Maternal folic acid supplementation does not impact skeletal muscle function and metabolism in male and female CD-1 mouse offspring.

Folic acid fortification of all white flour, enriched pasta, and cornmeal products became mandatory in Canada to reduce risk of neural tube defects at birth. Furthermore, Health Canada and the Society of Obstetricians and Gynaecologists of Canada recommend women take daily prenatal folic acid supplements in addition to folic acid fortified foods during pregnancy. However, the influence of maternal folic acid supplementation on offspring development, specifically the highly abundant and metabolically active skeletal muscle, is currently unknown. Thus, the purpose of this study was to determine the effect of supplemental folic acid (four times higher than normal dietary consumption), in utero and throughout suckling on muscle size, function, and metabolism in male and female CD-1 mouse offspring. The major findings were that maternal exposure to supplemental folic acid (i) had no impact on postpartum growth rates or muscle mass in female and male offspring, (ii) had no impact on skeletal muscle contractile kinetics in females and male offspring, and (iii) increased maximal phosphofructokinase activity in extensor digitorum longus of female and male offspring. These findings suggest that exposure to folic acid supplementation in utero and throughout suckling at levels four times higher than recommended had minimal effect on skeletal muscle size, function, and metabolism regardless of sex. Future research is needed explore the underlying biological pathways and mechanisms affected by folic acid supplementation during pregnancy and lactation on offspring skeletal muscle tissue, specifically in humans.

Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation.

Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK-/-) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK-/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK-/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK-/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK-/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK-/- muscles without RLC phosphorylation.

Myosin phosphorylation potentiates steady-state work output without altering contractile economy of mouse fast skeletal muscles.

Skeletal myosin light chain kinase (skMLCK)-catalyzed phosphorylation of the myosin regulatory light chain (RLC) increases (i.e. potentiates) mechanical work output of fast skeletal muscle. The influence of this event on contractile economy (i.e. energy cost/work performed) remains controversial, however. Our purpose was to quantify contractile economy of potentiated extensor digitorum longus (EDL) muscles from mouse skeletal muscles with (wild-type, WT) and without (skMLCK ablated, skMLCK-/-) the ability to phosphorylate the RLC. Contractile economy was calculated as the ratio of total work performed to high-energy phosphate consumption (HEPC) during a period of repeated isovelocity contractions that followed a potentiating stimulus (PS). Consistent with genotype, the PS increased RLC phosphorylation measured during, before and after isovelocity contractions in WT but not in skMLCK-/- muscles (i.e. 0.65 and 0.05 mol phosphate mol-1 RLC, respectively). In addition, although the PS enhanced work during repeated isovelocity contractions in both genotypes, the increase was significantly greater in WT than in skMLCK-/- muscles (1.51±0.03 versus 1.10±0.05, respectively; all data P<0.05, n=8). Interestingly, the HEPC determined during repeated isovelocity contractions was statistically similar between genotypes at 19.03±3.37 and 16.02±3.41 µmol P; respectively (P<0.27). As a result, despite performing significantly more work, the contractile economy calculated for WT muscles was similar to that calculated for skMLCK-/- muscles (i.e. 5.74±0.67 and 4.61±0.71 J kg-1 µmol-1 P, respectively (P<0.27). In conclusion, our results support the notion that myosin RLC phosphorylation enhances dynamic contractile function of mouse fast skeletal muscle but does so without decreasing contractile economy.

Influence of longitudinal radiation exposure from microcomputed tomography scanning on skeletal muscle function and metabolic activity in female CD-1 mice.

