Jeffrey J. Widrick

In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

Effect of a 17 day spaceflight on contractile properties of human soleus muscle fibres

1 Soleus biopsies were obtained from four male astronauts 45 days before and within 2 h after a 17 day spaceflight. 2 For all astronauts, single chemically skinned post‐flight fibres expressing only type I myosin heavy chain (MHC) developed less average peak Ca2+ activated force (Po) during fixed‐end contractions (0.78 ± 0.02 vs. 0.99 ± 0.03 mN) and shortened at a greater mean velocity during unloaded contractions (Vo) (0.83 ± 0.02 vs. 0.64 ± 0.02 fibre lengths s−1) than pre‐flight type I fibres. 3 The flight‐induced decline in absolute Po was attributed to reductions in fibre diameter and/or Po per fibre cross‐sectional area. Fibres from the astronaut who experienced the greatest relative loss of peak force also displayed a reduction in Ca2+ sensitivity. 4 The elevated Vo of the post‐flight slow type I fibres could not be explained by alterations in myosin heavy or light chain composition. One alternative possibility is that the elevated Vo resulted from an increased myofilament lattice spacing. This hypothesis was supported by electron micrographic analysis demonstrating a reduction in thin filament density post‐flight. 5 Post‐flight fibres shortened at 30 % higher velocities than pre‐flight fibres at external loads associated with peak power output. This increase in shortening velocity either reduced (2 astronauts) or prevented (2 astronauts) a post‐flight loss in fibre absolute peak power (μN (fibre length) s−1). 6 The changes in soleus fibre diameter and function following spaceflight were similar to those observed after 17 days of bed rest. Although in‐flight exercise countermeasures probably reduced the effects of microgravity, the results support the idea that ground‐based bed rest can serve as a model of human spaceflight. 7 In conclusion, 17 days of spaceflight decreased force and increased shortening velocity of single Ca2+‐activated muscle cells expressing type I MHC. The increase in shortening velocity greatly reduced the impact that impaired force production had on absolute peak power.

Muscle Mechanics: Adaptations with Exercise-Training

Based on the MHC isoform pattern, adult mammalian limb skeletal muscles contain two and, in some species, three types of fast fibers (Type IIa, IIx, and IIb), and one slow fiber (Type I). Slow muscles, such as the soleus, contain primarily the slow Type I fiber, whereas fast-twitch muscles are composed primarily of a mixture of the fast myosin isozymes. Force generation involves cross-bridge interaction and transition from a weakly bound, low-force state (AM-ADP-P(i)) to the strongly bound, high-force state (AM-ADP). This transition is thought to be rate limiting in terms of dP/dt, and the high-force state is the dominant cross-bridge form during a peak isometric contraction. Intact fast and slow skeletal muscles generate approximately the same amount of peak force (Po) of between 200 and 250 kN.m-2. However, the rate of transition from the low- to high-force state shows Ca2+ sensitivity and is 7-fold higher in fast-twitch, as compared to slow-twitch, skeletal muscle fibers. Fiber Vo or the maximal cross-bridge cycle rate is highly correlated with and thought to be dependent on the specific activity of the myosin or myofibrillar ATPase. The hierarchy for Vo is the Type IIb > IIx > IIa > I. This functional difference for the fast fiber types explains the higher Vo observed in the predominantly Type IIb SVL vs. the mixed fast Type IIa and IIb EDL muscle. A plot of Vo vs. species size demonstrates that an inverse relationship exists between Vo and body mass. From the standpoint of work capacity, the important property is power output. An analysis of individual muscles indicates that peak power is obtained at loads considerably below 50% of Po. Individuals with a high percentage of fast-twitch fibers generate a greater torque and higher power at a given velocity than those with predominantly slow-twitch fibers. In humans, mean peak power occurred in a ratio of 10:5:1 for the Type IIb, IIa, and I fibers. The in vivo measurement of the torque-velocity relationship and Vmax in human muscle is difficult because of limitations inherent in the equipment used and the inability to study the large limb muscles independently. Nevertheless, the in vivo torque-velocity relationships are similar to those measured in vitro in animals. This observation suggests that little central nervous system inhibition exists and that healthy subjects are able to achieve maximal activation of their muscles. Although peak isometric tension is not dependent on fiber type distribution, a positive correlation exists between the percentage of fast fibers and peak torque output at moderate-to-high angular isokinetic velocities. Consequently, peak power output is substantially greater in subjects possessing a predominance of fast fibers. The mechanical properties of slow and fast muscles do adapt to programs of regular exercise. Endurance exercise training has been shown to increase the Vo of the slow soleus by 20%. This increase could have been caused by either a small increase in all, or most, of the fibers, or to a conversion of a few fibers from slow to fast. Recently, the increase was shown to be caused by the former, as the individual slow Type I fibers of the soleus showed a 20% increase in Vo, but there was little or no change in the percentage of fast fibers. The increased Vo was correlated with, and likely caused by, an increased fiber ATPase. We hypothesize that the increased ATPase and cross-bridge cycling speed might be attributable to an increased expression of fast MLCs in the slow Type I fibers (Fig. 14.10). This hypothesis is based on the fact that light chains have been shown to be involved in the power stroke, and removal of light chains depresses force and velocity. Regular endurance exercise training had no effect on fiber size, but with prolonged durations of daily training it depressed Po and peak power. When the training is maintained over prolonged periods, it may even induce atrophy of the slow Type I and fast Type IIa fibers. (ABSTRACT TRUNCATED)

