Optimization of post-activation potentiation in girls and women.

BACKGROUND: Maximal conditioning contractions (CCs) can lead to the enhancement of evoked-twitch characteristics in human skeletal muscle. This phenomenon is termed post-activation potentiation (PAP). In the knee extensors, PAP is greater in men compared with boys. In adults, the optimal CC duration for PAP is ~ 10 s. We examined child-adult differences in PAP among females and aimed to determine the optimal CC duration in girls and women. METHODS: Eleven girls (9.3 ± 1.4 years) and 13 women (23.4 ± 2.7 years) participated in this study. Maximal isometric evoked twitches were recorded in the knee extensors before and after 4 maximal CCs of different durations (5, 10, 20, and 30 s), in a random order. PAP was calculated as the percent-change in peak torque (Tpeak) and peak rate of torque development (RTDpeak) after each CC. RESULTS: There was a group-by-duration interaction (p < 0.001), reflecting greater Tpeak PAP in women compared with girls following 5 and 10 s CCs, and lower RTDpeak PAP in women following the 30 s CC. The 5 and 10 s CCs lead to the greatest Tpeak and RTDpeak PAP amongst the women while there were no differences between CC durations in girls. CONCLUSION: After both a 5 and 10 s CC, women have greater PAP compared with girls. The optimal CC duration for the knee extensors in women appears to be ~ 5-10 s, while CC durations between 5 and 30 s do not appear to affect levels of PAP in girls.

Characterizing the effects of muscle-specific GSK3α/ß reduction on murine muscle contractility and metabolism in female mice.

Dysregulation of skeletal muscle morphology and metabolism is associated with chronic diseases such as obesity and type 2 diabetes. The enzyme glycogen synthase kinase 3 (GSK3) is highly involved in skeletal muscle physiology and metabolism, acting as a negative regulator of muscle size, strength, adaptive thermogenesis, and glucose homeostasis. Correspondingly, we have shown that partial knockdown (∼40%) of GSK3 specifically in skeletal muscle increases lean mass, reduces fat mass, and activates muscle-based adaptive thermogenesis via sarco(endo)plasmic reticulum Ca2+ (SERCA) uncoupling in male mice. However, the effects of GSK3 knockdown in female mice have yet to be investigated. Here, we examined the effects of muscle-specific GSK3 knockdown on body composition, muscle size and strength, and whole body metabolism in female C57BL/6J mice. Our results show that GSK3 content is higher in the female soleus versus the male soleus; however, there were no differences in the extensor digitorum longus (EDL). Furthermore, muscle-specific GSK3 knockdown did not alter body composition in female mice, nor did it alter daily energy expenditure, glucose/insulin tolerance, mitochondrial respiration, or the expression of the SERCA uncouplers sarcolipin and neuronatin. We also did not find any differences in soleus muscle size, strength, or fatigue resistance. In the EDL, we found that an increase in absolute and specific force production, but there were no differences in fatigability. Therefore, our study highlights sex differences in the response to genetic reduction of gsk3, with most of the effects previously observed in male mice being absent in females.NEW & NOTEWORTHY Here we show that partial GSK3 knockdown has minimal effects on whole body metabolism and muscle contractility in female mice. This is partly inconsistent with previous results found in male mice, which reveal a potential influence of biological sex.

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.

Post-activation potentiation and potentiated motor unit firing patterns in boys and men.

