Sex-related resistance to myocardial ischemia-reperfusion injury is associated with high constitutive ARC expression

Abstract

The female sex has been associated with improved myocardial salvage after ischemia and reperfusion (I/R). Estrogen, specifically 17beta-estradiol, has been demonstrated to mediate this phenomenon by limiting cardiomyocyte apoptosis. We sought to quantitatively assess the effect of sex, ovarian hormone loss, and I/R on myocardial Bax, Bcl-2, and apoptosis repressor with caspase recruitment domain (ARC) expression. Male (n = 48), female (n = 26), and oophorectomized female (n = 20) rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. The myocardial area at risk and infarct size were determined using a double-staining technique and planimetry. In situ oligo ligation was used to assess apoptotic cell death. Western blot analysis was used to determine proapoptotic (Bax) and antiapoptotic (Bcl-2 and ARC) protein levels in all three ischemic groups and, additionally, in three nonischemic groups. Infarct size (43.7 +/- 3.2%) and apoptotic cell death (0.51 +/- 0.10%) were significantly attenuated in females compared with males (56.4 +/- 1.6%, P < 0.01, and 4.29 +/- 0.95%, P < 0.01) and oophorectomized females (55.7 +/- 3.4%, P < 0.05, and 4.36 +/- 0.51%, P < 0.01). Females expressed significantly higher baseline ARC levels (3.62 +/- 0.29) compared with males (1.78 +/- 0.18, P < 0.01) and oophorectomized females (1.08 +/- 0.26, P < 0.01). Males expressed a significantly higher baseline Bax-to-Bcl-2 ratio (4.32 +/- 0.99) compared with females (0.65 +/- 0.13, P < 0.01) and oophorectomized females (0.42 +/- 0.10, P < 0.01). I/R significantly reduced Bax-to-Bcl-2 ratios in males. In all other groups, ARC levels and Bax-to-Bcl-2 ratios did not significantly change. These results support the conclusion that in females, endogenous estrogen limits I/R-induced cardiomyocyte apoptosis by producing a baseline antiapoptotic profile, which is associated with estrogen-dependent high constitutive myocardial ARC expression.

Fig. 1.

Area at risk (AAR) expressed as a percentage of the left ventricle (LV) and infarct size (I) expressed as a percentage of the AAR in all ischemic groups (males: n = 38, females: n = 16, and oophorectomized females: n = 10). Values are means ± SE. Error bars represent SE. *P < 0.01 vs. males and P < 0.05 vs. oophorectomized females.

Fig. 2.

In situ oligo ligation (ISOL)-positive nuclei in the AAR. A: ISOL reaction in the myocardial AAR (specimen taken from the AAR of an oophorectomized female). Original magnification: ×10. Arrows indicate dark red ISOL-positive nuclei of apoptotic cells. Arrowheads indicate blue ISOL-negative nuclei. B: comparison of apoptosis (expressed as the percentage of ISOL-positive nuclei in the myocardial AAR) among ischemic groups (n = 8 for all groups) after 30 min of myocardial ischemia and 180 min of reperfusion. Values are means ± SE. Error bars represent SE. *P < 0.01 vs. males and oophorectomized females.

Fig. 3.

Western blot analysis. A: representative immunoblots. B: Bax, Bcl-2, Bax/Bcl-2, and apoptosis repressor with caspase recruitment domain (ARC) levels in the AAR of the ischemic groups (n = 10 for all groups) and in the corresponding segment of the LV of the nonischemic groups (n = 10 for males and females and n = 5 for oophorectomized females) as determined by the Western blot technique. Bax-to-Bcl-2 ratios were determined for individual rabbits followed by the calculation of group means. Integrated optical density values are reported as means ± SE. Error bars represent SE. *P < 0.01 vs. Bax-to-Bcl-2 ratios in ischemic males, nonischemic females, and nonischemic oophorectomized females; **P < 0.01 vs. ARC levels in nonischemic males and nonischemic oophorectomized females.