Gene Transfer of Skeletal Muscle-Type Myosin Light Chain Kinase via Adeno-Associated Virus 6 Improves Muscle Functions in an Amyotrophic Lateral Sclerosis Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients' quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.

X-ray Diffraction Analysis to Explore Molecular Traces of Eccentric Contraction on Rat Skeletal Muscle Parallelly Evaluated with Signal Protein Phosphorylation Levels.

We performed X-ray diffraction analyses on rat plantaris muscle to determine if there are strain-specific structural changes at the molecular level after eccentric contraction (ECC). ECC was elicited in situ by supramaximal electrical stimulation through the tibial nerve. One hour after a series of ECC sessions, the structural changes that remained in the sarcomere were evaluated using X-ray diffraction. Proteins involved in cell signaling pathways in the muscle were also examined. ECC elicited by 100, 75, and 50 Hz stimulation respectively developed peak tension of 1.34, 1.12 and 0.79 times the isometric maximal tetanus tension. The series of ECC sessions phosphorylated the forkhead box O proteins (FoxO) in a tension-time integral-dependent manner, as well as phosphorylated the mitogen-activated protein kinases (MAPK) and a protein in the mammalian target of rapamycin (mTOR) pathway in a maximal tension dependent manner. Compared to isometric contractions, ECC was more efficient in phosphorylating the signaling proteins. X-ray diffraction revealed that the myofilament lattice was preserved even after intense ECC stimulation at 100 Hz. Additionally, ECC < 75 Hz preserved the molecular alignment of myoproteins along the myofilaments, while 75-Hz stimulation induced a slight but significant decrease in the intensity of meridional troponin reflection at 1/38 nm-1, and of myosin reflection at 1/14.4 nm-1. These two reflections demonstrated no appreciable decrease with triple repetitions of the standard series of ECC sessions at 50 Hz, suggesting that the intensity decrease depended on the instantaneous maximal tension development rather than the total load of contraction, and was more likely linked with the phosphorylation of MAPK and mTOR signaling proteins.

Crossbridge Recruitment Capacity of Wild-Type and Hypertrophic Cardiomyopathy-Related Mutant Troponin-T Evaluated by X-ray Diffraction and Mechanical Study of Cardiac Skinned Fibers.

X-ray diffraction and tension measurement experiments were conducted on rat left ventricular skinned fibers with or without "troponin-T treatment," which exchanges the endogenous troponin T/I/C complex with exogenous troponin-T. These experiments were performed to observe the structural changes in troponin-T within a fiber elicited by contractile crossbridge formation and investigate the abnormality of hypertrophic cardiomyopathy-related troponin-T mutants. The intensity of the troponin reflection at 1/38.5 nm-1 was decreased significantly by ATP addition after treatment with wild-type or mutant troponin-T, indicating that crossbridge formation affected the conformation of troponin-T. In experiments on cardiac fibers treated with the hypertrophic cardiomyopathy-related mutants E244D- and K247R-troponin-T, treatment with K247R-troponin-T did not recruit contracting actomyosin to a greater extent than wild-type-troponin-T, although a similar drop in the intensity of the troponin reflection occurred. Therefore, the conformational change in K247R-troponin-T was suggested to be unable to fully recruit actomyosin interaction, which may be the cause of cardiomyopathy.

Myosin and tropomyosin-troponin complementarily regulate thermal activation of muscles.

Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy. Rapid heating (25 to 41.5°C) within 2 s induced reversible sarcomere shortening in relaxing solution. Further, we investigated the temperature-dependence of the sliding velocity of reconstituted fast skeletal or cardiac thin filaments on fast skeletal or ß-cardiac myosin in an in vitro motility assay within the body temperature range. We found that (a) with fast skeletal thin filaments on fast skeletal myosin, the temperature dependence was comparable to that obtained for sarcomere shortening in fast skeletal myofibrils (Q10 ∼8), (b) both types of thin filaments started to slide at lower temperatures on fast skeletal myosin than on ß-cardiac myosin, and (c) cardiac thin filaments slid at lower temperatures compared with fast skeletal thin filaments on either type of myosin. Therefore, the mammalian striated muscle may be fine-tuned to contract efficiently via complementary regulation of myosin and tropomyosin-troponin within the body temperature range, depending on the physiological demands of various circumstances.