Analysis of mechanisms of myofibril assembly by force-expression of DNA

通过在培养的心肌、骨骼肌细胞和成纤维细胞中强制表达表位标记的cDNA，分析肌球蛋白碱性轻链(LC)和肌钙蛋白I(TnI)在肌原纤维组装过程中的等位蛋白的分类。在具有心肌肌球蛋白重链(MHC)的心肌细胞中，LC亚型对肌节的分选特异性由大到小依次为：非肌肉LC_3 nm>慢肌LC_1Sa>慢/室肌LC_1sb>快肌LC_1f和LC_3f。在表达非肌肉MHC的成纤维细胞中，心肌细胞中Lc1sa和Lc1sb的顺序颠倒。因此，LC的分选取决于MHC亚型和LO亚型。对力表达嵌合LC的亚细胞分布分析表明，LC的第二个EF-手负责这种异构体特异性的分选。在心肌和骨骼肌的肌原纤维中分别掺入了FORCE表达的心肌和快肌TnI。然而，心脏TnI没有被整合到SK…中更多的骨骼肌肌原纤维，尽管快速肌肉TnI被组装到心肌肌原纤维上。嵌合TnI的表达表明，TnI亚型的不同行为取决于同功蛋白的C-末端部分的不同。通过注射罗丹明标记的肌动蛋白和光漂白后的荧光恢复(FR)检测心肌细胞和成纤维细胞肌动蛋白的交换性。应激纤维的FR速率始终快于肌原纤维，表明应激纤维和非横纹肌原纤维虽然在外观和组成上相似，但在肌动蛋白稳定性上是不同的。在肌原纤维中，非纹状部的FR速度快于纹状部，肌肉肌动蛋白的FR率快于非粘液型。在活的心肌细胞中检测到融合了绿色荧光蛋白(LC3f-GFP)的LC3f掺入肌原纤维。LC3f-GFP最初在A带的两端积累，然后整合到除H带外的整个A带中。较少

The sorting of isoproteins of myosin alkali light chain (LC) and troponin I (TnI) during myofibril assembly was analyzed by force-expression of epitope-tagged cDNAs in cultured cardiac and skeletal muscle cells and fibroblasts. In cardiomyocytes possessing cardiac myosin heavy chain (MHC), sorting specificity of LC isoforms to the sarcomeres was increased in the order from nonmuscle LC3nm, to slow muscle LC1sa, to slow/ventricular muscle LC1sb, and to fast muscle LC1f and LC3f. In fibroblasts expressing nonmuscIe MHC, the order of LC1sa and LC1sb in cardiomyocytes was reversed. Thus, the sorting of LC occurred depending on MHC isoforms as well as LO isoforms. Analysis of subcellular distribution of force-expressed chimeric LCs revealed that the second EF-hand of LC is responsible for such isoform specific sorting. Force-expressed cardiac and fast muscle TnI were incorporated into myofibrils of cardiac and skeletal muscles, respectively. However, cardiac TnI was not incorporated into sk … More eletal muscle myofibrils, although fast muscle TnI was assembled on to cardiac myofibrils. Expression of chimeric TnI revealed that the differential behavior of TnI isoforms depends on difference in the C-terminal halves of the isoproteins. The exchangeability of actin was examined by injection of rhodamine-labeled actin and fluorescence recovery (FR) after photobleaching in cardiomyocytes and fibroblasts. The FR rate in stress fibers was consistently faster than that in myofibrils, indicating that stress fibers and non-striated myofibrils are different in actin stability, although they are similar in appearance and composition. In myofibrils, non-striated parts exhibited faster FR rate than striated portions, and the FR rate of muscle actin was faster than that of nonmucle type. Incorporation of LC3f fused with green fluorescence protein (LC3f-GFP) into myofibrils was monitored in living cardiomyocytes. LC3f-GFP was initially accumulated at both ends of A-bands, and then incorporated into the whole of A-bands except the H-zone. Less