title: Targeted disruption of N-RAP gene function by RNA interference: a role for N-RAP in myofibril organization.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ashwini
authors.initials: A
authors.lastname: Dhume
authors.forename: Shajia
authors.initials: S
authors.lastname: Lu
authors.forename: Robert
authors.initials: R
authors.lastname: Horowits
mesh_terms: D000185:Actinin / Q000378:metabolism; D000818:Animals; D001381:Azepines / Q000378:metabolism; D024722:Cardiac Myosins / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D017353:Gene Deletion; D015870:Gene Expression; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000096:biosynthesis / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D024501:Nonmuscle Myosin Type IIB / Q000096:biosynthesis / Q000378:metabolism; D010641:Phenotype; D020033:Protein Isoforms; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
chemical_list: D001381:Azepines; D009124:Muscle Proteins; D009281:Naphthalenes; C491567:Nrap protein, mouse; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C070571:ML 7; D000185:Actinin; D024722:Cardiac Myosins; D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains