**6. Estrogen, mitochondria, and cardiovascular disease**

Cardiovascular disease (CVD) is the leading cause of death in men and women, and American Heart Association statistics reveal a significant increase in CVD mortality in women compared to men [47]. Mitochondrial dysfunction has been implicated as a causative factor in CVD, with mitochondrial DNA damage being significantly increased in the heart and aorta of patients with CVD compared to healthy controls [25, 48–50]. Women do not generally present with CVD until the seventh decade, while the incidence of death due to CVD is high in men throughout life. This age-dependent increase in CVD in women has been linked to the onset of menopause and a reduction in circulating estrogen levels. The "Free Radical Theory of Aging" proposes that, with increased age, an increase in free radical formation initiates a vicious cycle of ROS formation that causes progressive cell injury [51]. Data suggest that loss of the antioxidant and anti-inflammatory effects of estrogen after menopause contributes to the development of mitochondrial injury [52, 53]. Thus, maintaining high levels of estrogen may increase lifespan and/or health in postmenopausal women.

Studies using experimental animal models of CVD show that OVX increases vascular inflammation/injury in a manner that is prevented by estrogen treatment [54, 55]. Analysis of mitochondria isolated from hearts of OVX rats reveals increased levels of apoptotic markers compared to mitochondria of intact animals. Administration of estrogen to these animals significantly attenuated apoptosis [56]. Since mitochondrial damage and apoptosis can be mediated by ROS, it has been hypothesized that the estrogen can decrease ROS by activating the antioxidant pathway. Treatment of human aortic endothelial cells (HAECs) with estradiol upregulates the mitochondrial antioxidant MnSOD by an ERα-dependent mechanism. The ability of estrogen to increase MnSOD levels is ablated in ERα KO mice but not in ERβ KO mice. Interestingly, while ERβ does not regulate MnSOD expression, it was shown to be essential for preventing atherosclerotic progression *in vivo* [57, 58]. These data show that ERs modulate mitochondrial antioxidant production and have distinctive vasoprotective mechanisms.

Gender differences have been identified in mitochondrial genes isolated from rat hearts [59]. Whole genome microarray analysis showed that expression of genes associated with mitochondrial apoptosis pathways is significantly elevated in male mice compared to females. In contrast, genes associated with fatty acid and glucose metabolism were upregulated in females. Female rats also displayed higher transcription levels for mitochondrial Complexes I and IV. These data suggest that genes related to cellular metabolism, including mitochondrial respiration, are upregulated in cardiac mitochondria from female rats while genes associated with mitochondrial apoptosis are increased in males. Whether this difference is directly related to the circulating levels of estrogen *in vivo* is unclear.

Mitochondrial structure in the heart is influenced by estrogen. The hearts of OVX rats that underwent I/R injury had lower levels of mitochondrial respiratory function and increased myocardial cell death compared to intact animals. Transmission electron microscopy showed that the mitochondria in cardiomyocytes from OVX rats were more disordered within the cell and structurally damaged compared to mitochondria from intact animals [41]. Interestingly, even male ERα KO mice that underwent cardiac I/R injury display lower coronary blood flow rates, increased calcium accumulation, and reduced nitrite production compared to non-ischemic hearts. Further, electron microscopic analysis revealed that the mitochondria from ERα KO mice were abnormally shaped [60]. These studies suggest that estrogen signaling plays a role in regulating mitochondrial structure in both females and males. In another model of I/R injury, female wild-type mouse hearts were shown to have better functional recovery and an attenuated inflammatory response compared to female ERα KO mice and wild-type male mice [61]. These data further suggest the importance of ER signaling as a cardioprotective mechanism in females.

Effects of estrogen on mitochondrial function have been tested in a genetic model of hypertrophic cardiomyopathy (cTnT-Q92). Estrogen treatment improved ATP production, the mitochondrial respiratory ratio, and diastolic function in OVX cTnT-Q92 mice compared to untreated OVX mice [62]. OVX in cTnT-Q92 mice attenuated the expression of the mitochondrial biogenesis genes PGC1α, peroxisome proliferator-activated receptor alpha (PPARα), mitochondrial transcription factor A (tFAM), and the antioxidant protein nuclear respiratory factor 1 (NRF-1). Estrogen treatment improved cardiac mitochondrial organization and cristae structure and increased mitochondrial biogenesis. These data directly show that estrogen exerts cytoprotective effects at the level of the mitochondrion that translate into an improvement in cardiac function.