BACKGROUND: Post-activation potentiation (PAP) describes the enhancement of twitch torque following a conditioning contraction (CC) in skeletal muscle. In adults, PAP may be related to muscle fibre composition and is accompanied by a decrease in motor unit (MU) firing rates (MUFRs). Muscle fibre composition and/or activation is different between children and adults. This study examined PAP and MU firing patterns of the potentiated knee extensors in boys and men. METHODS: Twenty-three boys (10.5 ± 1.3 years) and 20 men (23.1 ± 3.3 years) completed familiarization and experimental sessions. Maximal isometric evoked-twitch torque and MU firing patterns during submaximal contractions (20% and 70% maximal voluntary isometric contraction, MVIC) were recorded before and after a CC (5 s MVIC). PAP was calculated as the percent-increase in evoked-twitch torque after the CC. MU firing patterns were examined during submaximal contractions before and after the CC using Trigno Galileo surface electrodes (Delsys Inc) and decomposition algorithms (NeuroMap, Delsys Inc). MU action potential amplitudes (MUAPamp) and MUFRs were calculated for each MU and exponential MUFR-MUAPamp relationships were calculated for each participant and trial. RESULTS: PAP was higher in men than in boys (98.3 ± 37.1% vs. 68.8 ± 18.3%, respectively; p = 0.002). Following potentiation, the rate of decay of the MUFR-MUAPamps relationship decreased in both contractions, with a greater decrease among boys during the high-intensity contractions. CONCLUSION: Lower PAP in the boys did not coincide with smaller changes in potentiated MU firing patterns, as boys had greater reductions in MUFRs with potentiation compared with men in high-intensity contractions.

Potentiation of force by extracellular potassium is not dependent on muscle length in mouse EDL muscle.

Increases in myofiber extracellular potassium with prolonged contractile activity can potentiate twitch force. Activity-dependent potentiation, another mechanism of force increase in skeletal muscle, has a strong dependence on muscle or sarcomere length. Thus, potassium-mediated twitch potentiation could also be length-dependent. However, this has not been previously investigated. To this end, we used isolated C57BL/6 mouse extensor digitorum longus (EDL) muscles and elicited twitches at 0.9 Lo, Lo, and 1.1 Lo (Lo refers to optimal length) in normal (5 mM) and high (10 mM) potassium solutions. Potentiation magnitude was similar to previous observations and was not significantly different between lengths (0.9 Lo: 12.3 ± 4.4%, Lo: 12.2 ± 3.6%, 1.1 Lo: 11.8 ± 4.8%, values are means ± SD). Exposure to dantrolene sodium, a compound that attenuates calcium release, reduced twitch force across lengths by ∼70%. When dantrolene-affected muscles were subsequently exposed to high potassium, potentiation was similar to that observed in the absence of the former. In total, these findings provide novel information on potassium-mediated twitch potentiation.NEW & NOTEWORTHY Here, we investigated the length-dependence of twitch force potentiation by extracellular potassium in mouse extensor digitorum longus (EDL) in vitro, at 25°C. Potentiation magnitude did not display a statistically significant difference between the examined muscle lengths. These results describe, for the first time, the relationship of this form of potentiation with muscle length, thus furthering the understanding of how it is integrated in in vivo muscle function.

Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target.

We examined the effects of ∼30 days of spaceflight on glycogen synthase kinase 3 (GSK3) content and inhibitory serine phosphorylation in murine muscle and bone samples from four separate missions (BION-M1, rodent research [RR]1, RR9, and RR18). Spaceflight reduced GSK3ß content across all missions, whereas its serine phosphorylation was elevated with RR18 and BION-M1. The reduction in GSK3ß was linked to the reduction in type IIA fibers commonly observed with spaceflight as these fibers are particularly enriched with GSK3. We then tested the effects of inhibiting GSK3 before this fiber type shift, and we demonstrate that muscle-specific Gsk3 knockdown increased muscle mass, preserved muscle strength, and promoted the oxidative fiber type with Earth-based hindlimb unloading. In bone, GSK3 activation was enhanced after spaceflight; and strikingly, muscle-specific Gsk3 deletion increased bone mineral density in response to hindlimb unloading. Thus, future studies should test the effects of GSK3 inhibition during spaceflight.

Posttetanic potentiation improves neuromuscular efficiency of mouse muscle in vitro.

