Dhume Ashwini, Lu Shajia, Horowits Robert
Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health (Department of Health and Human Services), Bethesda, MD 20892, USA.
Cell Motil Cytoskeleton. 2006 Aug;63(8):493-511. doi: 10.1002/cm.20141.
N-RAP is a muscle-specific protein concentrated in myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by approximately 70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching approximately 50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that alpha-actinin organization into myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms.
N-RAP是一种肌肉特异性蛋白，在肌节组装过程中集中于肌原纤维前体，在成年心脏的闰盘中也有表达。我们利用RNA干扰技术，有针对性地降低了胚胎小鼠心肌细胞原代培养物中N-RAP转录本和蛋白水平。用N-RAP特异性小干扰RNA转染后2天内，N-RAP转录本水平下降了约70%。N-RAP蛋白水平在数天内稳步下降，6天内降至对照水平的约50%。通过α-辅肌动蛋白组织成成熟条纹评估，N-RAP蛋白敲低与肌原纤维组装减少有关。在N-RAP蛋白敲低期间，与组装或成熟肌原纤维相关的N-RAP结合蛋白的转录本，如α-辅肌动蛋白、Krp1和肌肉LIM蛋白，表达水平正常，α-辅肌动蛋白和Krp-1蛋白水平也未改变。编码肌肉肌球蛋白重链和非肌肉肌球蛋白重链IIB的转录本也以相对正常的水平表达。然而，N-RAP蛋白水平降低与编码的肌球蛋白蛋白的显著变化有关，肌肉肌球蛋白重链水平增加，非肌肉肌球蛋白重链IIB水平降低。N-RAP转录本和蛋白水平分别在第6天和第7天恢复正常，肌原纤维组织和肌球蛋白重链同工型水平的变化也得到逆转。我们的数据表明，利用RNA干扰技术可以实现N-RAP蛋白的瞬时敲低，并且心肌细胞中α-辅肌动蛋白组织成肌原纤维与N-RAP蛋白水平密切相关。最后，N-RAP蛋白水平通过转录后机制调节非肌肉肌球蛋白IIB和肌肉肌球蛋白之间的平衡。
