Mechanisms involved in male-female differences in cardioprotection

本研究的目的是了解缺血再灌注损伤和心脏保护的性别差异的机制。为了确定膜结合雌激素受体在心脏保护中的作用，我们使用了附着在树枝状二聚体（EDC）上的雌激素。 我们用雌激素、EDC结合的雌激素和载体治疗卵巢切除的雌性小鼠。 我们发现，内分泌干扰素结合的雌激素表现出减少缺血和再灌注后的梗死面积类似于雌激素治疗的小鼠中观察到的。 我们也有兴趣研究参与保护女性的信号通路，我们使用树脂辅助捕获方法（SNO-RAC）来测量蛋白质S-亚硝基化。 我们观察到一些蛋白质，男性和女性之间的差异S-亚硝基化。 此外，我们还测试了这一假设，即女性的心脏保护作用是由线粒体蛋白水平的改变和/或翻译后修饰介导的。使用在体内和离体心脏模型的缺血和再灌注（I/R），我们发现，女性比男性有更少的损伤。使用蛋白质组学方法，我们发现女性心脏的醛脱氢酶（ALDH）2的磷酸化和活性增加，ALDH 2是一种对活性氧（ROS）产生的醛加合物进行解毒的酶，并且ALDH 2的激活剂减少了男性的I/R损伤，但对女性没有显着影响。磷脂酰肌醇3-激酶抑制剂渥曼青霉素阻断了雌性动物的保护作用和ALDH 2磷酸化的增加，但对雄性动物没有影响。此外，我们发现在女性心脏中α-酮戊二酸脱氢酶（α-KGDH）的磷酸化增加。α-KGDH是ROS产生的主要来源，特别是在I/R期间发生的高NADH/NAD比率。我们发现在给予α-KGDH底物和NADH的透化雌性线粒体中ROS产生减少，表明α-KGDH的磷酸化增加可能减少α-KGDH产生的ROS。我们感兴趣的是检查翻译后修饰α-酮戊二酸脱氢酶（α-KGDH）在雌性大鼠心脏中与雄性大鼠相比增加的潜在作用。α-KGDH是一种高度调节的克雷布斯循环酶，已被认为是产生活性氧（ROS）的来源之一，特别是在高NADH/NAD+的条件下。我们确定α-KGDH的翻译后修饰是否可能改变酶的ROS产生。在不存在NADH的情况下，在透化线粒体中由α-KGDH产生的ROS在性别之间没有差异（M：1.0 ± 0.01 pmol/min/mg蛋白质对F：0.9 ± 0.05，p=ns），然而，在添加NADH的情况下，M产生显著更多的ROS（M：5.2 ± 0.5; F：2.6 ± 0.1 p<0.01）。我们通过用活性蛋白激酶C-激酶（PKCe）处理纯化的α-KGDH来检测α-KGDH的磷酸化对ROS产生的影响。与非磷酸化a-KGDH相比，磷酸化a-KGDH El亚基的增加导致在NADH存在下产生的ROS减少（p-aKGDH：47.60.8 vs aKGDH：64.23.5; p<0.05）。在NAD存在下，由磷酸化和非磷酸化的a-KGDH产生的ROS是相似的。为了支持这一假设，我们发现蛋白激酶C依赖的纯化的α-KGDH的磷酸化减少了ROS的产生。此外，来自女性心脏的肌细胞在I/R后产生的ROS比男性少，并且添加渥曼青霉素使女性的ROS产生增加到与男性相同的水平。这些数据表明，翻译后修饰可以改变ROS处理，并在女性心脏保护中发挥重要作用。 我们也开始研究microRNA是否存在男性和女性的差异。基因调控的一种机制是通过microRNA的表达。MicroRNA是小的（20-24个核苷酸）非编码RNA的单链片段，其结合mRNA并破坏翻译。因此，这项研究的假设是，雄性和雌性小鼠心脏之间的microRNA表达会有所不同。使用Affyscore microRNA阵列，我们证明了男性和女性microRNA之间主成分分析的显着差异。

The goals of this study are to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. To determine the role of membrane bound estrogen receptors in cardioprotection we used an estrogen that was attached to a dendimer (EDC). We treated ovariectomized female mice with estrogen, the EDC conjugated estrogen and vehicle. We found that EDC-conjugated estrogen showed a reduction in infarct size following ischemia and reperfusion similar to that observed in estrogen treated mice. We were also interested in examining signaling pathways involved in the protection in females and we used a resin-assisted capture method (SNO-RAC) to measure protein S-nitrosylation. We observed a number of proteins that were differentially S-nitrosylated between males and females. In addition we also tested the hypothesis that cardioprotection in females is mediated by altered mitochondrial protein levels and/or posttranslational modifications. 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 (alpha-KGDH) in female hearts. Alpha-KGDH 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 alpha-KGDH substrates and NADH, suggesting that increased phosphorylation of alpha-KGDH might reduce ROS generation by alpha-KGDH. We were interested in examining the potential role of the increase in post-translational modifications alpha-ketoglutarate dehydrogenase (a-KGDH) in female rat heart compare to male. Alpha-KGDH is a highly regulated Krebs cycle enzyme, which has been suggested to be one source for generation of reactive oxygen species (ROS), especially under conditions of high NADH/NAD+. We determine whether the post-translational modification of alpha-KGDH might alter ROS production of the enzyme. The production of ROS by alpha-KGDH in permeabilized mitochondria in the absence of NADH was not different between sexes (M: 1.0 0.01 pmols/min/mg of protein vs. F:0.9 0.05, p=ns), however with addition of NADH, M generated significantly more ROS (M: 5.2 0.5; F: 2.6 0.1 p<0.01). We examined the effect of phosphorylation of -KGDH on ROS production by treating purified a-KGDH, with active protein kinase C-epsilon (PKCe). The resulting increase in phosphorylated a-KGDH E1 subunit resulted in a reduction of ROS generated in the presence of NADH compared to non-phosphorylated a-KGDH (p-aKGDH: 47.60.8 vs aKGDH: 64.23.5; p<0.05). ROS generated from phosphorylated and non-phosphorylated a-KGDH in the presence of NAD was similar. In support of this hypothesis, we found that protein kinase C dependent phosphorylation of purified alpha-KGDH 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. These data suggest that posttranslational modifications can modify ROS handling and play an important role in female cardioprotection. We have also begun to examine whether there are male-female differences in microRNAs. One mechanism of gene regulation is through the expression of microRNAs. MicroRNAs are small (20-24 nucleotides) single stranded pieces of non-coding RNA that bind to mRNA and disrupt translation. Accordingly, the hypothesis of this study was that microRNA expression would differ between male and female mice hearts. Using an Affymetrix microRNA array, we demonstrated a significant difference in the principle component analysis between male and female microRNA.