Mutant lamins cause nuclear envelope rupture and DNA damage in skeletal muscle cells.

Mutations in the LMNA gene, which encodes the nuclear envelope (NE) proteins lamins A/C, cause Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy and other diseases collectively known as laminopathies. The mechanisms responsible for these diseases remain incompletely understood. Using three mouse models of muscle laminopathies and muscle biopsies from individuals with LMNA-related muscular dystrophy, we found that Lmna mutations reduced nuclear stability and caused transient rupture of the NE in skeletal muscle cells, resulting in DNA damage, DNA damage response activation and reduced cell viability. NE and DNA damage resulted from nuclear migration during skeletal muscle maturation and correlated with disease severity in the mouse models. Reduction of cytoskeletal forces on the myonuclei prevented NE damage and rescued myofibre function and viability in Lmna mutant myofibres, indicating that myofibre dysfunction is the result of mechanically induced NE damage. Taken together, these findings implicate mechanically induced DNA damage as a pathogenic contributor to LMNA skeletal muscle diseases.

Cell Mechanical and Physiological Behavior in the Regime of Rapid Mechanical Compressions that Lead to Cell Volume Change.

Cells respond to mechanical forces by deforming in accordance with viscoelastic solid behavior. Studies of microscale cell deformation observed by high speed video microscopy have elucidated a new cell behavior in which sufficiently rapid mechanical compression of cells can lead to transient cell volume loss and then recovery. This work has discovered that the resulting volume exchange between the cell interior and the surrounding fluid can be utilized for efficient, convective delivery of large macromolecules (2000 kDa) to the cell interior. However, many fundamental questions remain about this cell behavior, including the range of deformation time scales that result in cell volume loss and the physiological effects experienced by the cell. In this study, a relationship is established between cell viscoelastic properties and the inertial forces imposed on the cell that serves as a predictor of cell volume loss across human cell types. It is determined that cells maintain nuclear envelope integrity and demonstrate low protein loss after the volume exchange process. These results define a highly controlled cell volume exchange mechanism for intracellular delivery of large macromolecules that maintains cell viability and function for invaluable downstream research and clinical applications.

Starring or Supporting Role? Satellite Cells and Skeletal Muscle Fiber Size Regulation.

Recent loss-of-function studies show that satellite cell depletion does not promote sarcopenia or unloading-induced atrophy, and does not prevent regrowth. Although overload-induced muscle fiber hypertrophy is normally associated with satellite cell-mediated myonuclear accretion, hypertrophic adaptation proceeds in the absence of satellite cells in fully grown adult mice, but not in young growing mice. Emerging evidence also indicates that satellite cells play an important role in remodeling the extracellular matrix during hypertrophy.

Integrative mRNA-microRNA analyses reveal novel interactions related to insulin sensitivity in human adipose tissue.

Adipose tissue has profound effects on whole-body insulin sensitivity. However, the underlying biological processes are quite complex and likely multifactorial. For instance, the adipose transcriptome is posttranscriptionally modulated by microRNAs, but the relationship between microRNAs and insulin sensitivity in humans remains to be determined. To this end, we utilized an integrative mRNA-microRNA microarray approach to identify putative molecular interactions that regulate the transcriptome in subcutaneous adipose tissue of insulin-sensitive (IS) and insulin-resistant (IR) individuals. Using the NanoString nCounter Human v1 microRNA Expression Assay, we show that 17 microRNAs are differentially expressed in IR vs. IS. Of these, 16 microRNAs (94%) are downregulated in IR vs. IS, including miR-26b, miR-30b, and miR-145. Using Agilent Human Whole Genome arrays, we identified genes that were predicted targets of miR-26b, miR-30b, and miR-145 and were upregulated in IR subjects. This analysis produced ADAM22, MYO5A, LOX, and GM2A as predicted gene targets of these microRNAs. We then validated that miR-145 and miR-30b regulate these mRNAs in differentiated human adipose stem cells. We suggest that use of bioinformatic integration of mRNA and microRNA arrays yields verifiable mRNA-microRNA pairs that are associated with insulin resistance and can be validated in vitro.

Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy.

Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.

Ribosome biogenesis: emerging evidence for a central role in the regulation of skeletal muscle mass.

