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An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2 D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . We show that myoblasts not only responded to 1,25(OH)2 D3 , but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2 D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2 D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2 D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2 D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . J. Cell. Physiol. 231: 2517-2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc., (© 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

It has been suggested that the number of myonuclei in a muscle fibre changes in proportion to the change in fibre size, resulting in a constant myonuclear domain size, defined as the cytoplasmic volume per myonucleus. The myonuclear domain size varies, however, between fibre types and is inversely related with the oxidative capacity of a fibre. Overall, the observations of an increase in myonuclear domain size during both maturational growth and overload-induced hypertrophy, and the decrease in myonuclear domain size during disuse- and ageing-associated muscle atrophy suggest that the concept of a constant myonuclear domain size needs to be treated cautiously. It also suggests that only when the myonuclear domain size exceeds a certain threshold during growth or overload-induced hypertrophy acquisition of new myonuclei is required for further fibre hypertrophy.

Vitamin D deficiency is associated with muscle weakness. It is unknown, however, how supra-physiological levels of vitamin D affect skeletal muscle. To investigate the effects of increased serum vitamin D (1,25 (OH)₂D₃ or 1,25D) levels on the contractile properties of the medial gastrocnemius muscle, adult and old female Fischer₃₄₄ x Brown Norway F1 rats were orally treated with vehicle or the vitamin D analogue alfacalcidol for 1 or 6 weeks. Alfacalcidol treatment resulted in elevated 1,25D serum levels. This was accompanied by hypercalcaemia and a reduction in body mass, the latter largely attributable to a reduced food intake. However, kidney function, as reflected by normal creatinine serum levels, as well as heart mass were unaffected. The 17% reduction in maximal isometric force and power was explicable by a similar loss of muscle mass. The force-frequency relationship of the 6-week-treated old rats was shifted to the left, but neither the shape of the force-velocity relationship nor the fatigability of the muscle were altered. Supra-physiological doses of vitamin D were accompanied by significant reductions in body and muscle mass, but not by an improvement in muscle functioning. Weight loss was largely due to a reduced food intake, while the left shift in the force-frequency relation may be due to increased 1,25D levels.

Intervention with the continuity of the tendon and part of the muscle fibers allows investigation of myofascial force transmission. The present study investigates the effects of proximal aponeurotomy on length-force characteristics and the geometry of the extensor digitorum longus (EDL) muscle, and compares those effects with the effects of both distal tenotomy (TT) and intramuscular fasciotomy (IF) of the EDL. After proximal aponeurotomy, the intramuscular connective tissue ruptured spontaneously below the location of intervention. Due to this rupturing, a gap developed within the proximal aponeurosis. The fibers that were continuous with the tendon at only one end were substantially shorter than before the intervention. Optimum muscle force was reduced by 29%. After distal TT (of heads II-IV) a gap developed within the muscle belly. This gap increased at higher muscle lengths. However, the length of the gap was much smaller than after aponeurotomy. Despite the TT-related gap, there was no rupturing of intramuscular connective tissue at the interface between heads IV and V, as there was after proximal aponeurotomy. The effects of TT on length-force characteristics and on lengths of fibers continuous with the tendon at only one end were much less compared to the effects of aponeurotomy. Subsequent IF for two-thirds the length of the interface between heads IV and V resulted in changes similar to the effects of proximal aponeurotomy plus rupture. The contrast regarding the occurrence of intramuscular connective tissue rupture indicates increased failure strength of the intramuscular connective tissue at distal locations. It is hypothesized that for multitendoned muscles in vivo, local shear and stress deformations will initiate local adaptation of the intramuscular connective tissue., (Copyright 2002 Wiley-Liss, Inc.)

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 microm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.

Intramuscular aponeurotic lengthening of muscles or intramuscular tenotomy involves bisecting the connective tissue fibers of the aponeurosis or tendon within the muscle belly. Because of its superficial location in the muscle, the aponeurosis may be bisected without damaging muscle fibers. Despite the existence of common operative methods for gaining length in short muscles, the effects on force and muscle function have not been studied. For this purpose animal experiments were performed. The medial gastrocnemius muscle of six male Wistar rats was lengthened by cutting the proximal aponeurosis at 50% of its length perpendicularly to the collagen fibers. The length gain was maintained by 3 days of cast immobilization at maximal dorsiflexion of the ankle. The long-term effect of the treatment was studied after 6 weeks and compared with 10 untreated controls and with six sham operated animals. The muscle was isolated in situ, and the force length characteristics were determined. In the untreated controls, the aponeurotomy was performed and the length force experiment was repeated to study the acute effects. The aponeurotic lengthening led acutely to a temporary loss of force because of an incomplete connection of the distal part of the muscle to the proximal insertion, but force recovered completely within 6 weeks. Although results from animal experiments cannot be transferred directly to humans, the principles of physiology are similar. Thus, for clinical use, aponeurotic lengthening should be considered if muscle force needs to be preserved. However, the drop of muscle force after surgery must be respected when mobilizing the patient during the postoperative rehabilitation program.