Microcomputed tomography (µCT) is an imaging technology to assess bone microarchitecture, a determinant of bone strength. When measured in vivo, µCT exposes the skeletal site of interest to a dose of radiation, in addition to nearby skeletal muscles as well. Therefore, the aim of this study was to determine the effects of repeated radiation exposure from in vivo µCT on muscle health - specifically, muscle morphometrics, contractile function, and enzyme activity. This study exposed the right hind limb of female mice to either a low (26 cGy) or moderate (46 cGy) dose, at 2, 4, and 6 months of age, while the left hind limb of the same animal was exposed to a single dose at 6 months to serve as a nonirradiated control. Muscle weight, cross-sectional area, isometric contractile function, and representative maximal enzyme activities of amino acid, fatty acid, glucose, and oxidative metabolism in extensor digitorum longus (EDL) and soleus were assessed. Low-dose radiation had no effect. In contrast, moderate-dose radiation resulted in a 5% increase in time-to-peak tension and 16% increase in half-relaxation time of isometric twitches in EDL, although these changes were not seen when normalized to force. Moderate-dose radiation also resulted in an ~33% decrease in citrate synthase activity in soleus but not EDL, with no changes to the other enzymes measured. Thus, three low doses of radiation over 6 months had no effect on contractile function or metabolic enzyme activity in soleus and EDL of female mice. In contrast, three moderate doses of radiation over 6 months induced some effects on metabolic enzyme activity in soleus but not EDL Future studies that wish to investigate muscle tissue that is adjacent to scanned bone should take radiation exposure dose into consideration.

Shortening speed dependent force potentiation is attenuated but not eliminated in skeletal muscles without myosin phosphorylation.

We investigated the influence of shortening speed on concentric force potentiation at different frequencies in muscles devoid of skeletal myosin light chain kinase (skMLCK-/-) and unable to phosphorylate myosin. EDL muscles from skMLCK-/- mice were activated in vitro (25 °C) across a range of stimulation frequencies (10-100 Hz) during shortening ramps at 0.10, 0.30, or 0.50 of maximum shortening velocity (Vmax) before and after a potentiating stimulus (PS). When collapsed across all frequencies, the PS increased relative (post/pre) concentric force to 1.27 ± 0.02 and 1.17 ± 0.02 of pre-PS values at 0.50 and 0.30 Vmax, respectively (n = 4, P < 0.05 for all speeds). In addition, potentiation was significantly greater at low and intermediate-than at high stimulus frequencies at both speeds. In contrast, during shortening at 0.10 Vmax, a posttetanic depression was observed as mean concentric forces were reduced to 0.85 ± 0.02 of pre-PS values. Thus, although reduced compared to published values for wildtype muscles (Gittings et al., J Muscle Res Cell Motil 33:359-368, 2012), skMLCK-/- muscles displayed a speed dependent potentiation of concentric force during moderate and fast shortening speed at all frequencies tested. Our data support the presence of a myosin phosphorylation-independent mechanism(s) for concentric force potentiation at moderate speeds of shortening, and also suggests that myosin phosphorylation may be necessary to prevent the concentric force depression that may be present at slow speeds of shortening. Although additive in nature, further work is needed to parse out the relative influence of myosin phosphorylation-independent and dependent potentiation mechanisms on wildtype contractile function during dynamic conditions.

Interaction of posttetanic potentiation and the catchlike property in mouse skeletal muscle.

INTRODUCTION: Posttetanic potentiation (PTP) and the catchlike property (CLP) enhance contractile function in skeletal muscle. We investigated the CLP during dynamic performance in mouse hindlimb muscles with (wild-type) and without (skMLCK(-/-) ) the primary mechanism for PTP (myosin phosphorylation) (in vitro, 25°C). METHODS: Extensor digitorum longus muscles of both genotypes were stimulated with constant frequency and catchlike trains (CFT and CLT), before and after a potentiating stimulus (PS). RESULTS: Before the PS, the CLT increased concentric force/work relative to the CFT, but this effect was greater for skMLCK(-/-) than wild-type muscles. After the PS, the catchlike effect was reduced in wild-type muscles but unchanged in skMLCK(-/-) muscles that did not display PTP. CONCLUSIONS: These data suggest that PTP interferes with the CLP during concentric force development at moderate speeds of shortening. We conclude that the physiological utility of each mechanism and their interactions provide important modulations to fast skeletal muscle function. Muscle Nerve 54: 308-316, 2016.