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Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

心脏缺血再灌注损伤机制及心脏保护作用

基本信息

批准号：
10929086
负责人：
Elizabeth Murphy
金额：
$ 197.55万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10929086
关键词：
Ablation Acceleration Address Adrenergic Agents Bioenergetics Calcium Cardiac Cardiac Myocytes Cardiovascular Diseases Catecholamines Cell Death Cell membrane Cellular Structures Complex Coupled Data Dioxygenases Disparity EF Hand Motifs Electric Stimulation Electron Transport Exhibits Family member Fatigue Fiber Fluorescence Future Generations Genetic Transcription Glycogen Goals Heart Heart Diseases Heart Hypertrophy Heart Injuries Heart Mitochondria Heart failure Homeostasis Human Hydroxylation Hypertrophy Impairment Infarction Ion Pumps Ions Ischemia Isoproterenol Knock-out Knockout Mice Label Liquid Chromatography Liver Liver Mitochondria Location Measures Mediating Metabolism Methods Mitochondria Modeling Mus Muscle Muscle Contraction Myocardial Ischemia Myocardium NADH Nerve Tissue Neurons Optics Oxygen Oxygenases Pathology Perfusion Permeability Phosphorylation Physiological Plasma Cells Play Post-Translational Protein Processing Procollagen-Proline Dioxygenase Protein Biosynthesis Proteins Proteomics RNA Reactive Oxygen Species Reperfusion Injury Reperfusion Therapy Reporting Ribosomal Proteins Role Rupture Signal Pathway Signal Transduction Skeletal Muscle Spectrum Analysis Testing Therapeutic Time Tissues Transcript Transcriptional Regulation Translations Uniport Wild Type Mouse adeno-associated viral vector alpha ketoglutarate aorta constriction calcium uniporter cardioprotection delivery vehicle ex vivo perfusion exercise capacity falls heart function human disease improved insight interest ischemic injury knockout animal mitochondrial permeability transition pore mouse model muscle physiology oxidation pharmacologic preconditioning pressure prevent pyruvate dehydrogenase ribosome profiling tandem mass spectrometry uptake

项目摘要

The long-term goals of this project are to understanding mechanisms involved in cardioprotection. We have focused on the role of mitochondrial calcium and the permeability transition pore. An increase in mitochondrial calcium is a well-known trigger of cell death. We were therefore interested in investigating the mitochondrial calcium uniport (MCU) complex, which is responsible for mitochondrial calcium uptake. Mitochondrial calcium regulates bioenergetics but also serves as a trigger for cell death. With a sustained increase in catecholamine or a large increase in cytosolic calcium as occurs with ischemia, mitochondrial calcium can rise to high levels leading to activation of the mitochondrial permeability transition pore, thus initiating cell death. Calcium uptake into mitochondria occurs via the mitochondrial calcium uniporter (MCU), which is regulated by three EF-hand proteins, mitochondrial calcium uptake (MICU) 1, 2, and 3. MICU1/MCU ratios vary in different tissues, and alterations in substrate have been shown to regulate MICU1 levels altering MCU-mediated calcium uptake. Mitochondrial Ca2+ overload is proposed to regulate cell death via opening of the mitochondrial permeability transition pore. It is hypothesized that inhibition of the mitochondrial Ca2+ uniporter (MCU) will prevent Ca2+ accumulation during ischemia/reperfusion and thereby reduce cell death. To address this, we evaluate mitochondrial Ca2+ in ex vivo perfused hearts from germline MCU-KO and WT mice using transmural spectroscopy. Matrix Ca2+ levels are measured with the genetically encoded, red fluorescent Ca2+ indicator (R-GECO1) using an adeno-associated viral vector (AAV9) for delivery. Due to the pH sensitivity of R-GECO1 and known fall in pH during ischemia, hearts are glycogen depletion to decrease the ischemic fall in pH. At 20 mins of ischemia, there is significantly less mitochondrial Ca2+ in MCU-KO hearts compared to MCU-WT controls. However, an increase in mitochondrial Ca2+ is present in MCU-KO hearts suggesting that mitochondrial Ca2+ overload during ischemia is not solely dependent on MCU. MICU3 is a regulator of MCU which has generally been thought to function primarily in neuronal tissue where it is highly expressed, and thus has been largely ignored in other tissues such as the heart. We performed quantitative proteomics using Tandem Mass Tag labelling coupled to liquid chromatography and tandem mass spectrometry to analyze MCU and MCU regulators in cardiac and hepatic mitochondria. Normalizing MICU levels to MCU in heart and liver mitochondria, we found that the MICU1/MCU ratio was 0.75 in liver and 0.25 in heart which is consistent with previous studies3; however, the MICU3/MCU ratio in heart is more than three-fold higher than that found