Development of a single-stage submaximal treadmill walking test

An equation was developed to estimate maximal oxygen uptake (VO2max, ml.kg-1.min-1) based on a single submaximal stage of a treadmill walking test. Subjects (67 males, 72 females) aged 20-59 yr completed 4-min stages at 0, 5, and 10% grades walking at a constant speed (2.0-4.5 mph) and then performed a VO2max test. Heart rate and respiratory gas exchange variables were measured during the test. Multiple regression analysis (N = 117) to estimate VO2max from the 4-min stage at 5% grade yielded the following model (R2 = 0.86; SEE = 4.85 ml.kg-1.min-1): VO2max = 15.1 + 21.8*SPEED (mph) -0.327*HEART RATE (bpm) -0.263*SPEED*AGE (yr) + 0.00504*HEART RATE*AGE + 5.98*GENDER (0 = Female; 1 = Male). The constant and all coefficients were highly significant (P less than 0.01). To assess the accuracy of the model in a cross-validation group (N = 22), an estimated VO2max value was obtained using the above model. Estimated VO2max then was regressed on observed VO2max yielding the following equation (R2 = 0.92): ESTIMATED VO2max = 0.15 + 1.03*OBSERVED VO2max. The intercept and slope of this equation were not significantly different from 0 and 1, respectively. For 90.9% of the subjects in the cross-validation group, residual scores were within the range of +/- 5 ml.kg-1.min-1. In conclusion, this submaximal walking test based on a single stage of a treadmill protocol provides a valid and time-efficient method for estimating VO2max.

Dry-land resistance training for competitive swimming

To determine the value of dry-land resistance training on front crawl swimming performance, two groups of 12 intercollegiate male swimmers were equated based upon preswimming performance, swim power values, and stroke specialties. Throughout the 14 wk of their competitive swimming season, both swim training group (SWIM, N = 12) and combined swim and resistance training group (COMBO, N = 12) swam together 6 d a week. In addition, the COMBO engaged in a 8-wk resistance training program 3 d a week. The resistance training was intended to simulate the muscle and swimming actions employed during front crawl swimming. Both COMBO and SWIM had significant (P < 0.05) but similar power gains as measured on the biokinetic swim bench and during a tethered swim over the 14-wk period. No change in distance per stroke was observed throughout the course of this investigation. No significant differences were found between the groups in any of the swim power and swimming performance tests. In this investigation, dry-land resistance training did not improve swimming performance despite the fact that the COMBO was able to increase the resistance used during strength training by 25-35%. The lack of a positive transfer between dry-land strength gains and swimming propulsive force may be due to the specificity of training.

