The role of alpha-actinin-2 in sarcomere development and function

Cardiac and skeletal muscles feature a structure known as the sarcomere, which facilitates contraction. alpha-Actinin-2 is a vital component of the sarcomere. As many muscle diseases are characterized by a breakdown of the sarcomere, it is important to address the role alpha-actinin-2 plays in sarcomere development and function.; RNA interference was performed against Actn2 and its homolog Actn3 in cultured C2C12 cells. A selectable retroviral system was used to generate C2C12 cell cultures stably expressing short hairpin RNAs against either Actn2 or Actn3. In addition, cell cultures stably expressing a short RNA that does not target any gene were generated as negative controls. These cell cultures showed specific reduction of the targeted mRNA and protein, compared to controls. C2C12 cells differentiated into contracting myofibers in the absence of either Actn2 or Actn3. Expression profiling analysis of mRNA harvested from these cultures demonstrated that reduction of Actn2 promoted increased expression of genes enriched in fast muscle fibers and decreased expression of genes characteristic of slow muscle fibers. The converse may hold for reduction of Actn3 mRNA. This study indicates that Actn2 is involved in slow muscle fiber type maintenance.; A mouse was generated to eliminate the actin-binding domain of Actn2. A targeting vector was designed to replace exons 3 and 4 of Actn2 with the neomycin resistance gene. No viable mouse homozygous for the neomycin insertion into Actn2 was identified. Staged pregnancies were set up and embryos were examined in utero to determine the point of lethality. No Actn2-/- mouse was found beyond E12. At stages E10 and E11 some homozygous mutant embryos were identified, but not at the expected frequency of 25%. Histological analysis of homozygous mutant embryos did not reveal a gross morphological defect.; The ultra structure of the heart was analyzed by electron microscopy. Abnormalities were found in sarcomeres of hearts of homozygous mutants. Aberrant Z lines and sarcomere disarray were observed in developing cardiomyocytes. These defects cannot be visualized by conventional microscopy and most likely explain lethality. It seems that Actn2 is necessary to stabilize the sarcomere during contraction or myofiber maturation.