Neuromuscular efficiency is defined as the ratio of work output to stimulation rate. The purpose of these experiments was to test the hypothesis that neuromuscular efficiency would be increased in proportion to posttetanic potentiation, that is, the stimulation-induced increase in work output displayed by rodent fast-twitch muscle. To this end, extensor digitorum longus muscles from wild-type and skeletal myosin light chain kinase knockout (skMLCK-/- ) mice were surgically isolated and suspended in vitro (25°C). Concentric force development during shortening at 70% of maximal unloaded shortening velocity was tested at stimulus frequencies between 10 and 80 Hz both before and after a potentiating tetanus. A strong genotype-dependent difference in the potentiation of concentric work output was observed; concentric work output of wild-type muscles was increased by 51%-88% while that of skMLCK-/- muscles was increased by only 20%-34% across the frequencies tested. As a result, comparison of work - frequency plots revealed that the frequency required for peak and 50% peak unpotentiated work of wild-type muscles was decreased from ~80 to 52 Hz and from ~48 to 21 Hz, respectively. By contrast, the frequency required for peak and 50% peak unpotentiated work of skMLCK-/- muscles was decreased from ~80 to 68 Hz and from ~51 to 41 Hz, respectively. Thus, wild-type muscles with the ability to phosphorylate myosin displayed larger increases in neuromuscular efficiency than skMLCK-/- muscles (25-30 vs 10-15 Hz, respectively). This suggests that the presence of myosin phosphorylation may ameliorate or mitigate fatigue mechanisms associated with high-frequency stimulation rates.

Low-dose lithium supplementation promotes adipose tissue browning and sarco(endo)plasmic reticulum Ca2+ ATPase uncoupling in muscle.

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) uncoupling in skeletal muscle and mitochondrial uncoupling via uncoupling protein 1 (UCP1) in brown/beige adipose tissue are two mechanisms implicated in energy expenditure. Here, we investigated the effects of glycogen synthase kinase 3 (GSK3) inhibition via lithium chloride (LiCl) treatment on SERCA uncoupling in skeletal muscle and UCP1 expression in adipose. C2C12 and 3T3-L1 cells treated with LiCl had increased SERCA uncoupling and UCP1 protein levels, respectively, ultimately raising cellular respiration; however, this was only observed when LiCl treatment occurred throughout differentiation. In vivo, LiCl treatment (10 mg/kg/day) increased food intake in chow-fed diet and high-fat diet (HFD; 60% kcal)-fed male mice without increasing body mass-a result attributed to elevated daily energy expenditure. In soleus muscle, we determined that LiCl treatment promoted SERCA uncoupling via increased expression of SERCA uncouplers, sarcolipin and/or neuronatin, under chow-fed and HFD-fed conditions. We attribute these effects to the GSK3 inhibition observed with LiCl treatment as partial muscle-specific GSK3 knockdown produced similar effects. In adipose, LiCl treatment inhibited GSK3 in inguinal white adipose tissue (iWAT) but not in brown adipose tissue under chow-fed conditions, which led to an increase in UCP1 in iWAT and a beiging-like effect with a multilocular phenotype. We did not observe this beiging-like effect and increase in UCP1 in mice fed a HFD, as LiCl could not overcome the ensuing overactivation of GSK3. Nonetheless, our study establishes novel regulatory links between GSK3 and SERCA uncoupling in muscle and GSK3 and UCP1 and beiging in iWAT.

Mechanisms of Estrogen Influence on Skeletal Muscle: Mass, Regeneration, and Mitochondrial Function.

Human menopause is widely associated with impaired skeletal muscle quality and significant metabolic dysfunction. These observations pose significant challenges to the quality of life and mobility of the aging population, and are of relevance when considering the significantly greater losses in muscle mass and force-generating capacity of muscle from post-menopausal females relative to age-matched males. In this regard, the influence of estrogen on skeletal muscle has become evident across human, animal, and cell-based studies. Beneficial effects of estrogen have become apparent in mitigation of muscle injury and enhanced post-damage repair via various mechanisms, including prophylactic effects on muscle satellite cell number and function, as well as membrane stability and potential antioxidant influences following injury, exercise, and/or mitochondrial stress. In addition to estrogen replacement in otherwise deficient states, exercise has been found to serve as a means of augmenting and/or mimicking the effects of estrogen on skeletal muscle function in recent literature. Detailed mechanisms behind the estrogenic effect on muscle mass, strength, as well as the injury response are beginning to be elucidated and point to estrogen-mediated molecular cross talk amongst signalling pathways, such as apoptotic signaling, contractile protein modifications, including myosin regulatory light chain phosphorylation, and the maintenance of muscle satellite cells. This review discusses current understandings and highlights new insights regarding the role of estrogen in skeletal muscle, with particular regard to muscle mass, mitochondrial function, the response to muscle damage, and the potential implications for human physiology and mobility.