Effect of 17 days of bed rest on peak isometric force and unloaded shortening velocity of human soleus fibers

The purpose of this study was to examine the effect of prolonged bed rest (BR) on the peak isometric force (Po) and unloaded shortening velocity ( V o) of single Ca2+-activated muscle fibers. Soleus muscle biopsies were obtained from eight adult males before and after 17 days of 6° head-down BR. Chemically permeabilized single fiber segments were mounted between a force transducer and position motor, activated with saturating levels of Ca2+, and subjected to slack length steps. V owas determined by plotting the time for force redevelopment vs. the slack step distance. Gel electrophoresis revealed that 96% of the pre- and 87% of the post-BR fibers studied expressed only the slow type I myosin heavy chain isoform. Fibers with diameter >100 μm made up only 14% of this post-BR type I population compared with 33% of the pre-BR type I population. Consequently, the post-BR type I fibers ( n = 147) were, on average, 5% smaller in diameter than the pre-BR type I fibers ( n = 218) and produced 13% less absolute Po. BR had no overall effect on Po per fiber cross-sectional area (Po/CSA), even though half of the subjects displayed a decline of 9-12% in Po/CSA after BR. Type I fiber V oincreased by an average of 34% with BR. Although the ratio of myosin light chain 3 to myosin light chain 2 also rose with BR, there was no correlation between this ratio and V o for either the pre- or post-BR fibers. In separate fibers obtained from the original biopsies, quantitative electron microscopy revealed a 20-24% decrease in thin filament density, with no change in thick filament density. These results raise the possibility that alterations in the geometric relationships between thin and thick filaments may be at least partially responsible for the elevated V o of the post-BR type I fibers.

Disproportionate loss of thin filaments in human soleus muscle after 17-day bed rest

Previously we reported that, after 17‐day bed rest unloading of 8 humans, soleus slow fibers atrophied and exhibited increased velocity of shortening without fast myosin expression. The present ultrastructural study examined fibers from the same muscle biopsies to determine whether decreased myofilament packing density accounted for the observed speeding. Quantitation was by computer‐assisted morphometry of electron micrographs. Filament densities were normalized for sarcomere length, because density depends directly on length. Thick filament density was unchanged by bed rest. Thin filaments/μm2 decreased 16–23%. Glycogen filled the I band sites vacated by filaments. The percentage decrease in thin filaments (Y) correlated significantly (P < 0.05) with the percentage increase in velocity (X), (Y = 0.1X + 20%, R2 = 0.62). An interpretation is that fewer filaments increases thick to thin filament spacing and causes earlier cross‐bridge detachment and faster cycling. Increased velocity helps maintain power (force × velocity) as atrophy lowers force. Atrophic muscles may be prone to sarcomere reloading damage because force/μm2 was near normal, and force per thin filament increased an estimated 30%.

Whole-body vibration slows the acquisition of fat in mature female rats.

