MECHANISM OF CHRONIC ALCOHOL INDUCED CARDIOMYOPATHY

这笔赠款将通过纵向评估左心室(LV)和分离的心肌细胞对慢性酒精摄入的结构和功能反应，来研究慢性酒精诱导扩张型心肌病(ADCM)的机制。此外，还将确定肾素-血管紧张素系统(RAS)的作用及其细胞内信号在产生ADCM过程中的变化。我们将检验以下特定假设：[H1]长期饮酒导致心肌细胞收缩、松弛、[Ca~(2+)]i瞬时和肌膜钙通道活动直接降低(ICa，L)，从而导致[H2]左心室收缩和舒张期功能进行性损害，并导致充血性心力衰竭，[H3]促进向ADCM过渡的最重要因素是酒精诱导的循环和心脏RAS的持续激活。这导致左心室和心肌细胞AT1受体偶联、蛋白激酶C和/或抑制性G蛋白(GI)介导的对血管紧张素II(Ang II)的反应发生变化。因此，用慢性血管紧张素转换酶抑制剂或血管紧张素ⅡAT1受体阻断剂阻断RAS，可以钝化或阻止慢性酒精所致的功能和结构改变。这些研究将在三个对照组和四个实验组中进行，研究持续八个月：1)非仪表性酒精喂养(22%酒精每天一次，提供总卡路里摄入量的33%)；2)仪表性酒精喂养；3)仪表性酒精-雷米普利(ACE抑制剂，0.1 mg/kg/d)喂养；4)仪表性酒精-厄贝沙坦(Ang II AT1阻滞剂，5 mg/kg/d)-饲料。控制组将由年龄匹配的狗在相同的条件下组成，除了控制组的狗将不接受酒精。在这些清醒的狗身上，心脏收缩和舒张期功能以及RAS激活的一系列变化将被量化，长期使用仪器测量左室压和容量。对新鲜分离的心肌细胞进行细胞收缩功能、[Ca~(2+)]i瞬变和ICa、L的连续测定。心肌细胞将在1)钙、2)异丙肾上腺素、3)匹莫本丹、4)酒精和5)Ang II的灌流条件下进行研究。此外，在ADCM发展之前、期间和之后，用AT1受体阻滞剂、蛋白激酶C抑制剂或GI蛋白抑制剂预先孵育后，将重复研究心肌细胞对Ang II的反应。这些研究将是第一次详细的纵向研究心脏和心肌细胞的结构，功能，[钙]i瞬变，ICA，L在慢性酒精摄入。此外，这些研究将提供关于RAS的作用和ADCM机制的独特信息。这些研究对于扩大我们对ADCM发生机制的认识和帮助ADCM的靶向治疗是必要的。

This grant will investigate the mechanism of chronic alcohol- induced dilated cardiomyopathy (ADCM), by longitudinally assessing the structural and functional responses of the left ventricle (LV) and isolated cardiomyocytes to chronic alcohol intake. In addition, the role of the renin-angiotensin system (RAS) and the alterations in its intracellular signaling in producing ADCM will be determined. We will test the following specific Hypotheses: [H1] Chronic alcohol intake produces direct depressions in cardiomyocyte contraction, relaxation, [Ca2+]i transient and sarcolemmal Ca2+ channel activity (ICa,L), and thus causes [H2] a progressive impairment of LV systolic, diastolic function, and leads to congestive heart failure, and [H3] the most important factor in promoting the transition to ADCM is the alcohol-induced, sustained activation of circulating and cardiac RAS. This results in alterations in the AT1 receptor-coupled, protein kinase C and/or inhibitory G protein (Gi)-mediated responses of the LV and myocytes to angiotensin II (ANG II). Thus, [H4] blocking the RAS with chronic ACE inhibition or ANG II AT1 receptor blocker will blunt or prevent the chronic alcohol- induced functional and structural changes. The studies will be conducted in three control and four experimental groups studied over eight months: 1) uninstrumented alcohol-fed (22 percent alcohol once per day, providing 33 percent of total daily caloric intake); 2) instrumented alcohol-fed; 3) instrumented alcohol- ramipril (ACE inhibitor, 0.1 mg/kg/day)-fed; 4) instrumented alcohol-irbesartan (ANG II AT1 blocker, 5 mg/kg/day)-fed. The control groups will consist of age-matched dogs under identical conditions except dogs in the control groups will not receive alcohol. Serial changes of cardiac systolic and diastolic function and RAS activation will be quantitated in these conscious dogs, chronically instrumented to measure LV pressures and volume. Cell contractile function, [Ca2+]i transient and ICa,L will be serially measured in freshly isolated cardiomyocytes from the LV obtained by biopsy from the same animals. Myocytes will be studied under conditions with superfusion of: 1) calcium, 2) isoproterenol, 3) pimobendan, 4) alcohol, and 5) ANG II. In addition, the studies of myocytes response to ANG II will be repeated after preincubation with an AT1 receptor blocker, a protein kinase C inhibitor, or a Gi protein inhibitor, before, during and after the development of ADCM. These studies will be the first detailed longitudinal studies of cardiac and myocyte structure, function, [Ca2+]i transient, ICa,L during chronic alcohol intake. In addition, these studies will provide unique information on the role of the RAS and the mechanism of ADCM. These studies are necessary to extend our knowledge about the mechanism in the development of ADCM and help target therapy for ADCM.