Opposing effects of age and calorie restriction on molecular determinants of myocardial ischemic tolerance

Abstract

We test the hypothesis that moderate calorie restriction (CR) reverses negative influences of age on molecular determinants of myocardial stress resistance. Postischemic contractile dysfunction, cellular damage, and expression of regulators of autophagy/apoptosis and of prosurvival and prodeath kinases were assessed in myocardium from young adult (YA; 2- to 4-month-old) and middle-aged (MA; 12-month-old) mice, and MA mice subjected to 14 weeks of 40% CR (MA-CR). Ventricular dysfunction after 25%±2%), as was cell death indicated by troponin I (TnI) efflux (1,701±214 ng vs. 785±102 ng in YA). MA hearts exhibited 30% and 65% reductions in postischemic Beclin1 and Parkin, respectively, yet 50% lower proapoptotic Bax and 85% higher antiapoptotic Bcl2, increasing the Bcl2/Bax ratio. Age did not influence Akt or p38-mitogen-activated protein kinase (MAPK) expression; reduced expression of increasingly phosphorylated ribosomal protein S6 kinase (p70S6K), increased expression of dephosphorylated glycogen synthase kinase 3β (GSK3β) and enhanced postischemic p38-MAPK phosphorylation. CR countered the age-related decline in ischemic tolerance, improving contractile recovery (60%±4%) and reducing cell death (123±22 ng of TnI). Protection was not associated with changes in Parkin or Bax, whereas CR partially limited the age-related decline in Beclin1 and further increased Bcl2. CR counteracted age-related changes in p70S6K, increased Akt levels, and reduced p38-MAPK (albeit increasing preischemic phosphorylation), and paradoxically reduced postischemic GSK3β phosphorylation. In summary, moderate age worsens cardiac ischemic tolerance; this is associated with reduced expression of autophagy regulators, dysregulation of p70S6K and GSK3β, and postischemic p38-MAPK activation. CR counters age effects on postischemic dysfunction/cell death; this is associated with reversal of age effects on p70S6K, augmentation of Akt and Bcl2 levels, and preischemic p38-MAPK activation. Age and CR thus impact on distinct determinants of ischemic tolerance, although p70S6K signaling presents a point of convergence.

FIG. 1.

Age reduces whereas calorie restriction (CR) enhances functional tolerance to ischemia-reperfusion. Recovery of contractile function and coronary flow was assessed at the end of 45 min reperfusion following 25 min of global ischemia in hearts from young, middle-aged, and middle-aged CR mice. Data are shown for recoveries of: (A) Left ventricular end-diastolic and developed pressures; and (B) +dP/dt and −dP/dt. Data are means±standard error of the mean (SEM). (*) p<0.05 versus young; (†) p<0.05 middle-aged versus middle-aged CR.

FIG. 2.

Age increases whereas calorie reduction (CR) reduces cardiac cell death (postischemic troponin I [TnI] efflux). Coronary washout of TnI throughout the 45-min reperfusion period following 25 min of global ischemia in hearts from young, middle-aged, and middle-aged CR mice. Data are means±standard error of the mean (SEM). (*) p<0.05 versus young; (†) p<0.05 middle-aged versus middle-aged CR.

FIG. 3.

Age and calorie restruction (CR) modify markers of autophagy and apoptosis in postischemic myocardium. Expression of Beclin1 (mammalian ortholog of yeast Atg6/Vps30, regulating autophagosome formation), Parkin (an E3 ubiquitin ligase targeting damaged mitochondria for mitophagy), and proapoptotic Bax and antiapoptotic Bcl2 was assessed in postischemic cardiac homogenates from young, middle-aged, and middle-aged CR mice (relative densities normalized to young). The ratio of Bcl2/Bax is also shown in the right panel. Left ventricular tissue was sampled after 25 min of global ischemia and 45 min of reperfusion. Data are means±standard error of the mean (SEM). (*) p<0.05 versus young; (†) p<0.05 middle-aged versus middle-aged CR.

FIG. 4.

Age and calorie restriction (CR) modify cardiac expression of prosurvival and prodeath kinases. Cardiac expression of Akt, GSK3β, p70S6K, and p38-mitogen-activated protein kinase (MAPK) was assessed in left ventricular homogenates from normoxic hearts from young, middle-aged, and middle-aged CR mice (relative densities all normalized to young). Data are means±standard error of the mean (SEM). (*) p<0.05 versus young; (†) p<0.05 middle-aged versus middle-aged CR.

FIG. 5.

Age and calorie restriction (CR) modify phosphoregulation of prosurvival and prodeath kinases in normoxic and postischemic myocardium. Relative phosphorylation of Akt, p70S6K, p38-mitogen-activated protein kinase (MAPK), and GSK3β was assessed in homogenates from normoxic and postischemic hearts from young, middle-aged, and middle-aged CR mice (ratios normalized to values for young). Left ventricular tissue was sampled after normoxic perfusion or following 25 min of global ischemia and 45 min of reperfusion. Data are means±standard error of the mean (SEM). (*) p<0.05 versus young; (†) p<0.05 middle-aged versus middle-aged CR.