in liver. To confirm that the MICU3 we observed in heart mitochondria was not due to contamination by mitochondria from nerve tissue in heart, we isolated cardiomyocytes and compared the ratio of MICU3 to MCU in cardiomyocytes and heart mitochondria and found similar ratios. These data suggest that MICU3 might play a role in regulating MCU in heart and skeletal muscle. We generated a mouse model of systemic MICU3 ablation and examined its physiological role in skeletal muscle. We found that loss of MICU3 led to impaired exercise capacity. When the muscles were directly stimulated there was a decrease in time to fatigue. MICU3 ablation significantly increased the maximal force of the KO muscle and altered fiber type composition with an increase in the ratio of type IIb (low oxidative capacity) to type IIa (high oxidative capacity) fibers. Furthermore, MICU3-KO mitochondria have reduced uptake of Ca2+ and increased phosphorylation of pyruvate dehydrogenase (PDH), indicating that KO animals contain less Ca2+ in their mitochondria. Skeletal muscle from MICU3-KO mice exhibit a net oxidation of NADH during electrically stimulated muscle contractions, These data demonstrate that MICU3 plays an essential role in skeletal muscle physiology by setting the proper threshold for Ca2+m uptake, which is important for matching energy demand and supply in muscle. We also measured Ca2+m in MICU3-KO and WT perfused hearts loaded with Rhod-2-AM using optical transmural spectroscopy in an integrating sphere as described above. We perfused hearts for 5 min with Isoproterenol (Iso) to increase Ca2+m uptake. Transmural Rhod-2 fluorescence, a measure of Ca2+m, was collected by a spectrometer. Data were analyzed by subtraction of tissue background and correction for inner filter effects. MICU3-/- hearts exhibited significantly less increase in Ca2+m during the 5 minute stimulation with Iso. WT hearts showed a 2.500.11 fold increase in fluorescence with Iso treatment, whereas MICU3-/- hearts showed a 1.250.05 fold increase. As cardiac injury after ischemia-reperfusion is thought to be associated with increased mitochondrial calcium leading to mitochondria-initiated cell death, we tested whether Micu3-/- hearts were protected against ischemia-reperfusion injury. Ex vivo Langendorff-perfused hearts from WT and Micu3-/- mice were subjected to 20 minutes of global ischemia followed by 90 minutes of reperfusion. Micu3-/- hearts had reduced infarct size normalized to the entire LV and improved contractile function following reperfusion. Protection in ex vivo Micu3-/- hearts strongly supports a role for MICU3 in cardiac mitochondria. Interestingly, germline ablation of MCU or EMRE was not cardioprotective, likely due to adaptations in these germline knockout mice. We speculate that loss of MICU3 does not lead to adaptation and under conditions of prolonged elevated calcium, the loss of MICU3 is protective. We also performed some studies addressing the role of transcriptional regulation in cardiovascular disease. Transcriptional changes in heart failure have been the focus of numerous studies. Disparities between transcript and protein abundances highlight the need to better understand translation in heart failure. One mechanism for achieving proteomic diversity through protein synthesis and stability is through the activity of prolyl hydroxylases. 2-oxoglutarate- and Fe2+-dependent dioxygenases modify target proteins to regulate protein stability, turnover, and activity. A lesser-known member of this family is 2-oxoglutarate- and Fe2+-dependent oxygenase domain-containing protein 1 (OGFOD1). OGFOD1 regulates translation by catalyzing prolyl hydroxylation of ribosomal protein S23 (RPS23), but its cardiac functions are largely unexplored. To define the role of OGFOD1 in heart disease, we measured OGFOD1 RNA and protein in failing and non-failing human hearts, and found that OGFOD1 was significantly higher in failing hearts. Because heart failure can result from many pathologies, including hypertrophy, we investigated the role of OGFOD1 in cardiac hypertrophy. We induced hypertrophy via pharmacological -adrenergic stimulation by treating wildtype (WT) and OGFOD1-knockout (KO) mice with isoproterenol (ISO). In an independent study, we induced pressure-overload-mediated hypertrophy using transverse aortic constriction (TAC). KO mice showed greater than 10% reduction in hypertrophy, regardless the mode of hypertrophy induction (P = 0.0132 for TAC, P = 0.0101 for ISO). Future studies will utilize ribosome profiling to identify transcript pools whose synthesis are regulated by OGFOD1 to confer protection in cardiac hypertrophy. Results from these studies will provide important mechanistic insight into the therapeutic potential of OGFOD1 in human disease.

