Differential 02 Sensing in Right and Left hearts: Crosstalk with Ca-Signaling

描述（由申请人提供）：急性缺氧导致心肌发生一系列事件，包括缺血性梗死、心律失常、心肌病和心力衰竭。缺氧期间的内在心脏保护作用是通过替代能量处理和Ca 2+信号传导降低需氧量而发生的。然而，左和右心脏在受伤时感知O2并激活这种保护机制的确切机制大多是未知的。虽然目前的心脏保护疗法主要防止心肌梗死后疾病的复发或进展，但它们无法对抗缺氧损伤期间的细胞死亡。此外，左心室和右心室对损伤的反应不同。尽管目前的治疗改善了左心室功能，但右心病理没有得到有效治疗。我们的长期目标是确定缺氧时左、右心氧传感和保护的不同机制，以帮助开发针对早期缺氧损伤的特异性治疗方法。我们的主要假设是，缺氧/缺氧差异改变钙信号在右心室与左心室。这一假设基于以下观察：1）急性缺氧导致单个离体心室肌细胞中电诱发钙瞬变的瞬间丧失2）肌浆网（SR）的Ca含量响应于10 mM咖啡因溶液的应用以可逆的方式继发于缺氧而降低3）SR抑制和恢复的时间轴在右心室和左心室之间是不同的4）已经表明，氧传感器缺氧诱导因子-la（HIF-1a）基因的缺失改变了心肌中的Ca信号传导，并且我们已经发现该蛋白在右心室中的基线mRNA水平较高。根据这些意见，本建议的重点是：1。表征右心室与左心室心肌细胞对缺氧/缺氧反应的膜电流和钙信号传导。我们将使分离的心肌细胞暴露于缺氧/低氧并监测i）K、Na、Ca的电流大小和动力学ii）Ca释放和摄取的大小。2.确定缺氧时左右心室差异SR调节的机制。在这里，我们将研究活性氧和活化的激酶（PKA，Ca/钙调蛋白激酶II）对i）离子电流和SR Ca释放/摄取的氧化还原调节ii）通过兰尼碱受体（RyR）/SERCA-磷蛋白磷酸化3调节SR Ca释放/摄取的作用。确定瞬时HIF-1a过表达对左右心室钙信号传导的影响。我们将使用腺病毒载体在心肌细胞中过表达HIF-1a基因，并评估i）钙电流和钙释放/摄取的幅度和动力学ii）RyR和SERCA-2A表达的程度。

DESCRIPTION (provided by applicant): Acute oxygen deprivation leads to an array of events in the myocardium that include ischemic infarction, arrhythmia, cardiomyopathy, and heart failure. Intrinsic cardioprotection during hypoxia occurs by decreasing oxygen demand through alternative energy handling and Ca2+ signaling. However, the exact mechanisms by which the left and right hearts sense O2 and activate such protective mechanisms at the time of injury are mostly unknown. While current cardio-protective therapies mainly prevent reoccurrence or progression of disease post myocardial infarction, they fail to combat cell death during hypoxic injury. Moreover, the left and right ventricles differ in their response to injury. Although, current therapies improve left ventricular function, right heart pathologies are not effectively treated. Our long-term objective is to determine differential mechanisms of O2 sensing and protection during hypoxia in left and right hearts to help develop specific therapies against early phase hypoxic injury. Our main hypothesis is that anoxia/hypoxia differentially alters calcium signaling in right versus left ventricles. This hypothesis is based on the following observations: 1) acute anoxia leads to instant loss of electrically evoked calcium transients in single isolated ventriclular myocytes 2) the Ca content of the sarcoplamic reticulum (SR) in response to application of 10mM caffeine solutions decreases secondary to anoxia in a reversible manner 3) The timeline of SR suppression and recovery is different between right and left ventricles 4) It has been shown that loss of oxygen-sensor Hypoxia Inducible Factor-la (HIF-1a) gene alters Ca-signaling in the myocardium and we have found higher baseline mRNA levels of this protein in the right ventricle. Based on these observations, the focus of this proposal is to: 1. Characterize membrane currents and calcium signaling in right versus left ventricular myocytes in response to anoxia/hypoxia. We will expose isolated cardiomyocytes to anoxia/hypoxia and monitor i) current magnitude and kinetics of K, Na, Ca ii) magnitude of Ca release and uptake. 2. Determine mechanisms of differential SR regulation in right and left ventricles during hypoxia. Here we will examine the role of reactive oxygen species and activated kinases (PKA, Ca/Calmodulin Kinase II) on i) redox regulation of ionic currents and SR Ca release/uptake ii) regulation of SR Ca release/uptake by ryanodine receptor (RyR)/SERCA-Phospholamban phosphorylation 3. Determine the effect of transient HIF-1a overexpression on calcium signaling in right and left ventricles. We will overexpress HIF-1a gene in cardiomyocytes using adenoviral vectors and evaluate the i) magnitude and kinetics of calcium currents and calcium release/uptake ii) degree of RyR and SERCA-2A expression.