Sex differences in the phosphorylation of mitochondrial proteins result in reduced production of reactive oxygen species and cardioprotection in females

Abstract

Rationale: Although premenopausal females have a lower risk for cardiovascular disease, the mechanism(s) are poorly understood.

Objective: We tested the hypothesis that cardioprotection in females is mediated by altered mitochondrial protein levels and/or posttranslational modifications.

Methods and results: Using both an in vivo and an isolated heart model of ischemia and reperfusion (I/R), we found that females had less injury than males. Using proteomic methods we found that female hearts had increased phosphorylation and activity of aldehyde dehydrogenase (ALDH)2, an enzyme that detoxifies reactive oxygen species (ROS)-generated aldehyde adducts, and that an activator of ALDH2 reduced I/R injury in males but had no significant effect in females. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked the protection and the increased phosphorylation of ALDH2 in females, but had no effect in males. Furthermore, we found an increase in phosphorylation of alpha-ketoglutarate dehydrogenase (alphaKGDH) in female hearts. alphaKGDH is a major source of ROS generation particularly with a high NADH/NAD ratio which occurs during I/R. We found decreased ROS generation in permeabilized female mitochondria given alphaKGDH substrates and NADH, suggesting that increased phosphorylation of alphaKGDH might reduce ROS generation by alphaKGDH. In support of this hypothesis, we found that protein kinase C-dependent phosphorylation of purified alphaKGDH reduced ROS generation. Additionally, myocytes from female hearts had less ROS generation following I/R than males and addition of wortmannin increased ROS generation in females to the same levels as in males.

Conclusions: These data suggest that posttranslational modifications can modify ROS handling and play an important role in female cardioprotection.

Figure 1

Female hearts exhibit less ischemia-reperfusion injury. Recovery of rate pressure product (A) and infarct size (B) in Male and Female hearts after 30 min of ischemia and 90min of reperfusion; C) Female and Ovx differences in recovery; D) Female and Ovx differences in infarct size; E) Male and Male+E2 differences in recovery; F) Male and Male+E2 differences in infarct size; G) Infarct size/area of risk after 45 min of LAD occlusion and 2hr of reperfusion. The area-at-risk was not different between males (40+/−3) and females (34+/−5); *p<0.05.

Figure 2

A representative overlay of equal amounts of protein between males (Cy5, red) and females (Cy3, green). Inset A shows a magnification of the PDH E1α region of this gel. Inset B shows the aldehyde dehydrogenase area taken from another gel where the difference is better illustrated.

Figure 3

Females have increase phosphorylation and activity of ALDH2. A) Representative gels for phosphorylated and total ALDH2 in male and female samples; B) Summarized quantitative data on the ratio of phosphorylated ALDH2 to total ALDH2; C) ALDH2 activity between male and female; D) Quantitative data on mitochondrial PKCε in male and female. *p<0.05.

Figure 4

Effect of agonists and inhibitors on I/R injury. RPP (A) and infartct size (B) after 30 min of ischemia and 90 minutes of reperfusion in hearts untreated or treated with Alda1; RPP (C) or infarct size (D) after 30 min ischemia and 90 minutes of reperfusion in hearts untreated or treated with DOG; RPP (E) and infarct size (F) after 30 min ischemia and 90 minutes of reperfusion in hearts untreated or treated with Ro-317549; RPP (G) or infarct size (H) after 30 min ischemia and 90 minutes of reperfusion in hearts untreated or treated with wortmannin. * p<0.05; ** p<0.01.

Figure 5

H₂O₂ production from male and female rat heart mitochondria measured by Amplex red. A) Permeabilized mitochondria with 0.12 mM HS-CoA of α-ketoglutarate as a substrate, NADH is added as indicated; B) Intact normoxia mitochondria with glutamate/malate (G/M) as substrates and C) Reoxygenated mitochondria with G/M as substrates (after 30 minutes of anoxia). *p<0.05

Figure 6

Effect of in vitro phosphorylation of α-ketoglutarate dehydrogenase. A) In vitro phosphorylation of α-KGDH by PKCε; B) ROS generated from α-KGDH in presence of NAD or NADH with or without pre-phosphorylation by PKCε; C) Plot of the slope of ROS generation from α-KGDH in presence of NADH with or without PKCε pretreatment. *p<0.05

Figure 7

ROS generation from cardiac myocytes after simulated ischemia.