Objective:To evaluate the effects of whole-body vibration on fat, bone, leptin and muscle mass.Methods/Design:Thirty 7-month-old female 344 Fischer rats were randomized by weight into three groups (baseline, vibration or control; n=8–10 per group). Rats in the vibration group were placed inside individual compartments attached to a Pneu-Vibe vibration platform (Pneumex, Sandpoint, ID, USA) and vibrated at 30–50 Hz (6 mm peak to peak) for 30 min per day, 5 days per week, for 12 weeks. The vibration intervention consisted of six 5-min cycles with a 1-min break between cycles.Results:There were significant body composition differences between the whole-body vibration and the control group. The whole-body vibration group weighed approximately 10% less (mean±s.d.; 207±10 vs 222±15 g, P<0.03) and had less body fat (20.8±3.8 vs 26.8±5.9 g, P<0.05), a lower percentage of body fat (10.2±1.7 vs 12±2.0%, P<0.05), and lower serum leptin levels (1.06±0.45 vs 2.27±0.57 ng ml−1, P<0.01) than the age-matched controls. No differences were observed for total lean mass, bone mineral content (BMC), bone mineral density (BMD), insulin-like growth factor-I (IGF-I) or soleus (SOL) and extensor digitorum longus (EDL) mass or function. Regional high-resolution dual-energy X-ray absoptiometry scans of the lumbar spine (L1-4) revealed that the whole-body vibration group had significantly greater BMC (0.33±0.05 vs 0.26±0.03 g, P<0.01) and BMD (0.21±0.01 vs 0.19±0.01 g cm−2, P<0.01) than the control group. No differences between the groups were observed in the amount of food consumed.Conclusion:These findings show that whole-body vibration reduced body fat accumulation and serum leptin without affecting whole body BMC, BMD or lean mass. However, the increase in vertebral BMC and BMD suggests that vibration may have resulted in local increases in bone mass and density. Also, whole-body vibration did not affect muscle function or food consumption.

Functional adaptability of muscle fibers to long-term resistance exercise.

PURPOSE We compared the functional properties of muscle fibers from two groups of subjects that differed widely in their training history to investigate whether long-term resistance exercise alters the intrinsic contractile properties of skeletal muscle fibers. METHODS Vastus lateralis muscle biopsies were obtained from six sedentary males (NT group, age = 23 +/- 1 yr) and six males who had participated in regular resistance exercise training over the preceding 7.6 +/- 1.6 yr (RT group, 22 +/- 1 yr). Chemically skinned muscle fiber segments were activated with a saturating free [Ca2+] to quantify fiber peak Ca2+-activated force (P(o)), unloaded shortening velocity (V(o)), and peak power. Fiber segment myosin heavy chain (MHC) isoform content was identified by gel electrophoresis. RESULTS Slow and fast fibers from the RT group were larger in CSA and produced greater absolute P(o) and absolute peak power in comparison with fibers from the NT group. However, these differences were no longer evident after P(o) and peak power were normalized to fiber CSA and fiber volume, respectively. V(o)/fiber length was dependent on fiber MHC content but independent of training status. CONCLUSION Fiber hypertrophy was sufficient to account for intergroup differences in P(o) and peak power of slow and fast fibers. There was no evidence that the intrinsic contractility of slow or fast fibers, as evaluated by force, shortening velocity, and power normalized to the appropriate fiber dimensions, differed between RT and NT groups.

Cross-bridge mechanisms of muscle weakness in multiple sclerosis

Vastus lateralis muscle biopsies were obtained from six individuals with multiple sclerosis (MS) having an Expanded Disability Status Score of 4.75 ± 0.28, and from six age‐ and gender‐matched individuals without MS. Biopsies from the MS group showed fewer fibers (31 ± 4 vs. 46 ± 4%) containing the type IIa myosin heavy chain (MHC) isoform exclusively. However, the percentage of fibers coexpressing type IIa and IIx MHC increased in direct proportion with MS disability status. The average unloaded shortening velocity of skinned fibers containing type I or IIa MHC did not differ between subject groups. Peak Ca2+‐activated force was 11–13% lower in fibers from the MS group due to atrophy (type I and IIa fibers) and reduced specific force (type I fibers). Increasing intracellular inorganic phosphate (0–30 mM) or hydrogen ion (pH 7.0–6.2) reduced Ca2+‐activated force in a manner that was independent of MS status. Thus, fibers from the MS group showed a subtle shift in fast MHC isoform coexpression and a modest reduction in cross‐bridge number, density, or average force, with no change in maximal cross‐bridge cycling rate or susceptibility to intracellular metabolites. These changes explain part of the muscle weakness and fatigue experienced by individuals with MS. Muscle Nerve 27: 456–464, 2003