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Short-term caloric restriction (CR) protects the young myocardium against ischemia/reperfusion (I/R) injury through a mechanism involving AMP-activated protein kinase (AMPK). Here we ask whether a life-long CR intervention can extend this protection to the aged myocardium, and whether AMP-activated protein kinase (AMPK) plays a role in that protection. Hearts from ad libitum fed (AL) and life-long calorically restricted (LCR) mice were examined at 30 months of age by 25/90 minute global I/R, with and without AMPK inhibition (AraA). LCR hearts were protected from infarction (AL, 28±4 % vs. LCR, 10±1 %, p < 0.01) and post-ischemic functional deficit (LVDP recovery: AL, 65±8 % vs. LCR, 93±7 %, p < 0.01). Pre-ischemic AraA impaired both of these protective effects (Infarct size: LCR+AraA, 22±4 %; LVDP recovery: LCR+AraA, 82±9 %, both p vs. AL > 0.1). AMPKα phosphorylation was dramatically increased in LCR hearts prior to I/R (AL, 1.18±0.01 vs. LCR, 1.68±0.04, ratio, p < 0.0001), and accompanied by a more modest increase in total AMPKα (AL, 2.18±0.03 vs. LCR, 2.39±0.08 ratio, p < 0.05). These results indicate that life-long caloric restriction profoundly protects the aged heart against I/R injury, and suggest that AMPK may play a role in that protection.
Recent estimates of global disease burden indicate that ischemic heart disease (IHD) accounts for 12.2 % of deaths worldwide (WHO, 2004), and among high-income nations, 89.8 % of IHD mortality occurs in individuals 60 years of age or older (WHO, 2004).
One biological contributor to this age-association of IHD mortality is a progressive impairment of myocardial resistance to ischemia/reperfusion (I/R) injury1. This impairment not only affects basal myocardial resistance to I/R, but also diminishes induction of I/R resistance by a variety of preconditioning strategies2–5. Together, these effects describe an aged myocardium that is basally susceptible to metabolic challenge, and less able to recruit cellular defenses after exposure to known protective treatments.
Caloric restriction (CR) is associated with enhanced physiological function in many species6,7, and recent evidence indicates that life-long CR can reduce cardiovascular disease (CVD) incidence by 50% in aged primates8. Shorter-term CR paradigms elicit protection against infarction5,9, post-ischemic functional deficit5,9,10, and susceptibility to arrhythmia3. However, none of these effects have been studied after life-long CR, or in an age range comparable to that at which human IHD manifests. To that end, we employ a well-described mouse model11 to test the primary hypothesis that life-long CR remains cardioprotective in animals aged to point comparable to that at which most human IHD occurs. Secondarily, we examine the role played by AMP-activated protein kinase (AMPK) in the I/R responses of the myocardium after life-long CR or ad libitum feeding. While this kinase is known to be involved in protection due to short-term CR9, some evidence suggests that its potential to protect the heart wanes over longer term interventions5.
B6D2F1 mice were purchased from the NIA at 24 months of age. At the time of delivery, all mice had undergone caloric restriction from 14 weeks of age (LCR), or been ad libitum (AL) fed according to the model developed for the Biomarkers of Aging Program (BAP)11. These treatments were replicated on-site until 30 months of age when mice were killed11. Global I/R (25/90 minute) was performed conventionally by the constant-pressure Langendorff method under normothermic conditions, as described previously12–14. The perfusate was modified slightly to better represent metabolic conditions in vivo through inclusion of 0.4 mM oleate and 10 μU.mL−1 insulin9,15. Hearts undergoing AMPK blockade were exposed to 100 μM 9-β-D-arabinofuranoside (AraA) for 20 minutes prior to ischemia, which has previously been shown to reduce myocardial AMPK phosphorylation9. Left ventricular pressure waveforms and terminal infarct size were measured by a pressure-transducing catheter and tetrazolium staining, respectively, as described previously by our laboratory12–14. Hearts from separate cohorts of AL and LCR were used for Western immunoblotting. Upon excision they were briefly rinsed, then immediately frozen in liquid nitrogen and stored at −80 °C. Further steps of tissue preparation and blotting were performed as previously described9. Sample sizes for I/R were: AL, n = 6; AL + AraA, n = 5; LCR, n = 6; LCR + AraA, n = 5. Blot data include six hearts from each group of animals. Post-ischemic cardiac function was statistically assessed by linear mixed model, where diet and AraA were included as a fixed factors and time was the sole random factor. Infarct size was treated by factorial ANOVA, and immunoblot data by Student's t-tests. An alpha level of 0.05 was assumed for all analyses.
Anticipated effects of CR were observed for morphological variables at the time of death (Table 1). While LV mass was higher in AL hearts (p < 0.001), in vivo cardiac dimension (measured by short-axis M-mode echocardiography one week prior to death), was unchanged by LCR (Table 1). LCR hearts were considerably more resistant (66 %) to infarction than AL hearts (p < 0.005; LCR vs. AL, p < 0.005), and this protection was susceptible to inhibition of AMPK (LCR vs. LCR + AraA, p < 0.05, Figure 1). Similar protection was observed in terms of post-ischemic recovery of left ventricular developed pressure (LVDP), which approached pre-ischemic levels among LCR hearts during minutes 30 – 75 of reperfusion. During the same time period AL hearts recovered to only 73 % (p < 0.05 vs. LCR, Figure 2) of pre-ischemic LVDP, and AraA treated LCR hearts recovered similarly to AL at 79 %, which was significantly less than LCR alone (p < 0.05 vs. LCR, Figure 2). Western blot data suggest that, after excision, LCR hearts exhibited much higher phosphorylation of the AMPK alpha subunit (p < 0.001, Figure 3), and that this catalytic subunit was also expressed at slightly higher levels compared to AL (p < 0.05, Figure 3).
The data presented here provide evidence that life-long CR elicits substantial myocardial resistance to I/R injury. This finding extends previous work by establishing CR as a strategy that remains cardioprotective after life-long intervention, and most importantly, that the conferred protection is pronounced in the otherwise I/R-susceptible aged murine heart. While the mechanisms underlying this protection are likely to involve many genes and substantial pleiotropy, our results suggest that AMPK is one active constituent of the protected myocardial phenotype accompanying life-long CR. Inhibition of AMPK by AraA markedly reduced infarct resistance and functional recovery in our LCR mice, and AraA has previously been shown to simultaneously decrease AMPKα phosphorylation and remove myocardial protection due to short-term CR9.
The means by which AMPK exerts its cardioprotective effects are unclear, in large part because the many roles that AMPK plays during I/R have not been reconciled in combination. Certainly AMPK orchestrates responses that are protective in isolation, such as translocation of GLUT4 and sarcolemmal KATP channels16,17, but it has also been proposed that AMPK contributes to I/R injury by shifting myocardial metabolism to favor less efficient fatty acid oxidation18. We cannot comment on these possibilities directly, but have attempted to permit for the latter by including physiologically representative concentrations of fatty acid and insulin in our perfusion system.
In conclusion, life-long caloric restriction elicits a myocardial phenotype that is profoundly protected against I/R injury, even at an age comparable to that at which most human ischemic events occur. AMPK activation may contribute to the mechanism by which life-long CR protects the myocardium against ischemia late in life.
This work was supported by the American Heart Association [AHA 0715537Z to AGE]; the National Heart, Lung, and Blood Institute [R01 HL072790 to R.M.]; and the National Institute on Aging [AG013038 to D.S., KO1-AG029337 to A.D., and KO1-AG033196-01 to L.L.].
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CONFLICT OF INTEREST: None declared.