The ribosome is a supramolecular ribonucleoprotein complex that functions at the heart of the translation machinery to convert mRNA into protein. Ribosome biogenesis is the primary determinant of translational capacity of the cell and accordingly has an essential role in the control of cell growth in eukaryotes. Cumulative evidence supports the hypothesis that ribosome biogenesis has an important role in the regulation of skeletal muscle mass. The purpose of this review is to, first, summarize the main mechanisms known to regulate ribosome biogenesis and, second, put forth the hypothesis that ribosome biogenesis is a central mechanism used by skeletal muscle to regulate protein synthesis and control skeletal muscle mass in response to anabolic and catabolic stimuli. The mTORC1 and Wnt/ß-catenin/c-myc signaling pathways are discussed as the major pathways that work in concert with each of the three RNA polymerases (RNA Pol I, II, and III) in regulating ribosome biogenesis. Consistent with our hypothesis, activation of these two pathways has been shown to be associated with ribosome biogenesis during skeletal muscle hypertrophy. Although further study is required, the finding that ribosome biogenesis is altered under catabolic states, in particular during disuse atrophy, suggests that its activation represents a novel therapeutic target to reduce or prevent muscle atrophy. Lastly, the emerging field of ribosome specialization is discussed and its potential role in the regulation of gene expression during periods of skeletal muscle plasticity.

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in lamin A/C-deficient mice.

Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Nuclear damage in LMNA mutant iPSC-derived cardiomyocytes is associated with impaired lamin localization to the nuclear envelope.

The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy (LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of LMNA-DCM remains incompletely understood. Using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared to healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and suggest that defects in nuclear lamina organization may contribute to the nuclear and cellular dysfunction in LMNA-DCM.

Satellite cell depletion does not inhibit adult skeletal muscle regrowth following unloading-induced atrophy.

Resident muscle stem cells, known as satellite cells, are thought to be the main mediators of skeletal muscle plasticity. Satellite cells are activated, replicate, and fuse into existing muscle fibers in response to both muscle injury and mechanical load. It is generally well-accepted that satellite cells participate in postnatal growth, hypertrophy, and muscle regeneration following injury; however, their role in muscle regrowth following an atrophic stimulus remains equivocal. The current study employed a genetic mouse model (Pax7-DTA) that allowed for the effective depletion of >90% of satellite cells in adult muscle upon the administration of tamoxifen. Vehicle and tamoxifen-treated young adult female mice were either hindlimb suspended for 14 days to induce muscle atrophy or hindlimb suspended for 14 days followed by 14 days of reloading to allow regrowth, or they remained ambulatory for the duration of the experimental protocol. Additionally, 5-bromo-2'-deoxyuridine (BrdU) was added to the drinking water to track cell proliferation. Soleus muscle atrophy, as measured by whole muscle wet weight, fiber cross-sectional area, and single-fiber width, occurred in response to suspension and did not differ between satellite cell-depleted and control muscles. Furthermore, the depletion of satellite cells did not attenuate muscle mass or force recovery during the 14-day reloading period, suggesting that satellite cells are not required for muscle regrowth. Myonuclear number was not altered during either the suspension or the reloading period in soleus muscle fibers from vehicle-treated or satellite cell-depleted animals. Thus, myonuclear domain size was reduced following suspension due to decreased cytoplasmic volume and was completely restored following reloading, independent of the presence of satellite cells. These results provide convincing evidence that satellite cells are not required for muscle regrowth following atrophy and that, instead, the myonuclear domain size changes as myofibers adapt.

Effect of leucine supplementation on indices of muscle damage following drop jumps and resistance exercise.

The purpose of this study was to determine the effect of leucine supplementation on indices of muscle damage following eccentric-based resistance exercise. In vitro, the amino acid leucine has been shown to reduce proteolysis and stimulate protein synthesis. Twenty-seven untrained males (height 178.6±5.5 cm; body mass 77.7±13.5 kg; age 21.3±1.6 years) were randomly divided into three groups; leucine (L) (n=10), placebo (P) (n=9) and control (C) (n=8). The two experimental groups (L and P) performed 100 depth jumps from 60 cm and six sets of ten repetitions of eccentric-only leg presses. Either leucine (250 mg/kg bm) or placebo was ingested 30 min before, during and immediately post-exercise and the morning of each recovery day following exercise. Muscle function was determined by peak force during an isometric squat and by jump height during a static jump at pre-exercise (PRE) and 24, 48, 72, and 96 h post-exercise (24, 48, 72, 96 h). Additionally, at these time points each group's serum levels of creatine kinase (CK) and myoglobin (Mb) along with perceived feelings of muscle soreness were determined. None of the C group dependent variables was altered by the recurring testing procedures. Peak force was significantly decreased across all time points for both experimental groups. The L group experienced an attenuated drop in mean peak force across all post-exercise time points compared to the P group. Jump height significantly decreased from PRE for both the L and P group at 24 h and 48 h. CK and Mb was significantly elevated from PRE for both experimental groups at 24 h. Muscle soreness increased across all time points for the both the L and P group, and the L group experienced a significantly higher increase in mean muscle soreness post-exercise. Following exercise-induced muscle damage, high-dose leucine supplementation may help maintain force output during isometric contractions, however, not force output required for complex physical tasks thereby possibly limiting its ergogenic effectiveness.

Effect of loading on peak power of the bar, body, and system during power cleans, squats, and jump squats.

