AMPK Activation Is Indispensable for the Protective Effects of Caloric Restriction on Left Ventricular Function in Postinfarct Myocardium

Figures

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  Figure 1. Effect of 8 weeks CR on body weight and plasma parameters. (A): Body weight at the beginning of the diet (left panel), end of the diet (middle panel), and LV weight normalized to tibia length at the end of the diet (right panel) in sham or infarct rats, following 8 weeks of control or CR diets. (B): Serum levels of acrp30, leptin, insulin, glucose, triglycerides, and free fatty acids (FFA) in these rats, obtained after overnight fasting. All data are mean ± SEM. n = 5–6 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

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  Figure 2. Effect of 8 weeks CR on LV systolic function and expression or release of brain natriuretic peptide (BNP). (A): LV fractional shortening (FS%) at the beginning of the diet (left panel), end of the diet (middle panel), and changes in FS during the feeding period (right panel) in sham or infarct rats following 8 weeks of control or CR diets. (B): LV mRNA expression (left panel) and serum levels of brain natriuretic peptide (right panel) in sham or infarct rats, following 8 weeks of control or CR diets. All data are mean ± SEM. n = 5–6 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

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  Figure 3. Effect of 8 weeks CR on AMPK activation, apoptosis, and mitochondrial biogenesis. (A): Representative Western blots and quantification of phosphorylation of AMPK (at Thr172), normalized to alpha AMPK, in the LV of sham or infarct rats, following 8 weeks of control or CR diets. (B): Representative Western blots and quantification of full-length and large fragment (17/19 kDa) of cleaved, activated caspase-3 in the LV of sham or infarct rats, following 8 weeks of control or CR diets. GAPDH served as loading control. (C): Representative Western blots and quantification of protein expression of Tfam, PGC-1alpha, mitochondrial components of complex IV (Cox 15 and I) and VDAC1, normalized to GAPDH, in LV tissue. (D): Relative copy number of mtDNA per diploid nuclear genome, as measured by real-time PCR. All data are mean ± SEM. n = 5–6 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001. Full size blots are provided in Supplementary Figure S2.

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  Figure 4. Effect of 8 weeks CR and 2 weeks of AMPK inhibition on LV systolic function, LV weight and expression or release of BNP. (A): LV fractional shortening (FS%) at the beginning of the diet (left panel), end of the diet (middle panel), and changes in FS during the feeding period (right panel) in sham or infarct rats, following 8 weeks of control or CR diets. (B): LV weight normalized to tibia length at the end of the study (left panel), LV mRNA expression (middle panel), and serum levels of brain natriuretic peptide (right panel) in sham or infarct rats following 8 weeks of control or CR diets. All data are mean ± SEM. n = 6–7 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

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  Figure 5. Effect of 8 weeks CR and 2 weeks of AMPK inhibition on AMPK activation, apoptosis, and mitochondrial biogenesis. (A): Representative Western blots and quantification of phosphorylation of AMPK (at Thr172), normalized to alpha AMPK, in the LV of sham or infarct rats, following 8 weeks of control or CR diets and AMPK inhibition. (B): Representative Western blots and quantification of full-length and large fragment (17/19 kDa) of cleaved, activated caspase-3 in the LV of sham or infarct rats, following 8 weeks of control or CR diets and AMPK inhibition. GAPDH served as loading control. (C): Representative Western blots and quantification of protein expression of Tfam, PGC-1alpha, mitochondrial components of complex IV (Cox 15 and I), and VDAC1, normalized to GAPDH, in LV tissue. All data are mean ± SEM. n = 6–7 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001. Full size blots are provided in Supplementary Figure S5.

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  Figure 6. Effect of 8 weeks CR and 2 weeks of AMPK inhibition on mitochondrial respiration. Active rates of respiration (state 3) were measured in saponin-skinned cardiac fibers in the presence of 5 mM ADP and either 10 mM pyruvate + 2 mM malate or 10 mM succinate in the presence of 5 µM rotenone in the LV of sham or infarct rats, following 8 weeks of control or CR diets and AMPK inhibition. All data are mean ± SEM. n = 5 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

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  Figure 7. Effect of 8 weeks CR on LV systolic function and expression or release of BNP. (A): LV fractional shortening (FS%) at the beginning of the diet (left panel) and changes in FS during the feeding period (right panel) in WT and KO sham or infarct mice, following 8 weeks of control or CR diets. (B): LV mRNA expression (left panel) and serum levels of brain natriuretic peptide (right panel) in WT and KO sham or infarct mice, following 8 weeks of control or CR diets. All data are mean ± SEM. n = 6–8 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

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  Figure 8. Effect of 8 weeks CR on AMPK activation, apoptosis, and mitochondrial biogenesis. (A): Representative Western blots and quantification of phosphorylation of AMPK (at Thr172), normalized to GAPDH, in WT and KO sham or infarct mice, following 8 weeks of control or CR diets. (B): Representative Western blots and quantification of full-length and cleaved, activated caspase-3. GAPDH served as loading control. (C): Representative Western blots and quantification of protein expression of Tfam, PGC-1alpha, mitochondrial components of complex IV (Cox 15 and I), and VDAC1, normalized to GAPDH, in LV tissue. All data are mean ± SEM. n = 6–8 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001. Full size blots are provided in Supplementary Figure S8.