Neurogranin inhibits calcineurin in murine soleus muscle: Effects of heterozygous knockdown on muscle adaptations to tenotomy and fatigue resistance.

Neurogranin (Ng) is a calmodulin (CaM) binding protein that negatively regulates calcineurin - a Ca2+/CaM-dependent phosphatase that can mitigate the slow-to-fast fibre type shift observed with muscle unloading. Here, we questioned whether heterozygous deletion of Ng (Ng+/-) would enhance calcineurin activity, thereby minimizing the slow-to-fast fibre type shift caused by muscle unloading. As expected, soleus muscles from young adult (3-4 months old) Ng± mice had lowered Ng content and enhanced calcineurin activity when compared to soleus muscles obtained from male age-matched wild-type (WT) mice. Two weeks after tenotomy surgery, where the soleus and gastrocnemius tendons were severed, soleus total fibre count were found to be similarly reduced across both genotypes. However, significant reductions in myofibre cross-sectional area were only found in WT mice and not Ng± mice. Furthermore, while soleus muscles from both WT and Ng± mice exhibited a slow-to-fast fibre type shift with tenotomy, soleus muscles from Ng± mice, in both sham and tenotomized conditions, had a greater proportion of oxidative fibres (type I and IIA) compared with that of WT mice. Corresponding well with this, we found that soleus muscles from Ng± mice were more fatigue resistant compared with those obtained from their WT counterparts. Collectively, these findings show that heterozygous Ng deletion increases calcineurin activation, preserves myofibre size in response to unloading, and promotes the oxidative fibre type to ultimately enhance fatigue resistance. This study demonstrates the role of Ng in regulating calcineurin in vivo and its influence on skeletal muscle form and function.

The effect of muscle length on post-tetanic potentiation of C57BL/6 and skMLCK-/- mouse EDL muscles.

Post-tetanic potentiation of fast-twitch skeletal muscle is dependent on muscle length, with greater potentiation observed at shorter compared to longer lengths. The structural effects of the primary potentiation mechanism, phosphorylation of the regulatory light chain (RLC) of myosin, are thought to explain this relationship. The purpose of these experiments was to determine whether the length-dependence of potentiation would be attenuated in the absence of RLC phosphorylation. To this end, we compared isometric twitch potentiation of mouse extensor digitorum longus (EDL) muscles with (wildtype, WT) and without (skeletal myosin light chain kinase knockout, skMLCK-/-) phosphorylation. Force was measured at five muscle lengths (0.90 Lo, 0.95 Lo, Lo, 1.05 Lo, 1.10 Lo, where Lo refers to optimal length) prior to and following a tetanic train. In accordance with prior findings, potentiation was dependent on muscle length, with greater values observed at short (e.g., 44.3 ± 4.6% for WT, 33.5 ± 6.2% for skMLCK-/-, at 0.90 Lo) compared to long lengths (e.g., 16.9 ± 1.3% for WT, 9.1 ± 1.8% for skMLCK-/-, at 1.10 Lo) in both genotypes. WT muscles displayed greater potentiation compared to their skMLCK-/- counterparts across lengths (e.g., 16.9 ± 1.6% vs 7.3 ± 1.5% at Lo). However, the relationship between potentiation and muscle length was not different between genotypes. Thus, the alternative mechanisms of potentiation, present in the skMLCK-/- EDL, display a length-dependence of post-tetanic potentiation similar to RLC phosphorylation-dominant potentiation. Additional mechanisms may be required to explain the length-dependence of potentiation.

Potentiation of force by extracellular potassium and posttetanic potentiation are additive in mouse fast-twitch muscle in vitro.

