N‐RAP expression during mouse heart development

N-RAP gene expression and N-RAP localization were studied during mouse heart development using semiquantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. N-RAP mRNA was detected at embryonic day (E) 10.5, significantly increased from E10.5 to E16.5, and remained essentially constant from E16.5 until 21 days after birth. In E9.5-10.5 heart tissue, N-RAP protein was primarily associated with developing premyofibril structures containing alpha-actinin, as well as with the Z-lines and M-lines of more-mature myofibrils. In contrast, N-cadherin was concentrated in patches at the periphery of the cardiomyocytes. N-RAP labeling markedly increased between E10.5 and E16.5; almost all of the up-regulated N-RAP was associated with intercalated disk structures, and the proportion of mature sarcomeres containing N-RAP decreased. In adult hearts, specific N-RAP staining was only observed at the intercalated disks and was not found in the sarcomeres. The results are consistent with N-RAP functioning as a catalytic scaffolding molecule, with low levels of the scaffold being sufficient to repetitively catalyze key steps in myofibril assembly.