SIGNALS OF OXIDANT INDUCED CARDIOMYOCYTE HYPERTROPHY

心肌细胞肥大是心脏衰老和许多心脏疾病的常见终点。目前尚不清楚是什么引发了这种变化，以及这种变化的机制。氧化剂是有氧代谢的副产物，在与缺血再灌注相关的病理条件下，在心脏中增加。利用H9C2和原代培养的新生大鼠心肌细胞，我们发现低浓度H2O2可引起心肌细胞肥厚。H2O2处理后的细胞体积增大4倍，细胞表面积增大4-10倍，每个细胞的蛋白质含量增加3倍。内分泌因子诱导的心肌细胞肥大涉及多个基因的表达升高、MAP激酶的激活和钙介导的NF-AT3的激活。由于H2O2已被报道激活MAP激酶并增加细胞质钙，我们假设H2O2激活MAP激酶或钙依赖性NF-AT3转录因子，从而改变肥厚基因的表达并诱导心肌细胞肥厚。采用Northern blot法检测H2O2处理的H9C2细胞心房利钠因子、β -肌球蛋白重链、骨骼α -肌动蛋白、α -肌球蛋白重链、心室特异性肌球蛋白轻链-2和肌钙蛋白C基因的表达。H2O2处理后，测定应激激活蛋白激酶、细胞外调节激酶和p38 MAP激酶的活性。这些激酶的抑制剂将测试其对H2O2诱导肥厚基因和细胞扩增的影响。同时，用H2O2处理细胞后，测定细胞内钙浓度和NF-AT3的DNA结合活性。钙螯合剂和钙调磷酸酶抑制剂将测试其阻止H2O2诱导的N-FAT3激活、肥厚基因激活和细胞扩大的能力。我们建议用新的和创新的方法来研究氧化应激的机制。这些结果将有助于了解心脏衰老的过程和心力衰竭的发病机制。

Cardiomyocyte hypertrophy is a common endpoint of heart aging and many cardiac diseases. It is not clear what triggers the change and the mechanism of the change. Oxidants are by-products of aerobic metabolism and increase in the heart under the pathological conditions associated with ischemia-reperfusion. Using H9C2 and primary cultured neonatal rat cardiomyocytes, we found that low concentrations of H2O2 can cause cardiomyocyte hypertrophy. H2O2 treated cells can be 4 times bigger in volume or 4-10 times larger in cell surface area and contain 3 times more proteins per cell. Cardiomyocyte hypertrophy induced by endocrine factors involves elevated expression of a number of genes, activation of MAP kinases and calcium mediated activation of NF-AT3. Since H2O2 has been reported to activate MAP kinases and increase cytosolic calcium, we hypothesize that H2O2 activates MAP kinases or calcium dependent NF-AT3 transcription factor to alter the expression of hypertrophic genes and to induce cardiomyocyte hypertrophy. The expression of the atrial natriuretic factor, beta-myosin heavy chain, skeletal alpha-actin, alpha-myosin heavy chain, ventricular-specific myosin light chain-2 and troponin C genes will be measured by Northern blot in H2O2 treated H9C2 cells. The activity of the stress activated protein kinase, extracellular regulated kinase and p38 MAP kinase will be determined after H2O2 treatment. The inhibitors of these kinases will be tested for their effect on induction of hypertrophic genes and cell enlargement by H2O2. In parallel, cytosolic calcium concentration and DNA binding activity of NF-AT3 will be measured after treating cells with H2O2. Calcium chelators and inhibitors of calcineurin will be tested for their ability to prevent H2O2 induced N-FAT3 activation, activation of hypertrophic genes and cell enlargement. We propose to study the mechanism of oxidant stress by novel and innovative approaches. The results will contribute to the understanding of the process of heart aging and the pathogenesis of heart failure.