson's test to identify a correlation between variables. R1 = Pearson's value for control TBARS in the four time points analyzed; R2 = Pearson's value for leptin TBARS in the four time points analyzed. Note that the correlation is strong and positive for the leptin group.