New In Vivo Apoptosis Model of Cardiomyocytes and Bcl-2 gene therapy

目前还没有足够的心肌细胞凋亡的体内模型。本研究旨在建立一种新的心脏细胞凋亡模型，并通过直接注射可溶性Fas配体（sFL）的方法，研究在体大鼠心肌细胞和心脏间质细胞对凋亡信号的敏感性，以及与肝细胞和肝间质细胞相比，心肌细胞和心脏间质细胞的凋亡清除率和超微结构。此外，还研究了Bcl-2基因治疗是否能抑制fas诱导的细胞凋亡。为了尽量减少对全身的影响，将可溶性Fas配体以0、0.5、2和5 μg/mL的浓度（C组、F0.5组、F2组和F5组）直接注射到体内大鼠的心脏和肝脏（作为对照）。心肌细胞凋亡和非心肌细胞凋亡仅在F5中有相似的发生率。其发生率在注射后12 h达到高峰（心肌细胞为2.0±0.09%），24 h后明显消失。Caspase-3仅在F5激活。panaspase抑制剂Boc-Asp-fmk抑制细胞凋亡，提示凋亡敏感性受caspase-3上游调控。非心肌细胞凋亡呈典型超微结构。然而，除了典型的超微结构，如细胞收缩、染色质凝聚和凋亡小体外，凋亡心肌细胞还表现出独特的特征：甜甜圈状，而不是半月形或新月形，染色质凝聚，甚至在早期就频繁出现质膜破裂；浓缩的线粒体，内部有褶皱的嵴；细胞质脂样液滴的出现；以及肌纤维紊乱。在肝脏中，即使在F0.5组，肝脏的肝细胞和非肝细胞也诱导了典型的凋亡，并在24小时后被清除。Bcl-2基因治疗可明显阻断凋亡细胞。与肝细胞相比，心脏的心肌细胞和非心肌细胞对凋亡信号的抵抗力更强，但清除速度同样快（在24小时内）。心肌细胞凋亡的超微结构是独特的。这些发现为心脏细胞死亡的动力学提供了新的见解。少

There is no adequate in vivo model of apoptotic cardiomyocyte. The purpose of the present study was to develop a new apoptosis model of heart, and to delineate sensitivity to the apoptotic signal and the clearance rate and ultrastructure of apoptosis in in vivo adult cardiomyocytes and interstitial cells of the heart in comparison with those of hepatocytes and interstitial cells of the liver by use of a direct injection method of soluble Fas ligand (sFL) into adult rat hearts and livers. In addition, it was studied whether Bcl-2 gene therapy can inhibit the Fas-induced apoptosis.To minimize the systemic influence, soluble Fas ligand was injected directly into in vivo rat hearts and livers (as the control) at concentrations of 0, 0.5, 2, and 5 μg/mL (groups C, F0.5, F2, and F5). Apoptotic cardiomyocytes and apoptotic noncardiomyocytes of the heart were identified with similar incidences only in F5. Their incidents peaked at 12 hours after injecction (2.0±0.09% in cardiomyocytes) and dim … More inished markedly 24 hours later. Caspase-3 was activated only in F5. Boc-Asp-fmk, a pancaspase inhibitor, inhibited apoptosis, suggesting that the apoptosis sensitivity was regulated upstream of caspase-3. Apoptotic noncardiomyocytes showed typical ultrastructure. In addition to the typical ultrastructure, such as cellular shrinkage, chromatin condensation, and apoptotic bodies, however, apoptotic cardiomyocytes showed unique features : doughnut-like, but not half-moon- or crescent-like, chromatin condensation : frequent plasma membrane rupture even during the early stage ; condensed mitochondria with wrinkled cristae inside ; the appearance of cytoplasmic lipid-like droplets ; and myofibrillar derangement. In the livers, typical apoptosis was induced in hepatocytes and nonhepatocytes of the liver even in the F0.5 group, which were cleared 24 hours later. Bcl-2 gene therapy blocked significantly apoptotic cells.Compared with liver cells, cardiomyocytes as well as noncardiomyocytes of the heart are more resistant against the apoptotic signal, but the clearance is similarly rapid (within 24 hours). The ultrastructure of apoptotic cardiomyocytes is unique. These findings provide new insights into the dynamics of cell death in the heart. Less