The influence of moderately elevated extracellular potassium concentration ([K+]) on muscle force has marked similarities to that of posttetanic potentiation (PTP) in that twitch force may be enhanced whilst high-frequency force is depressed. The purpose of this work was to test whether K+-induced potentiation is mechanistically related to PTP via skeletal myosin light-chain kinase (skMLCK)-catalyzed phosphorylation of the myosin regulatory light chains (RLC). To do this, we assessed the influence of elevated [K+] on the force response at various frequencies in extensor digitorum longus (EDL) muscles isolated from wild-type and skeletal myosin light-chain kinase (skMLCK-/-) absent mice. Changing [K+] of the incubation medium from 5 to 10 mmol increased isometric twitch force by a similar amount in wild-type and skMLCK-/- muscles (~ 13% in both genotypes) (all data n = 7-8, P < 0.05). In contrast, 100- and 200-Hz forces were depressed in both genotypes (by 5-7 and 15-18%, respectively). The isometric twitch potentiation caused by a tetanic stimulus series was similar at both [K+] levels for each genotype but was much greater for wild-type than for skMLCK-/- muscles (i.e., 23-25 and 8-9%, respectively). Thus, we conclude that [K+]- and stimulation-induced potentiation are additive and that [K+]-induced potentiation is independent of RLC phosphorylation.

GSK3 inhibition with low dose lithium supplementation augments murine muscle fatigue resistance and specific force production.

Calcineurin is a Ca2+ -dependent serine/threonine phosphatase that dephosphorylates nuclear factor of activated T cells (NFAT), allowing for NFAT entry into the nucleus. In skeletal muscle, calcineurin signaling and NFAT activation increases the expression of proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) and slow myosin heavy chain (MHC) I ultimately promoting fatigue resistance. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that antagonizes calcineurin by re-phosphorylating NFAT preventing its entry into the nucleus. Here, we tested whether GSK3 inhibition in vivo with low dose lithium chloride (LiCl) supplementation (10 mg kg-1  day-1 for 6 weeks) in male C57BL/6J mice would enhance muscle fatigue resistance in soleus and extensor digitorum longus (EDL) muscles by activating NFAT and augmenting PGC-1α and MHC I expression. LiCl treatment inhibited GSK3 by elevating Ser9 phosphorylation in soleus (+1.8-fold, p = .007) and EDL (+1.3-fold p = .04) muscles. This was associated with a significant reduction in NFAT phosphorylation (-50%, p = .04) and a significant increase in PGC-1α (+1.5-fold, p = .05) in the soleus but not the EDL. MHC isoform analyses in the soleus also revealed a 1.2-fold increase in MHC I (p = .04) with no change in MHC IIa. In turn, a significant enhancement in soleus muscle fatigue (p = .04), but not EDL (p = .26) was found with LiCl supplementation. Lastly, LiCl enhanced specific force production in both soleus (p < .0001) and EDL (p = .002) muscles. Altogether, our findings show the skleletal muscle contractile benefits of LiCl-mediated GSK3 inhibition in mice.

Lack of influence of estrogen on myosin phosphorylation and post-tetanic potentiation in muscles from young adult C57BL mice.

Estrogen influences myosin phosphorylation and post-tetanic potentiation in murine fast muscle. We tested the hypothesis that this influence is mediated by estrogen effects on skeletal myosin light chain kinase (skMLCK) activity. To this end, extensor digitorum longus muscles from female wildtype and skMLCK-absent (skMLCK-/-) mice were grouped as follows: ovariectomized with estrogen (E+), ovariectomized without estrogen (E-), sham surgery, and intact baseline. At 8 weeks of age, the ovariectomized groups were ovariectomized followed by implantation of either a 0.1 mg 17ß-estradiol (E+) or placebo pellet (E-). Two weeks later, muscles were isolated and suspended in vitro (25° C) for determination of regulatory light chain phosphorylation and post-tetanic potentiation. Regulatory light chain phosphorylation was not different across conditions within either genotype although wildtype values were significantly greater than skMLCK-/- values. Consistent with this, the potentiation of concentric twitch force was similar between E+ and E- groups within each genotype but wildtype values were greater than skMLCK-/- values. However, unaltered estradiol levels following ovariectomy, likely due to previously underappreciated confounds of mouse age, development, and growth during estrogen supplementation, prevented direct testing of the hypothesis. Future studies should note the importance of estrous cycles and continuing physiological developments of young adult mice when working with ovarian hormones.

