Myotilin cDNAs from human, mouse, and some other species have been cloned and sequenced [Salmikangas et al. 1999
; Strausberg et al., 2002
]. Although a predicted cDNA sequence of chicken myotilin cDNA is available from the databases, to the best of our knowledge, direct cDNA from chicken has not been sequenced. In this study, we have cloned and sequenced the cDNAs of myotilin from chicken heart and skeletal muscle. The nucleotide sequences of myotilin from heart and skeletal muscles are identical (data not shown) suggesting that they are the products of the same Myot gene. The deduced amino acid sequences of chicken myotilin contain 506 amino acid residues whereas human and mouse myotilin contain 498 and 496 amino acid residues respectively. The extra eight amino acid residues in chicken compared to human myotilin variant 1, are located at the N-terminal part of the myotilin peptides as shown in . The percent similarity of myotilin protein compared to human and mouse myotilin is 72 and 70 respectively. The percent identity between chicken and human or chicken and mouse myotilin proteins are 66 and 65 respectively.
Myotilin, together with palladin, and myopalladin, is a member of a family of muscle proteins with Ig domains that function as scaffolds and regulate actin organization [Bang et al., 2001
; Mykkanen et al., 2001
; Otey et al., 2005
; von Nadelstadh et al., 2009
]. Myotilin is localized in the Z-bands of cross-striated muscles with many binding partners among which are actin, alpha-actinin, ArgBP2, FATZ/myozenin and filamin [van der Ven et al. 2000
; Otey et al., 2005
; Gontier et al., 2005; Moza et al., 2007
; Moza, 2008
; Sanger and Sanger, 2008
; von Nadelstadh et al., 2005
; Sanger et al., 2010
;]. Although missense mutations in myotilin have been implicated in limb girdle muscular dystrophy type 1A and spheroid body myopathy in humans [Selcen, 2011
], it is not clear whether myotilin has any role in myofibrillogenesis. A targeted deletion of the myotilin gene (Myot) does not affect structure and function of muscle in Myot knockout mice [Moza et al., 2007
]. This may be due, however, to the ability of the other Z-band proteins with similar Ig-like domains, viz. palladin and myopalladin, to take over the role of the deleted myotilin [Ochala et al., 2009
Von Nandelstadh et al. 
studied the actin binding properties of wild type myotilin and the four myotilin mutants then known to cause myopathies (S55F, T57I, S60C, and S95I), but did not detect any differences between the actin binding properties of the wild type and myotilin mutants. More recently three of these myotilin mutants (S55F, T57I, and S60C) were shown to have slower degradation rates than wild type myotilin [Von Nandelstadh et al., 2011
]. How slower degradation rates of myotilin mutants lead to muscle diseases is unknown.
There are now seven different mutations of myotilin that lead to dystrophies in skeletal muscle cells [Salmikangas et al., 1999
; Selcen and Engel, 2004
; Foroud et al., 2005
; Shalaby et al., 2009
; Selcen, 2011
]. The seven YFP fusion proteins of these myotilin mutants located normally in the Z-bands of mature myofibrils, and we were unable to detect significant differences in dynamic behavior between these myotilin mutants and wild type myotilin measured with FRAP. Perhaps other approaches in the future (e.g., Fluorescence Resonance Energy Transfer or FRET, Stout et al., 2008
) may discover differences in the binding properties of the myotilin mutants and their binding partners that lead to myofibrillar instabilities, a hallmark of myotilinopathy [Selcen, 2011
FRAP is a powerful technique that allows the mobility of proteins between a cytoplasmic pool and a subcellular site to be measured in the setting of the live cell where multiple binding interactions influence a protein’s localization. Full length myotilin is a very dynamic protein recovering 80% of fluorescence within 5 minutes after bleaching. The C-terminal half of the molecule in which all the myotilin binding sites for Z-band proteins are found has a slower recovery of fluorescence indicating a tighter binding of the protein. The myofibril disassembly resulting from expression of myotilin-C may arise when the fragment binds tightly to Z-band partners and competes with full-length myotilin for Z-band localization, impairing the function that the intact myotilin provides and causing disruption of the myofibrils. It may also indicate that the disease-associated serine-rich region in N-terminus is important for normal dynamics and function of myotilin in maintenance of myofibrils.
We found that the C-terminal half of myotilin (aa 251–498) was much less dynamic than that of the full myotilin molecule and the N-terminal half of myotilin (aa 1–250). Its stronger Z-band affinity was accompanied by the formation of aggregates in the cytoplasm of the transfected muscle cells and loss of myofibrils (, , ). These results are similar to the effect of a truncated fragment of alpha-actinin: the 27 KD fragment responsible for the actin-binding property of alpha-actinin [Wang et al., 2005
]. This fragment of alpha-actinin initially exhibited normal binding to the Z-bands of mature myofibrils, but with time also led to myofibril disassembly. FRAP experiments in myotubes expressing this truncated alpha-actinin revealed a 50% reduction in the recovery of postbleach fluorescence intensity compared wild type YFP-alpha-actinin. We hypothesize that these myotilin and alpha-actinin truncated fragments compete with their full-length counterparts and prevent the normal dynamics of Z-band proteins that are required for the stability and maintenance of Z-bands of the myofibrils. In addition, these truncated fragments are missing parts of the intact molecules that may allow binding to other partners of the Z-bands. The inability to bind these other partners may weaken the Z-band structure and the stability of the mature myofibrils. These truncations of either alpha-actinin or myotilin would be incompatible with embryonic development due to the importance of contractile embryonic hearts and skeletal muscle cells.
In summary, chicken myotilin has properties that are very similar to those of human myotilin molecules. We did not detect any changes in either the localization or in the dynamics of the seven known myotilin mutations in muscle cells (S55F, S55I, T57I, S60C, S60F, S95I, R405K). The C-terminal half of the myotilin molecule, when expressed as a truncated fusion protein, exhibits decreased dynamics in the Z-bands of mature myofibrils in both skeletal and cardiac muscle cells, and induces the disassembly of myofibrils.