Calorie restriction combined with resveratrol induces autophagy and protects 26-month-old rat hearts from doxorubicin-induced toxicity

Abstract

The multiple beneficial effects of calorie restriction (CR) on several organs, including the heart, are widely known. Recently, the plant polyphenol resveratrol has been shown to possess several beneficial effects similar to those of CR. Among the host of effects on cardiac muscle, a cellular self-eating process called autophagy has been shown to be induced by both CR and resveratrol. Autophagy is vital in removing dysfunctional organelles and damaged proteins from the cell, thereby maintaining cellular quality control. In this study, we explored whether short-term moderate CR (20%), either alone or in combination with resveratrol, can induce autophagy in the hearts of 26-month-old Fischer 344 × Brown Norway rats. Autophagy stimulation was investigated by measuring the protein expression levels of the autophagy proteins beclin-1, Atg5, and p62 and the LC3-II/LC3-I ratio. We found that 20% CR or resveratrol alone for 6 weeks could not induce autophagy, but 20% CR in combination with 50 mg/kg/day resveratrol resulted in an induction of autophagy in the hearts of 26-month-old rats. Although oxidative stress has been proposed to be an inducer of autophagy, treatment with the chemotherapeutic drug doxorubicin was unable to stimulate autophagy. The enhanced autophagy due to CR + resveratrol was associated with protection from doxorubicin-induced damage, as measured by cardiac apoptotic levels and serum creatine kinase and lactate dehydrogenase activity. We propose that a combinatorial approach of low-dose CR and resveratrol has the potential to be used therapeutically to induce autophagy and provides protection against doxorubicin-mediated toxicity.

Keywords: Autophagy; Calorie restriction; Doxorubicin; Free radicals; Heart; Oxidative stress; Resveratrol.

Fig. 1. Effect of interventions on autophagy markers in the left ventricle

(A–D) Protein expression levels of autophagy markers LC3B (A), p62 (B), Beclin-1 (C) and Atg5–Atg12 conjugate (D) were measured by immunoblotting in the left ventricular homogenates of saline-treated AL, CR, CR + Resv-5 and CR + Resv-50 animals. GAPDH was used as a loading control. *p < 0.05 vs. AL. n = 4 to 12.

Fig. 2. Effect of doxorubicin on LC3-II/LC3-I ratio and p62 accumulation in the left ventricle

(A–C) Protein expression levels of autophagy markers LC3B (a), p62 (B), and Beclin-1 (C) were measured by immunoblotting in the left ventricular homogenates of saline or doxorubicin-treated animals. GAPDH was used as a loading control. **p < 0.01 vs. saline. n = 5 to 12.

Fig. 3. Effect of interventions on doxorubicin-mediated cardiac damage

(A) Apoptotic induction in left ventricular homogenates of saline-treated AL rats and doxorubicin-treated AL, CR, CR + Resv-5 and CR + Resv-50 groups was determined by measuring the release of mono- and oligo-nucleosomes in the cytosolic fraction. Values for the assay have been normalized to the total amount of protein. *p < 0.05 vs. AL + saline; ^#p < 0.05 vs. AL + doxorubicin; (B) Serum CK levels were measured in the same groups as in (A). **p < 0.01 vs. AL + saline, ^#p < 0.05 vs. AL + doxorubicin. n = 5 to 12.

Fig. 4. Serum LDH activity in saline- and doxorubicin-treated AL, CR, CR + Resv-5 and CR + Resv-50 FBN rats

(A) Serum LDH activity was determined in saline-treated AL and in doxorubicin-treated AL, CR, CR + Resv-5 and CR + Resv-50 animals. **p < 0.01 vs. AL + saline; ^#p < 0.05 vs. AL + doxorubicin. n = 5 to 12; (B) Serum LDH activity was determined in saline-treated AL, CR, CR + Resv-5 and CR + Resv-50 animals. n = 4 to 12.

Fig. 5. Proposed pathways through which CR and resveratrol induces autophagy

Both CR and resveratrol can activate autophagy by inhibiting the mTOR pathway and/or by activating of the AMPK and SIRT1 pathways. AMPK induces activating phosphorylation on the ULK1 – Atg13 – FIP200 complex, thereby stimulating autophagy. SIRT1 can decetylate and activate key autophagy genes Atg5, Atg7 and LC3, thereby inducing autophagy.