Depletion of zebrafish Tcap leads to muscular dystrophy via disrupting sarcomere–membrane interaction, not sarcomere assembly

Abstract

Tcap/telethonin encodes a Z-disc protein that plays important roles in sarcomere assembly, sarcomere-membrane interaction and stretch sensing. It remains unclear why mutations in Tcap lead to limb-girdle muscular dystrophy 2G (LGMD2G) in human patients. Here, we cloned tcap in zebrafish and conducted genetic studies. We show that tcap is functionally conserved, as the Tcap protein appears in the sarcomeric Z-disc and reduction of Tcap resulted in muscular dystrophy-like phenotypes including deformed muscle structure and impaired swimming ability. However, the observations that Tcap integrates into the sarcomere at a stage after the Z-disc becomes periodic, and that the sarcomere remains intact in tcap morphants, suggest that defective sarcomere assembly does not contribute to this particular type of muscular dystrophy. Instead, a defective interaction between the sarcomere and plasma membrane was detected, which was further underscored by the disrupted development of the T-tubule system. Pertinent to a potential function in stretch sensor signaling, zebrafish tcap exhibits a variable expression pattern during somitogenesis. The variable expression is inducible by stretch force, and the expression level of Tcap is negatively regulated by integrin-link kinase (ILK), a protein kinase that is involved in stretch sensing signaling. Together, our genetic studies of tcap in zebrafish suggested that pathogenesis in LGMD2G is due to a disruption of sarcomere-T-tubular interaction, but not of sarcomere assembly per se. In addition, our data prompted a novel hypothesis that predicts that the transcription level of Tcap can be regulated by the stretch force to ensure proper sarcomere-membrane interaction in striated muscles.

Distinct roles for telethonin N‐versus C‐terminus in sarcomere assembly and maintenance

“…In this study, knocking down endogenous telethonin resulted in paralyzed embryos, trunk curvature, myocyte defects, and significantly reduced sarcomere formation. Our results in Xenopus are consistent with a recent telethonin knockdown study in zebrafish, in which morphant embryos also displayed trunk curvature, loss of motility, and disordered myofibrils and sarcomeres (Zhang et al, 2009). In both studies, myofibrils appear wavy and thin, and sarcomeres are reduced in number and/ or are disordered.…”

“…Together, these studies clearly indicate telethonin is required for muscle development and/ or maintenance of muscle function. However, based on the presence of disordered, but otherwise relatively normal, sarcomeres in the morphant myocytes, Zhang et al (2009) conclude that telethonin is not required for de novo assembly. We believe this discrepancy in results can be explained by disparities in the degree of knockdown and other experimental differences.…”

“…On one hand, studies supporting the importance of the telethonin/titin interaction during de novo myofibrillogenesis include overexpression of N-terminal titin (residues 1-362), Z-disc titin (Z1-Z2 repeats, residues 1-200), telethonin, or C-terminal a-actinin missing its titin-binding domain, all of which result in severe myofibril disruption (Turnacioglu et al, 1997;Peckham et al, 1997;Gregorio et al, 1998;Lin et al, 1998). On the other hand, telethonin is incorporated into the Z-disc late during myofibrillogenesis (Wang et al, 2005;Sanger et al, 2009;Zhang et al, 2009), and once incorporated has very low mobility as assessed with FRAP measurements (Wang et al, 2005, Sanger et al, 2009). In addition, truncation mutations in telethonin are associated with a late-onset limb-girdle muscular dystrophy (LGMD 2G), and muscle biopsies from LGMD 2G patients are immunonegative for telethonin (Moreira et al, 2000;Olivé et al, 2008).…”

“…In C 2 C 12 cells, RNAi knockdown of telethonin significantly reduced expression of key myogenic regulatory factors, presumably mediated by telethonin/myostatin interactions (Markert et al 2008). In zebrafish, T-cap knockdown disrupted development of the T-tubule system and altered myofibril and myoseptum continuity (Zhang et al 2009). In Xenopus, transverse T-tubule development does not occur within the first 48 hr in culture (unpublished data), well after developed myofibrils and sarcomeres are present.…”

“…However, late onset Limb Girdle Muscular Dystrophy type 2G (LGMD 2G) is associated with telethonin truncation mutations, suggesting that telethonin is dispensable for early sarcomere formation and muscle development (Moreira et al, 2000;Olivé et al, 2008). Furthermore, a recent Tcap knockdown study in zebrafish concluded that telethonin is not required for sarcomere assembly (Zhang et al, 2009). It thus remains unclear whether telethonin has a role in de novo sarcomere assembly versus maintenance.…”