title: Role of nonmuscle myosin IIB and N-RAP in cell spreading and myofibril assembly in primary mouse cardiomyocytes.
abstract: We investigated the role of nonmuscle myosin heavy chain (NMHC) IIB in cultured embryonic mouse cardiomyocytes by specific knockdown using RNA interference. NMHC IIB protein levels decreased 90% compared with mock-transfected cells by 3 days post transfection. NMHC IIB knockdown resulted in a slow decrease in N-RAP protein levels over 6 days with no change in N-RAP transcript levels. N-RAP is a scaffold for alpha-actinin and actin assembly during myofibrillogenesis, and we quantitated myofibril accumulation by morphometric analysis of alpha-actinin organization. Between 3 and 6 days, NMHC IIB knockdown was accompanied by the abolishment of cardiomyocyte spreading. During this period the rate of myofibril accumulation steadily decreased, correlating with the slowly decreasing levels of N-RAP. Between 6 and 8 days NMHC IIB and N-RAP protein levels recovered, and cardiomyocyte spreading and myofibril accumulation resumed. Inhibition of proteasome function using MG132 led to accumulation of excess N-RAP, and the secondary decrease in N-RAP that otherwise accompanied NMHC IIB knockdown was abolished. The results show that NMHC IIB knockdown led to decreased N-RAP levels through proteasome-mediated degradation. Furthermore, these proteins have distinct functional roles, with NMHC IIB playing a role in cardiomyocyte spreading and N-RAP functioning in myofibril assembly.
authors.affiliation: Department of Health and Human Services, Laboratory of Muscle Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda 20892-8024, Maryland, USA.
authors.forename: Shajia
authors.initials: S
authors.lastname: Lu
authors.forename: Robert
authors.initials: R
authors.lastname: Horowits
mesh_terms: D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D024501:Nonmuscle Myosin Type IIB / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D009124:Muscle Proteins; C491567:Nrap protein, mouse; D010446:Peptide Fragments; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D024501:Nonmuscle Myosin Type IIB