Depletion of Zebrafish Titin Reduces Cardiac Contractility by Disrupting the Assembly of Z-Discs and A-Bands

Abstract

Abstract-The genetic study of titin has been notoriously difficult because of its size and complicated alternative splicing routes. Here, we have used zebrafish as an animal model to investigate the functions of individual titin isoforms. We identified 2 titin orthologs in zebrafish, ttna and ttnb, and annotated the full-length genomic sequences for both genes. We found that ttna, but not ttnb, is required for sarcomere assembly in the heart as well as the subsequent establishment of cardiac contractility. In fact, ttna is the earliest sarcomeric mRNA that is expressed in the heart, which makes it an early molecular marker for cardiomyocyte differentiation. Surprisingly, ttna is required for later steps of sarcomere assembly, including the assembly of Z-discs and A-bands, but not for early steps such as the assembly of Z-bodies and nonstriated myosin filaments. Reduction of individual titin isoforms in vivo using morpholino-modified antisense oligonucleotides indicated that (1) both N2B exon-containing and N2A exon-containing isoforms of ttna are required for sarcomere assembly in the heart; (2) N2A exon-containing isoforms of both ttna and ttnb are required for sarcomere assembly in the somites; and (3) the N2B exon-containing isoforms of ttnb are expressed later than other titin isoforms and are probably involved in modulating their expression; however, these isoforms of ttnb are not required for sarcomere assembly. Collectively, our results reveal distinct functions of different titin isoforms and suggest that various phenotypes in "titinopathies" may be attributable to the disruption of different titin isoforms. (Circ Res. 2007;100:238-245.)

Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis

“…Similar abnormalities of myofibrillogenesis have been reported in titin M-line deficient mice (Peng et al 2005;Weinert et al 2006) and in mouse skeletal myotubes that differentiated in culture following titin truncation (Miller et al 2003). Reduction of titin expression using morpholino antisense oligonucleotides disrupted the assembly of Z-discs and A-bands of myofibrils in zebrafish (Seeley et al 2007). Significant abnormalities of the Z-disk structure, shortened actin filaments, dysregulation of genes involved in calcium homeostasis and glycogen metabolism have been reported in nebulin-deficient mice (Witt et al 2006).…”

“…This is consistent with the localization of these proteins at different sarcomeric compartments and their signaling properties in addition to the structural involvement in the process of myofibril assembly. Of special interest is the fact that functional impairment of obscurin expression in cardiac ) and skeletal muscle ) results in similar effects on the structure of the contractile apparatus as the down-regulation of titin function (Person et al 2000;Musa et al 2006;Seeley et al 2007). …”

Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

“…Thus, it seems that ␣-actinin is not absolutely required for Z-disc formation and function, but it is needed to maintain Z-disc stability in this organism. The giant sarcomeric protein titin has also been implicated in the assembly and maintenance of the Z-disc structure, whereas the specific contributions of other Z-disc components are currently unknown (Zou et al, 2006;Seeley et al, 2007).…”

“…Thus, it seems that ␣-actinin is not absolutely required for Z-disc formation and function, but it is needed to maintain Z-disc stability in this organism. The giant sarcomeric protein titin has also been implicated in the assembly and maintenance of the Z-disc structure, whereas the specific contributions of other Z-disc components are currently unknown (Zou et al, 2006;Seeley et al, 2007).Z-discs contain the barbed-end capping protein CapZ. CapZ binds with high affinity (K d Ϸ 1 nM) to the barbed ends of actin filaments, in which it effectively inhibits actin polymerization and depolymerization (Caldwell et al, 1989).…”

Reduction of caldesmon expression induces apoptosis and causes disassembly of the sarcomeric protein complex in cardiomyocytes in vivo

“…Actin and tropomyosin are essential components of the thin filaments. 8,9 Alpha-actinin is an actin-binding protein and one of the major structural elements of the Z-disc, interacting with tropomyosin. 10 All of these proteins are mutually bound as a complex and reduced expression of one of them causes instability or degradation of the others.…”