In this study we report that females have less ischemia-reperfusion injury than males both in vivo and in an isolated perfused heart model. Because mitochondria play a central role in ischemia reperfusion injury, we examined male-female differences in the mitochondrial proteome and identified a number of mitochondrial proteins that have male-female differences in post-translational modification. In particular we find that males have increased phosphorylation of the PDH-E1α subunit and females have increased phosphorylation of ALDH2, and the E2 subunit of α-KGDH.
PDH catalyzes the conversion of pyruvate to acetyl-CoA. PDH activity is inhibited by phosphorylation of the PDH-E1α subunit. The sex differences in phosphorylation of PDH-E1α are consistent with a recent report that females have decreased mRNA expression of PDH kinase27
. The increased phosphorylation of PDH-E1 α in males is consistent with our previous finding that compared to females, males have a lower ratio of carbohydrate/fatty acid metabolism. Churchill et al.30
showed that δPKC translocation to the mitochondria during reperfusion resulted in inhibition of PDH and increase injury. Activation of PDH has been shown to be beneficial31
. Thus a decrease in phosphorylation (and an increase in activity) of PDH in females would be expected to reduce ischemic injury. In addition to being a key regulator of metabolism, PDH and α-KGDH are responsible for a significant amount of ROS generated by mitochondria28, 29, 32
. Interestingly we find male-female differences in phosphorylation of α-KGDH. In the absence of NAD, oxygen can act as an electron acceptor for α-KGDH, thereby generating superoxide. Also the lipoamide dehydrogenase that is present in α-KGDH and PDH is capable of functioning as an NADH oxidase leading to H2
. The generation of ROS by αKGDH is dependent on the NADH/NAD ratio; a high ratio enhances ROS formation. This observation led us to speculate that the modification of α-KGDH observed in females might attenuate the ROS generation by α-KGDH. Consistent with this hypothesis we found reduced ROS formation in female mitochondria after addition of α-ketoglutarate and CoA-SH with addition of NADH. To further test this hypothesis we phosphorylated purified α-KGDH by addition of active PKC and showed that addition of substrates and NADH to phosphorylated α-KGDH resulted in less ROS generation than was observed with non-phosphorylated α-KGDH. NADH levels at the start of reperfusion are higher and therefore might be expected to cause a larger increase in ROS via α-KGDH in males than in females. Studies in confirmed this hypothesis. We also find that WM blocks the reduction in ROS observed in females following ischemia-reperfusion ().
Our finding of less ROS generation in female mitochondria is also consistent with other reports in the literature21–24
. However, our data provide a mechanism for the reduced ROS generation in females. We propose that sex differences in post-translational modification of α-KGDH result in altered ROS generation, particularly under conditions of high NADH/NAD, conditions that occur at the start of reperfusion after ischemia, which is a key time for cardioprotection.
We also made the novel observation that females have increased phosphorylation and activation of ALDH2. ALDH2 and succinate-semialdehyde dehydrogenase are both involved in detoxifying toxic aldehydes such as 4-hydroxy-2-nonenal (HNE) which is an end product of lipid peroxidation. A recent study showed that PKCε activation induces increased phosphorylation of mitochondrial ALDH2 which results in decrease in ischemic injury25
. HNE has also been reported to regulate mitochondrial uncoupling, at least in part by interaction with the adenine nucleotide translocator33
Interestingly, a number of the mitochondrial male-female differences observed are involved in regulating ROS homeostasis. We report altered post-translational modification of α-KGDH, which is an important generator of mitochondrial ROS. We also report increased phosphorylation and activation of ALDH2, which has been shown to be cardioprotective. These data are consistent with a growing number of studies reporting that mitochondria from females generate less ROS and that hearts from females show less oxidative damage21–24
. Protein changes associated with oxidative stress were shown to be greater in aged males compared to aged females suggesting higher production of ROS in males34
The reduced ischemia-reperfusion injury that we observe in females appears to be mediated by the PI3-kinase pathway as the protection was blocked by treatment with WM. Mitochondria from females also have increased PKCε. Shinmura et al. also showed that PKCε dependent signaling is altered in ovx female mice and is responsible for the loss of ischemic preconditioning35
. Estrogen has been shown to activate PI3-kinase16
and activation of PI3-kinase has been shown to be important in cardioprotection18
; thus these data provide a plausible mechanism for the protection observed in females. The finding that the PI3-kinase pathway mediates protection is also consistent with the observation that a number of proteins exhibit male-female differences in post translational modification. Interestingly we find that WM blocked the increase in phosphorylation of ALDH2. These data support the conclusion that the increased phosphorylation of ALDH2 is modulated by the PI3-kinase pathway and is an important mediator of the cardioprotection observed in females.
In summary, female Sprague Dawley rat hearts have less ischemia-reperfusion injury than males. Consistent with reduced ischemia-reperfusion injury in females we find that mitochondria from females have a number of post-translational modifications in mitochondrial enzymes involved in regulating ROS generation and oxidative metabolism, and females have reduced ROS generation on reoxygenation. Figure V
in the online supplement presents a model in which estrogen activation of PI3K and PKC leads to phosphorylation of ALDH2 which decreases toxic aldehydes generated by ROS. Activation of PI3K also increases phosphorylation of α-KGDH which reduces ROS generation under conditions of high NADH which occur with ischemia and reperfusion. Taken together, these data provide a mechanistic basis for the protection observed in females.
Novelty and Significance
What is known?
- Females have less cardiovascular disease than males.
- Low levels of reactive oxygen species (ROS) are important in cell signaling whereas high levels of ROS can contribute to cell death and data suggest that female mitochondria produce less ROS than males.
- Phosphorylation of mitochondrial aldehyde dehydrogenase 2 (ALDH2), an enzyme that detoxifies ROS generated aldehyde adducts, has been shown to reduce ischemia-reperfusion injury.
What new information does this article contribute?
- This study provides novel information in support of the hypothesis that cardioprotection in females involves a PI3-kinase mediated decrease in ROS production and better detoxification of ROS by-products.
- Our data show male-female differences in phosphorylation of α-KGDH, a major source of ROS, and that α-KGDH from female mitochondria produces less ROS.
- Our data show male-female differences in phosphorylation and activity of ALDH2.
This paper examined the mechanistic basis for reduced ischemia-reperfusion injury in females. We provide novel data demonstrating male-female differences in phosphorylation of two proteins involved in ROS handling, ALDH2 and α-KGDH. We further show that phosphorylation of these proteins is mediated by PI3-kinase and that phosphorylation is important in the protection observed in females. We demonstrate that inhibition of PI3-kinase blocks both the protection in females and phosphorylation of ALDH2. We further show that females have increased activity of ALDH2 and that an activator of ALDH2 is more protective in males than in females. We also show that the increase in phosphorylation of α-KGDH reduces ROS generation by this enzyme and consistent with this playing an important role in protection in females, we find that females generate less ROS following ischemia than males and that inhibition of PI3K results in an increase in post-ischemic ROS production in females to a level similar to that observed in males. These studies provide important new insights into male-female differences in handling of ROS and show a role for altered enzyme activity in protection in females. These studies could have important clinical implications for understanding the basis of gender differences in cardiac disease.