N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about N-RAP?s role in myofibril assembly and its association with the ends of muscle cells, we investigated N-RAP gene expression and N-RAP localization during mouse heart development. Using semi-quantitative RT-PCR on RNA from dissected heart tissue, N-RAP expression was first detected on embryonic day 10.5 (E10.5). N-RAP expression significantly increased from E10.5 to E16.5, and remained essentially constant from E16.5 until 21 days after birth (P21). At no time during cardiac development did we detect expression of the skeletal muscle-specific exon 12 of N-RAP. Frozen sections of mouse embryos or isolated hearts were double immuno-labeled to localize N-RAP. We evaluated N-RAP?s association with myofibrillar structures marked with antibodies against sarcomeric alpha-actinin and developing intercalated disks labeled with antibodies against N-cadherin. In E9.5-10.5 heart tissue, N-RAP was primarily associated with developing premyofibril structures, 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. As development progressed from E14 to birth at E20-21, N-RAP fluoresence markedly increased, with almost all of the upregulated N-RAP associated with intercalated disk structures marked by N-cadherin antibodies. Low-level N-RAP staining was still observed in conjunction with myofibril precursors, but the proportion of mature sarcomeres containing N-RAP at the Z-line and M-line progressively decreased during this period. 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 assembly. The relatively large amounts of N-RAP that accumulate at the intercalated disks may serve as a reserve of molecules poised to quickly initiate assembly at the ends of the cell, or alternatively may serve a structural or signaling function at these key junctional regions. We previously identified 42 exons in the mouse and human N-RAP gene. RT-PCR analysis of adult tissues indicated that exon 12 is expressed in skeletal muscle, but not in the heart. We named the cardiac N-RAP isoform N-RAP-c and the predominant skeletal muscle isoform N-RAP-s. We analyzed mouse skeletal muscle for expression of N-RAP exon 12 during development, and found that the N-RAP-s isoform was predominant in skeletal muscle at all stages examined. N-RAP-s mRNA steadily increased From E17 to 21 days after birth. In contrast, the N-RAP-c mRNA was expressed at a constant, low level during this period. Both N-RAP isoforms were expressed in cultured undifferentiated C2C12 myoblasts, with N-RAP-c predominating. Upon serum withdrawal at 80% confluence, N-RAP-c mRNA steadily increased. In contrast, N-RAP-s mRNA remained low for ~24 hours, after which it was upregulated coincident with myoblast differentiation and fusion to form polynucleated myotubes. The results are consistent with a specialized role for N-RAP-s in myotubes. N-RAP is concentrated in myofibril precursors during sarcomere assembly in cultured cardiomyocytes. Distinct regions of N-RAP bind specific cytoskeletal proteins, and overexpression of these regions in cultured chick cardiomyocytes inhibits or arrests myofibril assembly. Thus, we hypothesize that N-RAP is a scaffolding protein that coordinates organization of alpha-actinin and actin in the initial premyofibril complexes that form at the cell periphery, and that deletion of N-RAP will yield cardiomyocytes incapable of assembling mature myofibrils. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful tool for analyzing gene knockdown phenotypes in living mammalian cells. Cocktails of double stranded siRNAs designed to target N-RAP mRNA were synthesized by in vitro transcription followed by cleavage with RNase III. Fetal mouse cardiomyocyte cultures were transfected with the siRNA cocktails and analyzed by fluorescence microscopy, quantitative RT-PCR and immunoblot. N-RAP transcript levels were decreased by ~70% 2-5 days after transfection, but recovered to normal levels on days 6-7. Changes in N-RAP protein lagged the decrease in mRNA, with N-RAP gradually decreasing between 3-6 days post-transfecton to ~50% of control levels. N-RAP protein expression recovered to control levels on days 7-8. These data show that N-RAP, like many other cytoskeletal proteins, is long-lived. Several other mRNAs were expressed at normal or increased levels following treatment with siRNAs targeted against N-RAP; these include muscle alpha-actinin, cardiac muscle myosin heavy chains, nonmuscle myosin heavy chain IIb, muscle LIM protein, and krp1. Alpha-actinin organization into mature myofibrillar striations paralleled N-RAP protein expression, with decreasing organization from days 3-6 and recovery to control levels on day 7. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNAi technique, that the technique is highly specific, and that myofibril organization in cardiomyocytes is closely linked to N-RAP protein levels.
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