Wang Yingcai, Szczesna-Cordary Danuta, Craig Roger, Diaz-Perez Zoraida, Guzman Georgianna, Miller Todd, Potter James D
Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, 1600 NW 10 Ave, Miami, FL 33136, USA.
FASEB J. 2007 Jul;21(9):2205-14. doi: 10.1096/fj.06-7538com. Epub 2007 Mar 13.
In skeletal muscle, the myosin molecule contains two sets of noncovalently attached low molecular weight proteins, the regulatory (RLC) and essential (ELC) light chains. To assess the functional and developmental significance of the fast skeletal isoform of the RLC (RLC-f), the murine fast skeletal RLC gene (Mylpf) was disrupted by homologous recombination. Heterozygotes containing an intronic neo cassette (RLC-/+) had approximately one-half of the amount of the RLC-f mRNA compared to wild-type (WT) mice but their muscles were histologically normal in both adults and neonates. In contrast, homozygous mice (RLC-/-) had no RLC-f mRNA or protein and completely lacked both fast and slow skeletal muscle. This was likely due to interference with mRNA processing in the presence of the neo cassette. These RLC-f null mice died immediately after birth, presumably due to respiratory failure since their diaphragms lacked skeletal muscle. The body weight of newborn RLC-f null mice was decreased 30% compared to heterozygous or WT newborn mice. The lack of skeletal muscle formation in the null mice did not affect the development of other organs including the heart. In addition, we found that WT mice did not express the ventricular/slow skeletal RLC isoform (RLC-v/s) until after birth, while it was expressed normally in the embryonic heart. The lack of skeletal muscle formation observed in RLC-f null mice indicates the total dependence of skeletal muscle development on the presence of RLC-f during embryogenesis. This observation, along with the normal function of the RLC-v/s in the heart, implicates a coupled, diverse pathway for RLC-v/s and RLC-f during embryogenesis, where RLC-v/s is responsible for heart development and RLC-f is necessary for skeletal muscle formation. In conclusion, in this study we demonstrate that the Mylpf gene is critically important for fast and slow skeletal muscle development.
在骨骼肌中，肌球蛋白分子包含两组非共价连接的低分子量蛋白质，即调节性轻链（RLC）和必需性轻链（ELC）。为了评估快速骨骼肌RLC亚型（RLC-f）的功能和发育意义，通过同源重组破坏了小鼠快速骨骼肌RLC基因（Mylpf）。与野生型（WT）小鼠相比，含有内含子新霉素盒的杂合子（RLC-/+）的RLC-f mRNA量约为其一半，但无论在成年还是新生小鼠中，其肌肉在组织学上均正常。相比之下，纯合小鼠（RLC-/-）没有RLC-f mRNA或蛋白质，并且完全缺乏快、慢骨骼肌。这可能是由于新霉素盒的存在干扰了mRNA加工。这些RLC-f基因敲除小鼠出生后立即死亡，推测是由于呼吸衰竭，因为它们的膈肌缺乏骨骼肌。与杂合子或WT新生小鼠相比，新生RLC-f基因敲除小鼠的体重下降了30%。基因敲除小鼠中骨骼肌形成的缺乏并不影响包括心脏在内的其他器官的发育。此外，我们发现WT小鼠直到出生后才表达心室/慢骨骼肌RLC亚型（RLC-v/s），而它在胚胎心脏中正常表达。在RLC-f基因敲除小鼠中观察到的骨骼肌形成缺乏表明，在胚胎发生过程中，骨骼肌发育完全依赖于RLC-f的存在。这一观察结果，连同RLC-v/s在心脏中的正常功能，暗示了在胚胎发生过程中RLC-v/s和RLC-f存在一条耦合的、多样的途径，其中RLC-v/s负责心脏发育，而RLC-f是骨骼肌形成所必需的。总之，在本研究中我们证明Mylpf基因对快、慢骨骼肌发育至关重要。