该项目的长期目标是了解心脏保护的机制。我们重点关注线粒体钙和通透性转换孔的作用。线粒体钙的增加是众所周知的细胞死亡触发因素。因此，我们对研究负责线粒体钙吸收的线粒体钙单向孔 (MCU) 复合体感兴趣。线粒体钙调节生物能，但也可作为细胞死亡的触发因素。随着儿茶酚胺的持续增加或缺血时胞质钙的大幅增加，线粒体钙会上升到高水平，导致线粒体通透性转换孔激活，从而引发细胞死亡。线粒体的钙摄取是通过线粒体钙单向转运蛋白 (MCU) 进行的，该蛋白由三种 EF 手蛋白（线粒体钙摄取 (MICU) 1、2 和 3）调节。MICU1/MCU 比率在不同组织中有所不同，并且底物的改变已被证明可以调节 MICU1 水平，从而改变 MCU 介导的钙摄取。线粒体 Ca2+ 超载被认为通过打开线粒体通透性转换孔来调节细胞死亡。据推测，抑制线粒体 Ca2+ 单向转运蛋白 (MCU) 将阻止缺血/再灌注期间 Ca2+ 的积累，从而减少细胞死亡。为了解决这个问题，我们使用跨壁光谱法评估了种系 MCU-KO 和 WT 小鼠离体灌注心脏中的线粒体 Ca2+。使用腺相关病毒载体 (AAV9) 进行递送，通过基因编码的红色荧光 Ca2+ 指示剂 (R-GECO1) 测量基质 Ca2+ 水平。由于 R-GECO1 的 pH 敏感性和已知的缺血期间 pH 值下降，心脏消耗糖原以减少缺血性 pH 值下降。缺血 20 分钟时，与 MCU-WT 对照相比，MCU-KO 心脏中的线粒体 Ca2+ 明显减少。然而，MCU-KO 心脏中线粒体 Ca2+ 增加，表明缺血期间线粒体 Ca2+ 超载不仅仅取决于 MCU。 MICU3 是 MCU 的调节因子，通常被认为主要在神经元组织中发挥作用，并在神经元组织中高度表达，因此在心脏等其他组织中很大程度上被忽视。我们使用串联质量标签标记结合液相色谱和串联质谱进行定量蛋白质组学，以分析心脏和肝脏线粒体中的 MCU 和 MCU 调节剂。将心脏和肝脏线粒体中的 MICU 水平标准化为 MCU，我们发现肝脏中的 MICU1/MCU 比率为 0.75，心脏中的 MICU1/MCU 比率为 0.25，这与之前的研究一致3；然而，心脏中的 MICU3/MCU 比率比肝脏中的比率高出三倍多。为了证实我们在心脏线粒体中观察到的MICU3不是由于心脏神经组织的线粒体污染造成的，我们分离了心肌细胞并比较了心肌细胞和心脏线粒体中MICU3与MCU的比率，发现了相似的比率。这些数据表明 MICU3 可能在调节心脏和骨骼肌中的 MCU 中发挥作用。我们生成了全身 MICU3 消融的小鼠模型，并检查了其在骨骼肌中的生理作用。我们发现 MICU3 的缺失会导致运动能力受损。当肌肉受到直接刺激时，疲劳时间就会缩短。 MICU3 消融显着增加了 KO 肌肉的最大力量，并改变了纤维类型组成，IIb 型（低氧化能力）与 IIa 型（高氧化能力）纤维的比例增加。此外，MICU3-KO 线粒体对 Ca2+ 的吸收减少，丙酮酸脱氢酶 (PDH) 的磷酸化增加，表明 KO 动物线粒体中 Ca2+ 含量较少。 