Mechanisms involved in male-female differences in cardioprotection

National Heart, Lung, And Blood Institute

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Abstract

Heart disease is the leading cause of death among women and well over 400,000 women in the USA die annually from this disease. Unfortunately, the treatment of heart disease in women is based primarily on research done on males. It is known that although pre-menopausal women are protected from heart disease, this protection is lost with menopause. A major hypothesis guiding our research is that in premenopausal females, estrogen leads to an increase in the levels and activity of an enzyme known as nitric oxide synthase (NOS). Activation of this enzyme leads to generation of nitric oxide (NO), which has several effects, one of which is to induce a modification known as S-nitrosylation (SNO) of several proteins which in turn leads to cardioprotection. We have previously published studies showing that young females (with normal estrogen levels) have an increase in SNO which is associated with cardioprotection. One of our goals is to identify which SNO proteins are responsible for cardioprotection. Identification of these proteins would allow us to directly target them. Our prior research has also shown that ischemia/reperfusion injury is mediated by an increase in mitochondrial calcium that activates cell death mechanisms, and our hypothesis is that SNO of a regulator of this process may be responsible for cardioprotection in females. Changes in mitochondrial calcium are proposed to be important regulators of bioenergetics and cell death; however, there are very few methods to measure mitochondrial calcium in an intact organ such as a beating perfused heart. To address this limitation, we utilized a combination of transmission spectroscopy and fluorescence to monitor free calcium in the mitochondrial matrix of a beating heart. Matrix calcium levels were measured with the genetically encoded, mitochondrial-targeted, red fluorescent calcium indicator (mt-R-GECO1). Mitochondrial localization of mt-R-GECO1 was confirmed by confocal microscopy as used previously to confirm Rhod-2 loading using a mouse with the mitochondrial membrane protein, TOMM20, tagged with a NeonGreen fluorescent protein. Using this method we have found that females have a less of an increase in mitochondrial calcium during ischemia which correlates with reduced ischemia/reperfusion mediated cell death in females. We also found that the attenuation of mitochondrial calcium uptake in females during ischemia does not occur if we knockout the mitochondrial calcium uniporter, which is the main mitochondrial calcium uptake mechanism. We examined whether NO signaling contributes to the sex differences observed in mitochondrial calcium accumulation. To test this hypothesis, we measured mitochondrial calcium levels in ex vivo perfused hearts from male and female mice using mt-R-GECO the previously described transmural spectroscopy method in heart perfused with modulators of NO/SNO. We found that the reduced rise in mitochondrial calcium observed in females was blocked by perfusion with an NO inhibitor and that the rise in mitochondrial calcium was enhanced in males with an NO donor. In parallel, we assessed the redox state of the mitochondrial calcium uniporter (MCU) by simultaneously measuring SNO and total protein oxidation. Our results indicate that SNO of MCU is higher in females contributing to a reduction in mitochondrial calcium accumulation, a key hallmark of ischemia/reperfusion (I/R) injury.