Glucose depletion is essential for the calorie-restriction-mediated cardiac angiogenesis via PKA/AMPK-dependent autophagy

Purpose: In last ESC meeting, we presented the impact of calorie restriction (CR) on cardiac remodeling observed in diet-induced obesity mice and pressure-over-loaded heart failure mouse model (ESC 2012, YIA runner of basic research); however, it remains unclear whether #1 the essential nutrient factor(s) that may be responsible for the CR-mediated enhanced cardiac angiogenesis and #2 the underlying molecular mechanism(s). Methods: Using both murine models of heart failure and cultured cardiomyocytes and endothelial cells, we examined mechanisms of CR-induced cardiac angiogenesis. Eighteen-week-old diet-induced obesity (DIO) mice, as a chronic heart failure model that exhibits characteristics of diabetic cardiomyopathy, were allocated into 4 week CR (DIO-CR) and ad libitum (DIO-AL) groups. Pressure overload (2 weeks) was generated by transaortic constriction (TAC) in mice, which were used as a acute heart failure induced by mechanical stress, and randomly allocated to a 4-week CR (TAC-CR and sham-CR) and ad libitum (TAC-AL and sham-AL) groups, and analyzed at 18 week-old. Cultured endothelial cells (ECs) were divided into 4 groups that exposed to culture media of 1) ordinary composition, 2) glucose-depleted, 3) amino acid-depleted, and 4) serum-depleted condition, respectively. Analyses for the changes in activities of autophagy (LC3-turnover assay and p62 level) and angiogenesis (tube formation and Akt/AMPK/eNOS activity) were evaluated. Results: Immunohistochemical analyses revealed that CR enhanced cardiac angiogenesis both in TAC and DIO mice. CR promoted increased in myocardial cyclic AMP concentration with concomitant PKA/AMPK/eNOS activation and the simultaneous increase in autophagic activity both in TAC and DIO hearts. Of note, cardiac Akt activity remains unchanged by CR. In vitro analysis revealed that glucose depletion, as well as forskolin (10 μM) and 8-bromo-cyclic AMP (1 mM), activated PKA/AMPK axis thereby facilitated angiogenesis and autophagy in ECs. The enhanced in vitro angiogenesis induced by glucose depletion and PKA enhancers was abrogated by autophagy inhibitor 3-MA treatment. PKA inhibitors (H89 and RP-cAMP) abrogated the increase in AMPK/eNOS phosphorylation levels induced by glucose depletion. Neither amino acid- nor serum-depletion had no effect on angiogenesis and autophagy. Conclusions: Glucose depletion is essential for the CR-mediated activation of cardiac angiogenesis and autophagy in a cAMP/PKA/AMPK-dependent fashion.