NO/STEM CELLS IN THE DIABETIC HEART

描述（由申请人提供）：糖尿病与冠状动脉疾病和心脏缺血相关，导致加速和侵袭性心脏病。在过去的3年里，我们一直专注于减少一氧化氮产生对糖尿病心脏的潜在作用。我们发现，四氧烷诱导的I型糖尿病的特征是eNOS mRNA增加，但蛋白质和NO的产生减少。我们还表明，心脏底物的使用有明显的变化，这与NO在控制心脏脂肪酸和葡萄糖摄取中的作用是一致的。我们还发现，一氧化氮产生的减少导致一氧化氮对心肌耗氧量的调节发生改变，这可能导致氧气输送和需求之间的不匹配。在目前的应用中，我们将继续关注NO在心脏功能和代谢控制中的作用。在具体目标1中，我们将研究磷酸化可能增加eNOS的活性，并补偿我们在糖尿病心脏中观察到的eNOS蛋白的减少。体外研究将梳理其信号机制，体内研究将采用Bezold-Jarisch反射冠状动脉扩张法。先前我们发现，尽管血浆葡萄糖显著增加，但糖尿病将心脏的底物摄取从脂肪酸转变为酮酸，没有葡萄糖摄取。我们还发现，这些变化发生在心脏一氧化氮产量下降的时候。我们将在本提案的具体目标2中研究糖尿病心脏中NO产生和底物使用之间的关系。虽然我们的大多数研究都集中在1型糖尿病上，但2型糖尿病的发病率正在以流行病的速度在人群中增加。为了研究一氧化氮生成改变的作用及其机制，我们已经开始在Zucker脂肪大鼠（II型）中进行研究，并将这些发现与使用链脲佐菌素（I型）或四氧嘧啶治疗的大鼠进行对比。有趣的是，我们发现eNOS蛋白在Zucker大鼠的心脏中是正常的，同时伴有NO生物活性的降低（不像Strep处理的大鼠，eNOS蛋白降低了50%）。这些数据暗示了超氧阴离子在II型糖尿病中的重要作用，并将成为本研究的重点。在具体目标4中，我们将确定用生长因子募集心脏干细胞治疗糖尿病狗心脏是否会导致梗死后心功能的改变。由于糖尿病的特点是侵袭性心肌缺血，我们假设糖尿病心脏的代偿机制如干细胞募集不足导致心肌缺血加剧。因此，本研究将继续研究NO在心脏功能和代谢控制中的作用，并将这些研究扩展到心脏干细胞在糖尿病缺血性心脏的发生和治疗中的作用。

DESCRIPTION (provided by applicant): Diabetes is associated with both coronary vascular disease and cardiac ischemia resulting in accelerated and aggressive cardiac disease. In the previous 3 years we have focused on the potential role of reduced NO production on the diabetic heart. We have found that alloxane induced type I diabetes is characterized by increased mRNA for eNOS but reduced protein and NO production. We have also shown that there are marked shifts in cardiac substrate use, consistent with the role of NO in the control of cardiac fatty acid and glucose uptake. We have also found that the decrease in NO production results in altered regulation of myocardial oxygen consumption by NO perhaps contributing to a mismatch between oxygen delivery and demand. In the current application we will continue to focus on the role of NO in the control of cardiac function and metabolism. In specific aim 1, we will examine the potential that phosphorylation can increase the activity of eNOS and compensate for the reduction in eNOS protein which we observed in the diabetic heart. In vitro studies will be used to sort out the signaling mechanism and the Bezold-Jarisch reflex coronary dilation will be used as method to study this in vivo. Previously we have found that diabetes shifts substrate uptake in the heart from fatty acids to keto acids with no glucose uptake despite the marked increase in plasma glucose. We have also found that these shifts occur at about the time that NO production by the heart falls. We will investigate the relationship between NO production and substrate use in the diabetic heart in specific aim 2 of this proposal. Whereas most of our studies have focused on type 1 diabetes, the incidence of type II diabetes is increasing in the population at epidemic rates. To investigate the role of altered NO production and the mechanism responsible we have begun studies in the Zucker fatty rat (Type II) and to contrast those findings with rats treated with Streptozotocin (Type I) or alloxan. Interestingly, and the focus of specific aim 3 we have found that eNOS protein is normal in the heart of the Zucker rat accompanied by a reduction in NO bioactivity (unlike the Strep treated rat where eNOS protein is reduced by 50%). These data imply an important role for superoxide anion in Type II diabetes and will be the focus of this specific aim. In specific aim 4 we will determine whether treatment of the diabetic dog heart with growth factors to recruit cardiac stem cells results in alterations in cardiac function after infarction. Because diabetes is characterized by aggressive myocardial ischemia we postulate that in the diabetic heart compensatory mechanisms such as stem cell recruitment are deficient resulting in exaggerated ischemia. Thus this proposal will continue work on the role of NO in the control of cardiac function and metabolism and extend these studies to the role of cardiac stem cells in the genesis and treatment of the diabetic ischemic heart.