Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation
Author:
Musa Hanny1, Meek Stephen2, Gautel Mathias3, Peddie Dianna2, Smith Andrew J. H.2, Peckham Michelle1
Affiliation:
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
Abstract
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 α-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.
Publisher
The Company of Biologists
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