MICU3-KO 小鼠的骨骼肌在电刺激肌肉收缩过程中表现出 NADH 的净氧化，这些数据表明，MICU3 通过设定适当的 Ca2+m 吸收阈值，在骨骼肌生理学中发挥着重要作用，这对于匹配肌肉的能量需求和供应非常重要。我们还在如上所述的积分球中使用光学透壁光谱法测量了负载有 Rhod-2-AM 的 MICU3-KO 和 WT 灌注心脏中的 Ca2+m。我们用异丙肾上腺素 (Iso) 灌注心脏 5 分钟，以增加 Ca2+m 的吸收。通过光谱仪收集透壁 Rhod-2 荧光（Ca2+m 的测量值）。通过减去组织背景并校正内部过滤效应来分析数据。在用 Iso 刺激 5 分钟期间，MICU3-/- 心脏的 Ca2+m 增加显着减少。 WT 心脏显示 Iso 处理后荧光增加了 2.500.11 倍，而 MICU3-/- 心脏显示荧光增加了 1.250.05 倍。由于缺血再灌注后的心脏损伤被认为与线粒体钙增加导致线粒体引发的细胞死亡有关，因此我们测试了 Micu3-/- 心脏是否能免受缺血再灌注损伤。来自WT和Micu3-/-小鼠的离体Langendorff灌注心脏经历20分钟的整体缺血，然后是90分钟的再灌注。 Micu3-/-心脏的梗塞面积缩小了，整个左心室正常化，并且再灌注后收缩功能得到改善。离体 Micu3-/- 心脏的保护有力地支持了 MICU3 在心脏线粒体中的作用。有趣的是，MCU 或 EMRE 的种系消融并不具有心脏保护作用，这可能是由于这些种系敲除小鼠的适应所致。我们推测 MICU3 的缺失不会导致适应，并且在钙长期升高的情况下，MICU3 的缺失具有保护作用。我们还进行了一些研究，探讨转录调控在心血管疾病中的作用。心力衰竭的转录变化一直是众多研究的焦点。转录本和蛋白质丰度之间的差异凸显了更好地理解心力衰竭翻译的必要性。通过蛋白质合成和稳定性实现蛋白质组多样性的一种机制是通过脯氨酰羟化酶的活性。 2-酮戊二酸和 Fe2+ 依赖性双加氧酶修饰靶蛋白以调节蛋白质稳定性、周转和活性。该家族的一个鲜为人知的成员是 2-氧化戊二酸和 Fe2+ 依赖性加氧酶结构域蛋白 1 (OGFOD1)。 OGFOD1 通过催化核糖体蛋白 S23 (RPS23) 的脯氨酰羟基化来调节翻译，但其心脏功能很大程度上尚未被探索。为了确定 OGFOD1 在心脏病中的作用，我们测量了衰竭和非衰竭人类心脏中的 OGFOD1 RNA 和蛋白质，发现 OGFOD1 在衰竭心脏中显着较高。由于心力衰竭可由多种病理引起，包括肥厚，因此我们研究了 OGFOD1 在心脏肥大中的作用。我们通过用异丙肾上腺素 (ISO) 治疗野生型 (WT) 和 OGFOD1 敲除 (KO) 小鼠，通过药理肾上腺素刺激诱导肥大。在一项独立研究中，我们使用横主动脉缩窄（TAC）诱导压力超载介导的肥大。无论诱导肥大的方式如何，KO 小鼠的肥大减少均超过 10%（TAC 的 P = 0.0132，ISO 的 P = 0.0101）。未来的研究将利用核糖体分析来识别其合成受 OGFOD1 调节的转录物池，以在心脏肥大中提供保护。这些研究的结果将为 OGFOD1 在人类疾病中的治疗潜力提供重要的机制见解。