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First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03198

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Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation

¹ Institute for Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK
² Gene Targeting Laboratory, Institute for Stem Cell Research, University of Edinburgh, Edinburgh, UK
³ Kings College London, Muscle Cell Biology, Cardiovascular and Randall Division for Cell and Molecular Biophysics, London, UK

^* Author for correspondence (e-mail: m.peckham{at}leeds.ac.uk)

Accepted 31 July 2006

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 -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.

Key words: Titin, Muscle, Myofibrillogenesis, Gene targeting

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