Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2

Abstract

Various hypotheses have been proposed to explain the molecule processes of sarcomere assembly, partially due to the lack of systematic genetic studies of sarcomeric genes in an in vivo model. Towards the goal of developing zebrafish as a vertebrate model for this purpose, we characterized myofibrillogenesis in a developing zebrafish heart and went on to examine the functions of cardiac troponin T (tnnt2). We found that sarcomere assembly in zebrafish heart was initiated from a non-striated actin filament network at the perimembrane region, whereas sarcomeric myosin is independently assembled into thick filaments of variable length before integrating into the thin filament network. Compared to Z-discs that are initially aligned to form shorter periodic dots and expanded longitudinally at a later time, M-lines assemble later and have a constant length. Depletion of full-length tnnt2 disrupted the striation of thin filaments and Z-bodies, which sequentially affects the striation of thick filaments and M-lines. Conversely, truncation of a C-terminal troponin complex-binding domain did not affect the striation of these sarcomere sub-structures, but resulted in reduced cardiomyocyte size. In summary, our data indicates that zebrafish are a valuable in vivo model for studying both myofibrillogenesis and sarcomere-based cardiac diseases.

Tropomyosin is required for cardiac morphogenesis, myofibril assembly, and formation of adherens junctions in the developing mouse embryo

“…Troponin T is essential for myofibril assembly and development of the mouse heart, where deletion of troponin T causes failure of myofibril assembly, severe cardiac dilation, and lack of heartbeat, leading to embryonic lethality at Ê10 (Nishii et al, 2008). Similar observations demonstrating essential functions for troponins in sarcomere formation, as well as exacerbation of defects by unregulated actomyosin activity during myofibril assembly, have been made in studies of zebrafish striated muscles (Sehnert et al, 2002;Huang et al, 2009;Ferrante et al, 2011). The primary direct evidence for a role of TM in myofibril assembly comes from the Mexican axolotl, in which the naturally occurring cardiac mutation eliminates TM expression and disrupts cardiac myofibril assembly via aberrant intracellular targeting of a host of thin filament and sarcomere-associated proteins and lack of coalescence of organized sarcomeres (Lemanski, 1973;Lemanski, 1979;Lemanski et al, 1980;Fuldner et al, 1984;Starr et al, 1989;Erginel-Unaltuna and Lemanski, 1994;La France and Lemanski, 1994;Zajdel et al, 1998;Zajdel et al, 1999;McLean et al, 2006;Zajdel et al, 2007).…”

“…Similarly, deletion of cardiac troponin T in mice results in embryonic lethality at Ê10 due to impaired cardiac myofibril assembly, sarcomere disruption, an abnormally thin cardiac wall with reduced trabeculae, and lack of heartbeat (Nishii et al, 2008), quite similar to the αTM1 −/− mouse heart. Troponin T is also essential for myofibril assembly in zebrafish striated muscles (Sehnert et al, 2002;Huang et al, 2009;Ferrante et al, 2011). It is noteworthy that the defects in myofibril assembly reported for the zebrafish troponin T mutants are accompanied by reduced assembly of αTM1 onto thin filaments (Sehnert et al, 2002;Ferrante et al, 2011), indicating that troponins are required for stable αTM1 association with thin filaments.…”

Troponin T is essential for sarcomere assembly in zebrafish skeletal muscle

“…In silent heart mutants, defective for the cardiac isoform troponin T2a, sarcomere assembly is impaired and dispersed thick filaments were reported (Sehnert et al, 2002). In that study, it was proposed that a common regulation of thin filament genes would be altered in the absence of troponin T and that lack of assembly is due to downregulation of thin filament genes and the consequent dearth of thin filament proteins (Huang et al, 2009;Sehnert et al, 2002). Similar observations, however, have not been reported for mouse troponin mutants (Nishii et al, 2008).…”

“…In D. melanogaster, downregulation of troponin I but not of other major thin filaments genes has been reported in flight muscles of a troponin T mutant (Nongthomba et al, 2007). The consequences of tnnt2a disruption in the zebrafish heart have been further investigated with confocal microscopy (Huang et al, 2009). These authors reported loss of periodicity for thin and thick filaments and for Z and M lines, similar to that observed in skeletal muscle.…”

Exon- and contraction-dependent functions of titin in sarcomere assembly

“…Zebrafish is a new vertebrate model for studying sarcomere assembly (Schoenebeck and Yelon, 2007;Huang et al, 2009;Sanger et al, 2009;Yang et al, 2014b). From a large-scale screen using N-ethyl-N-nitrosourea as a mutagen, a group of ttn mutants, named pickwick ( pik), have been identified (Xu et al, 2002).…”