Myotilin dynamics in cardiac and skeletal muscle cells

Abstract

Myotilin cDNA has been cloned for the first time from chicken muscles and sequenced. Ectopically expressed chicken and human YFP-myotilin fusion proteins localized in avian muscle cells in the Z-bodies of premyofibrils and the Z-bands of mature myofibrils. Fluorescence recovery after photobleaching experiments demonstrated that chicken and human myotilin were equally dynamic with 100% mobile fraction in premyofibrils and Z-bands of mature myofibrils. Seven myotilin mutants cDNAs (S55F, S55I, T57I, S60C, S60F, S95I, R405K) with known muscular dystrophy association localized in mature myofibrils in the same way as normal myotilin without affecting the formation and maintenance of myofibrils. N- and C-terminal halves of human myotilin were cloned and expressed as YFP fusions in myotubes and cardiomyocytes. N-terminal myotilin (aa 1-250) localized weakly in Z-bands with a high level of unincorporated protein and no adverse effect on myofibril structure. C-terminal myotilin (aa 251-498) localized in Z-bands and in aggregates. Formation of aggregated C-terminal myotilin was accompanied by the loss of Z-band localization of C-terminal myotilin and partial or complete loss of alpha-actinin from the Z-bands. In regions of myotubes with high concentrations of myotilin aggregates there were no alpha-actinin positive Z-bands or organized F-actin. The dynamics of the C-terminal-myotilin and N-terminal myotilin fragments differed significantly from each other and from full-length myotilin. In contrast, no significant changes in dynamics were detected after expression in myotubes of myotilin mutants with single amino acid changes known to be associated with myopathies.

Figure 1. Alignment of deduced amino acid sequences, and domain structure of chicken myotilin

A. Alignment of the deduced amino acid sequences with other known myotilin sequences. B. A schematic diagram of the protein structure shows the serine-rich region (blue box) containing a hydrophobic stretch (not shown) and the two Ig domains (loops) [Salmikangas et al., 1999]. There are eight extra amino acid residues (from 68–75) in chicken myotilin sequence.

Figure 1. Alignment of deduced amino acid sequences, and domain structure of chicken myotilin

A. Alignment of the deduced amino acid sequences with other known myotilin sequences. B. A schematic diagram of the protein structure shows the serine-rich region (blue box) containing a hydrophobic stretch (not shown) and the two Ig domains (loops) [Salmikangas et al., 1999]. There are eight extra amino acid residues (from 68–75) in chicken myotilin sequence.

Figure 2

Fluorescent images of developing myotubes from embryonic quail muscle in a culture transfected with YFP-chicken myotilin. (a) Myotilin is localized in the premyofibrils at the end of a myotube and (b) in the Z-bands of mature myofibrils at the central region of a myotube. Bar = 5 μm.

Figure 3

Comparison of the dynamics measured by FRAP of chicken and human myotilin localized in the Z-bands of quail myotubes. The rates and half-lives of exchange of the two isoforms of myotilin are the same in the Z-bands of quail myotubes.

Figure 4

Fluorescence images of quail skeletal muscle cells co-transfected with (a) the N-terminal half of myotilin, and (b) CeFP-alpha-actinin. (c) Merged images of (a) and (b). (a) The N-terminal half of human myotilin (myotilin-N) co-localizes in Z-bands with (b) alpha-actinin, but there is a high background of unincorporated myotilin in the sarcoplasm (a). Bar = 5 μm.

Figure 5

Fluorescence images of myotubes co-transfected the C-terminal half of YFP-myotilin (a, c, e) and CeFP-alpha-actinin (b, d, f). (a, b) Two to three days post-transfection, myotilin-C co-localized with alpha-actinin in Z-bands of some myotubes. (c, d) In other myotubes, aggregates of myotilin-C (c, arrow) were aligned along myofibrils in myotubes in which alpha-actinin was present in both aggregates (d, arrow) and Z-bands. (e, f) Four days or more after co-transfection, both myotilin-C and alpha-actinin were present as aggregates and Z-bands were absent. (f) Note that the myotube with alpha-actinin incorporation in Z-bands in the lower part of the image did not express myotilin-C. Bar = 5 μm.

Figure 6

Fluorescence images of myotubes transfected with (a) YFP-human myotilin-C and then fixed, and stained with (b) rhodamine-phalloidin. (c) Merged images of (a) and (b). The distribution of actin in a sarcomeric pattern was seen in the two myotubes that did not express myotilin-C and in the lower region of the center myotube where the concentration of myotilin-C aggregates was much less than in the disrupted region above. Bar = 5 μm.

Figure 7

Fluorescence images of cardiomyocytes transfected with plasmids encoding fragments of human myotilin. (a) YFP-myotilin, (b) N-terminal (YFP-myotilin-N), (c, d) C-terminal (YFP-myotilin-C) of myotilin, (e) YFP-myotilin-C, and (f) same cardiomyocyte as in (e) co-expressing CeFP-alpha-actinin. As in skeletal muscle myotubes, (a) the Z-band localization of full length YFP-myotilin contrasted with (b) a high background of unincorporated YFP-myotilin-N that was present together with Z-band localization of the protein fragment. (c) YFP-myotilin-C localized in Z-bands and also (d) in aggregates in cardiomyocytes in the same culture. (e) In cardiomyocytes with high concentrations of aggregated YFP-myotilin-C myofibrils appeared disrupted and (f) CeFP-alpha-actinin colocalized with myotilin. Bar = 5 μm.

Figure 8

Images from a FRAP experiment of myotubes expressing (a–e) full-length myotilin or (f–j) myotilin-C. Images (a, f) pre-bleach, (b, g) bleach, (c, h) recovery 1 minute post-bleach, (d, i) recovery 5 minutes post-bleach and (e, j) recovery 20 minutes post-bleach. Bar = 5 μm.

Figure 9

Recovery after photobleaching in myotubes and cardiomyocytes expressing full-length myotilin or Myotilin-N or Myotilin-C. (a) The average FRAP curve of full-length, N-terminal half and C-terminal half of myotilin in Z-bands of mature myofibrils in myotubes. (b) a comparison of the fast and slow mobile fractions of full-length, myotilin-N and myotilin-C in Z-bands of myofibrils in myotubes. Note that a slow mobile fraction is absent in the recovery of myotilin-N fluorescence. Both mobile fractions of myotilin-C are reduced compared with full-length myotilin. (c) The average FRAP curve of full-length, myotilin-N and myotilin-C in Z-bands of myofibrils in cardiomyocytes and (d) comparison of the fast and slow mobile fractions of full-length myotilin-N and myotilin-C in Z-bands of myofibrils in cardiomyocytes.

Figure 10

Myotubes expressing wild type myotilin (a) and myotilin with seven different known mutations (b–h). There are no differences between the localization of the wild type myotilin and the myotilin mutants; all are localized in the Z-bands. Bar = 5 μm.

Figure 11

FRAP curves of wild type myotilin and mutated versions of human myotilin. Seven single amino acid mutations of human myotilin (S55F, S55I, T57I, S60C, S60F, S95I, R405K) were expressed in quail myotubes and localized in the Z-bands of the mature myofibrils. The average curves of the myotilin mutants each fall within the error bars of the wild type myotilin molecules in the Z-bands of mature myofibrils. Two-population t-test analysis revealed that the recoveries for the seven mutations of myotilin were not significantly different from wild type myotilin (95% confidence intervals).