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

描述(申请人提供)：急性缺氧导致一系列心肌事件，包括缺血性脑梗塞、心律失常、心肌病和心力衰竭。低氧时的内在心脏保护是通过替代能量处理和钙信号转导来减少需氧量来实现的。然而，左心和右心在损伤时感知氧气并激活这种保护机制的确切机制大多尚不清楚。虽然目前的心脏保护疗法主要是防止心肌梗死后疾病的复发或进展，但它们无法对抗缺氧性损伤期间的细胞死亡。此外，左、右心室对损伤的反应也不同。虽然目前的治疗方法改善了左心功能，但右心病变并没有得到有效的治疗。我们的长期目标是确定左心和右心在缺氧过程中氧气感知和保护的不同机制，以帮助开发针对早期缺氧损伤的特定治疗方法。我们的主要假设是缺氧/缺氧不同地改变了右和左心室的钙信号。这一假说基于以下观察：1)急性缺氧导致单个分离的心室肌细胞瞬间失去电诱发的钙瞬变；2)应用10 mM咖啡因溶液后，肌浆网(SR)的钙含量可逆地降低；3)右室和左室肌浆网抑制和恢复的时间线不同；4)已经证明氧敏感缺氧诱导因子-1a(HIF-1a)基因的丢失改变了心肌中的钙信号，我们发现该蛋白在右心室中的基础mRNA水平较高。基于这些观察结果，本研究的重点在于：1.研究左、右室肌细胞对缺氧/缺氧的反应中的膜电流和钙信号。我们将使分离的心肌细胞暴露于缺氧/低氧中，并监测钾、钠、钙电流的大小和动力学，钙的释放和摄取的大小。2.探讨低氧时左、右室肌浆网差异调节的机制。在这里，我们将研究活性氧物种和激活的激酶(PKA，钙/钙调蛋白激酶II)在离子电流的氧化还原调节和肌浆网钙释放/摄取中的作用。2)兰诺定受体(RyR)/SERCA-磷蛋白磷酸化调节肌浆网钙的释放/摄取。我们将用腺病毒载体在心肌细胞中过表达HIF-1a基因，并评估其钙电流的大小和动力学以及钙释放/摄取的程度。

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.