Caffeine attenuates contraction-induced diminutions of the intracellular calcium transient in mouse lumbrical muscle ex vivo.

The amount of calcium released from the sarcoplasmic reticulum in skeletal muscle rapidly declines during repeated twitch contractions. In this study, we test the hypothesis that caffeine can mitigate these contraction-induced declines in calcium release. Lumbrical muscles were isolated from male C57BL/6 mice and loaded with the calcium-sensitive indicator, AM-furaptra. Muscles were then stimulated at 8 Hz for 2.0 s in the presence or absence of 0.5 mM caffeine, at either 30 °C or 37 °C. The amplitude and area of the furaptra-based intracellular calcium transients and force produced during twitch contractions were calculated. For each of these measures, the values for twitch 16 relative to twitch 1 were higher in the presence of caffeine than in the absence of caffeine at both temperatures. We conclude that caffeine can attenuate contraction-induced diminutions of calcium release during repeated twitch contractions, thereby contributing to the inotropic effects of caffeine.

Epinephrine augments posttetanic potentiation in mouse skeletal muscle with and without myosin phosphorylation.

Sympathetic tone may influence force potentiation, that is, the stimulation-induced increase in skeletal muscle mechanical function associated with myosin phosphorylation, although the mechanism for this effect remains unknown. The purpose of this study was to examine the influence of epinephrine on concentric twitch force potentiation of wild-type and skeletal myosin light-chain kinase devoid mouse muscle (skMLCK-/- ). To this end, concentric twitch force was assessed before and after a potentiating stimulus (PS) to determine the peak and the duration of potentiation in the absence (-EPI) and presence (+EPI) of 1 µmol/L epinephrine in both genotypes. Twitch force of wild-type and skMLCK-/- muscles was increased by up to 31 and 35% and 18 and 23% in the -EPI and EPI conditions, respectively (all data n = 8, P < 0.05). In wild-type muscles, the PS increased RLC phosphorylation from 0.14 ± 0.05 (rest) to 0.66 ± 0.08 mol phos mol RLC; by 480 sec RLC phosphorylation had returned to baseline (all data n = 4 each time point, P < 0.05). Neither resting nor peak levels of RLC phosphorylation were altered by +EPI, although the duration of RLC phosphorylation was prolonged. In skMLCK-/- muscles, RLC phosphorylation was not elevated above constituent levels by stimulation in either the -EPI or +EPI condition. Thus, given the similarity in potentiation responses between genotypes our data suggest that the influence of epinephrine on potentiation was independent of skMLCK catalyzed phosphorylation of the RLC, although the clinical significance of this pathway for skeletal muscle function remains to be identified.

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.

Contraction-induced enhancement of relaxation during high force contractions of mouse lumbrical muscle at 37°C.

Repeated stimulation of unfatigued rodent fast-twitch skeletal muscle accelerates the kinetics of tension relaxation through an unknown mechanism. This effect varies with muscle type and stimulation parameters, and has been observed at physiological temperatures for submaximal but not maximal contractions. The purpose of this study was to compare relaxation kinetics of C57BL/6 mouse lumbrical muscles ex vivo from maximal isometric force (500 Hz for 20 ms) when evoked before (pre) and after (post) an intervening tetanic contraction at 37°C. During post contractions, we noted significant increases in the rate of tension decline during both the slow linear phase and the fast exponential phase of relaxation, as well as a reduced duration of the slow phase of relaxation compared with pre contractions (all P<0.05). This is the first demonstration of enhanced slow and fast relaxation phases from maximal isometric tension induced by prior stimulation in intact muscle at a physiological temperature.