Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation.
Titin, a multifunctional protein that stretches from the Z-disk to the M-band in heart and skeletal muscle, contains a kinase domain, phosphorylation sites and multiple binding sites for structural and signalling proteins in the M-band. To determine whether this region is crucial for normal sarcomere development, we created mouse embryonic stem cell (ES) lines in which either one or both alleles contained a targeted deletion of the entire M-band-coding region, leaving Z-disk-binding and myosin-filament-binding sites intact. ES cells were differentiated into cardiomyocytes, and myofibrillogenesis investigated by immunofluorescence microscopy. Surprisingly, deletion of one allele did not markedly affect differentiation into cardiomyocytes, suggesting that a single intact copy of the titin gene is sufficient for normal myofibrillogenesis. By contrast, deletion of both alleles resulted in a failure of differentiation beyond an early stage of myofibrillogenesis. Sarcomeric myosin remained in non-striated structures, Z-disk proteins, such as alpha-actinin, were mainly found in primitive dot-like structures on actin stress fibres, M-band-associated proteins (myomesin, obscurin, Nbr1, p62 and MURF2) remained punctate. These results show that integration of the M-band region of titin is required for myosin filament assembly, M-band formation and maturation of the Z-disk.
Musa
Hanny
H
Institute for Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
Meek
Stephen
S
Gautel
Mathias
M
Peddie
Dianna
D
Smith
Andrew J H
AJ
Peckham
Michelle
M
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D064211:Connectin; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D017353:Gene Deletion; D006579:Heterozygote; D006720:Homozygote; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008957:Models, Genetic; D009124:Muscle Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009210:Myofibrils / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism*; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism
D064211:Connectin; C490981:Gtf2h1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; C486326:Nbr1 protein, mouse; D011506:Proteins; C577294:Tcap protein, mouse; D014157:Transcription Factors; C502755:muscle RING finger 2 protein, mouse; D051758:Transcription Factor TFIIH; D011494:Protein Kinases