Reduced thin filament length in nebulin-knockout skeletal muscle alters isometric contractile properties

Abstract

Gokhin DS, Bang ML, Zhang J, Chen J, Lieber RL. Reduced thin filament length in nebulin-knockout skeletal muscle alters isometric contractile properties. Am J Physiol Cell Physiol 296: C1123-C1132, 2009. First published March 18, 2009 doi:10.1152/ajpcell.00503.2008 is a large, rod-like protein believed to dictate actin thin filament length in skeletal muscle. NEB gene defects are associated with congenital nemaline myopathy. The functional role of NEB was investigated in gastrocnemius muscles from neonatal wild-type (WT) and NEB knockout (NEB-KO) mice, whose thin filaments have uniformly shorter lengths compared with WT mice. Isometric stress production in NEB-KO skeletal muscle was reduced by 27% compared with WT skeletal muscle on postnatal day 1 and by 92% on postnatal day 7, consistent with functionally severe myopathy. NEB-KO muscle was also more susceptible to a decline in stress production during a bout of 10 cyclic isometric tetani. Length-tension properties in NEB-KO muscle were altered in a manner consistent with reduced thin filament length, with length-tension curves from NEB-KO muscle demonstrating a 7.4% narrower functional range and an optimal length reduced by 0.13 muscle lengths. Expression patterns of myosin heavy chain isoforms and total myosin content did not account for the functional differences between WT and NEB-KO muscle. These data indicate that NEB is essential for active stress production, maintenance of functional integrity during cyclic activation, and lengthtension properties consistent with a role in specifying normal thin filament length. Continued analysis of NEB's functional properties will strengthen the understanding of force transmission and thin filament length regulation in skeletal muscle and may provide insights into the molecular processes that give rise to nemaline myopathy. neonatal mouse; isometric stress; myosin heavy chain; length-tension curve FORCE GENERATION in skeletal muscle results from the interdigitation of actin (thin) filaments and myosin (thick) filaments in the sarcomeres of the myofibrillar lattice. The magnitude of active force production is predicted by the sliding filament model, which states that active muscle force is proportional to the degree of thin and thick filament overlap (15, 16). The length-tension relationship quantifies the relationship between myofilament overlap and force production and is determined by eliciting isometric tetani at a discrete series of lengths (7,9,10,14). Myofilaments are polymeric, but their lengths are controlled precisely during sarcomere assembly and are highly uniform within a fiber (6, 24). Myofilament length varies across muscle type and species (11,13,43,44,47), with resultant functional consequences on the shape of the lengthtension curve (11). Therefore, the molecular basis of myofilament length regulation has substantial physiological implications.Previous studies have demonstrated that thin filament length is partly controlled by the action of "capping" proteins. At the thin filament barbed end (anchore...

Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation

“…reduced from ϳ80 mN/mm 2 in WT to ϳ40 mN/mm 2 in NEB KO fibers. This ϳ50% reduction is less than the Ͼ90% reduction (from ϳ60 to ϳ 5 mN/mm 2 ) that was recently reported for intact gastrocnemius muscle of NEB KO mice at postnatal day 7 (19). A possible explanation for the discrepancy is that depressed excitation-contraction coupling lowered the activating calcium level in the intact muscle experiments, an effect that would be absent in skinned fiber experiments where calcium is controlled at a saturating level.…”

Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

“…This property of skeletal muscle has been demonstrated directly in vivo in adult fish, in which fast-and slowtwitch muscles have distinctly different thin filament lengths . This property has also been demonstrated in nebulin-null mice, whose skeletal muscle thin filaments are substantially shorter and produce optimal force at a shorter sarcomere length compared with their wild-type counterparts (Gokhin et al, 2009;Ottenheijm et al, 2009). Based on these previous experiments, and the fact that thin filaments from Tmod1-null muscles are of normal length (Table I), we conclude that the optimal sarcomere length range for force production does not differ between Tmod1 +/+ and Tmod1 /Tg+ muscles.…”

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse

“…This suggests a role of the nebulin SH3 domain in excitation-contraction coupling, either through slower Ca 2+ release or faster Ca 2+ uptake. Ablation of nebulin has previously been shown to result in a dramatic upregulation of sarcolipin, a sarcoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor (Gokhin et al, 2009;Ottenheijm et al, 2008;Witt et al, 2006). However, no changes in sarcolipin mRNA levels were found in the gene expression analysis.…”

“…The majority of the protein is composed of repeating modules, which interact with thin filament components along the length of the thin filament, whereas its N-and C-terminal regions contain unique sequences that bind to proteins at the actin filament pointed end and the Z-line, respectively. Based on in vivo and in vitro studies, nebulin has recently been implicated in various important processes, including stabilization of thin filaments (Bang et al, 2006;Castillo et al, 2009;Gokhin and Fowler, 2013;Littlefield and Fowler, 2008;Pappas et al, 2010;Witt et al, 2006), myofibrillar force generation (Bang et al, 2006;Gokhin et al, 2009;Ochala et al, 2011;Ottenheijm et al, 2010;Ottenheijm et al, 2009), regulation of the actomyosin interaction Castillo et al, 2009;Ochala et al, 2011;Ottenheijm et al, 2011), sarcoplasmic Ca 2+ handling (Ottenheijm et al, 2008;Witt et al, 2006), maintenance of sarcomeric integrity during muscle contraction (Bang et al, 2006), as well as Z-line alignment, width and integrity (Bang et al, 2006;Pappas et al, 2010;Tonino et al, 2010;Witt et al, 2006). However, although recent studies have provided new insights into the role of nebulin in skeletal muscle, its function in the Z-line remains poorly understood.…”