Recordings of action potentials, charge movements, and sarcoplasmic reticulum Ca2+ release in isolated adult zebrafish fast skeletal muscle fibers reveals very fast kinetics of excitation–contraction coupling

The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation–contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2−/−) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2−/− mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2−/− mice. Small-angle X-ray diffraction of C2−/− EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2−/− EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2−/− mice. Finally, C2−/− muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

ECC meets CEU—New focus on the backdoor for calcium ions in skeletal muscle cells

In this issue, Michelucci et al. report the existence of specific sites acting as Ca2+ entry units (CEUs) in fast skeletal muscle of mice lacking calsequestrin (CASQ1), the major Ca2+ binding protein of the SR. The CEU provides constitutive and store-operated Ca2+ entry (SOCE) and resistance to force decline resulting from SR Ca2+ depletion during repetitive muscle activity.

Mutations in MYLPF cause a novel segmental amyoplasia that manifests as distal arthrogryposis

We identified ten persons in six consanguineous families with Distal Arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T, p.(Cys157Phe) or c.469T>C, p.(Cys157Arg)) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A, p.(Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly-inherited DA, a c.98C>T, p.(Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

CARDIAC TROPONIN T MEDIATED AUTOIMMUNE RESPONSE AND ITS ROLE IN SKELETAL MUSCLE AGING

Cardiac troponin T (cTnT), a key component of contractile machinery essential for muscle contraction, is also expressed in skeletal muscle under certain conditions (e.g. neuromuscular diseases and aging). We have reported that skeletal muscle cTnT regulates neuromuscular junction denervation preferentially in fast skeletal muscle of old mice. Here, we further report that cTnT is also enriched within some myofibers, and/or along microvascular walls in old mice fast skeletal muscle. Strikingly, immunoglobulin G (IgG), together with markers of complement system activation, cell death (necroptosis or apoptosis), and macrophage infiltration, were all found to be co-localized with cTnT and IgG in those areas. In addition, elevated cTnT and IgG are associated with lower dystrophin expression on muscle fiber membrane, lower muscle capillary density, and reduced muscle performance (wire hanging test). Using purified recombinant TnT proteins, we confirmed that only cTnT, but not slow or fast skeletal muscle TnT1 or TnT3, was detected by immunoblot using sera from old (but not young) mice with pre-determined elevated cTnT and IgG in their skeletal muscle, indicating the existence of anti-cTnT autoantibodies in sera (previously found in human blood) and skeletal muscle of old mice. Immunoblotting further revealed that the age related changes in skeletaI muscle cTnT and IgG are more prominent in fast skeletal muscle than in slow. Importantly, elevated cTnT and IgG were also detected in skeletal muscles from 4 older adults (65-70 yrs, IMFIT). Our finding suggests a novel autoimmune mechanism mediated by cTnT that underlies age related skeletal muscle abnormalities and dysfunction.

Functional Characterization of the Intact Diaphragm in a Nebulin-Based Nemaline Myopathy (NM) Model-Effects of the Fast Skeletal Muscle Troponin Activator tirasemtiv

Respiratory failure due to diaphragm dysfunction is considered a main cause of death in nemaline myopathy (NM) and we studied both isometric force and isotonic shortening of diaphragm muscle in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was found in nebulin-deficient intact muscle that is frequency dependent, with the largest deficits at low–intermediate stimulation frequencies (e.g., a deficit of 72% at a stimulation frequency of 20 Hz). The effect of the fast skeletal muscle troponin activator (FSTA) tirasemtiv on force was examined. Tirasemtiv had a negligible effect at maximal stimulation frequencies, but greatly reduced the force deficit of the diaphragm at sub-maximal stimulation levels with an effect that was largest in Neb cKO diaphragm. As a result, the force deficit of Neb cKO diaphragm fell (from 72% to 29% at 20 Hz). Similar effects were found in in vivo experiments on the nerve-stimulated gastrocnemius muscle complex. Load-clamp experiments on diaphragm muscle showed that tirasemtiv increased the shortening velocity, and reduced the deficit in mechanical power by 33%. Thus, tirasemtiv significantly improves muscle function in a mouse model of nebulin-based nemaline myopathy.