Nine males (age 24.7 ± 2.1 years, height 175.3 ± 5.5 cm, body mass 80.8 ± 7.2 kg, power clean 1-RM 97.1 ± 6.36 kg, squat 1-RM = 138.3 ± 20.9 kg) participated in this study. On day 1, the participants performed a one-repetition maximum (1-RM) in the power clean and the squat. On days 2, 3, and 4, participants performed the power clean, squat or jump squat. Loading for the power clean ranged from 30% to 90% of the participant's power clean 1-RM and loading for the squat and jump squat ranged from 0% to 90% of the participant's squat 1-RM, all at 10% increments. Peak force, velocity, and power were calculated for the bar, body, and system (bar + body) for all power clean, squat, and jump squat trials. Results indicate that peak power for the bar, body, and system is differentially affected by load and movement pattern. When using the power clean, squat or jump squat for training, the optimal load in each exercise may vary. Throwing athletes or weightlifters may be most concerned with bar power, but jumpers or sprinters may be more concerned with body or system power. Thus, the exercise type and load vary according to the desired stimulus.

A comparison of men's and women's strength to body mass ratio and varus/valgus knee angle during jump landings.

The purpose of this investigation was to compare valgus/varus knee angles during various jumps and lower body strength between males and females relative to body mass. Seventeen recreationally active females (age: 21.94 ± 2.59 years; height: 1.67 ± 0.05 m; mass: 64.42 ± 8.39 kg; percent body fat: 26.89 ± 6.26%; squat one-repetition maximum: 66.18 ± 19.47 kg; squat to body mass ratio: 1.03 ± 0.28) and 13 recreationally active males (age: 21.69 ± 1.65 years; height: 1.77 ± 0.07 m; mass: 72.39 ± 9.23 kg; percent body fat: 13.15 ± 5.18%; squat one-repetition maximum: 115.77 ± 30.40 kg; squat to body mass ratio: 1.59 ± 0.31) performed a one-repetition maximum in the squat and three of each of the following jumps: countermovement jump, 30 cm drop jump, 45 cm drop jump, and 60 cm drop jump. Knee angles were analysed using videography and body composition was analysed by dual-energy X-ray absorptiometry to allow for squat to body mass ratio and squat to fat free mass ratio to be calculated. Significant differences (P ≤ 0.05) were found between male and female one-repetition maximum, male and female squat to body mass ratio, and male and female squat to fat free mass ratio. Significant differences were found between male and female varus/valgus knee positions during maximum flexion of the right and left leg in the countermovement jump, drop jump from 30 cm, drop jump from 45 cm, and drop jump from 60 cm. Correlations between varus/valgus knee angles and squat to body mass ratio for all jumps displayed moderate, non-significant relationships (countermovement jump: r = 0.445; drop jump from 30 cm: r = 0.448; drop jump from 45 cm: r = 0.449; drop jump from 60 cm: r = 0.439). In conclusion, males and females have significantly different lower body strength and varus/valgus knee position when landing from jumps.

LINCing Nuclear Mechanobiology With Skeletal Muscle Mass and Function.

Skeletal muscle demonstrates a high degree of adaptability in response to changes in mechanical input. The phenotypic transformation in response to mechanical cues includes changes in muscle mass and force generating capabilities, yet the molecular pathways that govern skeletal muscle adaptation are still incompletely understood. While there is strong evidence that mechanotransduction pathways that stimulate protein synthesis play a key role in regulation of muscle mass, there are likely additional mechano-sensitive mechanisms important for controlling functional muscle adaptation. There is emerging evidence that the cell nucleus can directly respond to mechanical signals (i.e., nuclear mechanotransduction), providing a potential additional level of cellular regulation for controlling skeletal muscle mass. The importance of nuclear mechanotransduction in cellular function is evident by the various genetic diseases that arise from mutations in proteins crucial to the transmission of force between the cytoskeleton and the nucleus. Intriguingly, these diseases preferentially affect cardiac and skeletal muscle, suggesting that nuclear mechanotransduction is critically important for striated muscle homeostasis. Here we discuss our current understanding for how the nucleus acts as a mechanosensor, describe the main cytoskeletal and nuclear proteins involved in the process, and propose how similar mechanoresponsive mechanisms could occur in the unique cellular environment of a myofiber. In addition, we examine how nuclear mechanotransduction fits into our current framework for how mechanical stimuli regulates skeletal muscle mass.

Comparison of kinetic variables and muscle activity during a squat vs. a box squat.

The purpose of this investigation was to determine if there was a difference in kinetic variables and muscle activity when comparing a squat to a box squat. A box squat removes the stretch-shortening cycle component from the squat, and thus, the possible influence of the box squat on concentric phase performance is of interest. Eight resistance trained men (Height: 179.61 ± 13.43 cm; Body Mass: 107.65 ± 29.79 kg; Age: 24.77 ± 3.22 years; 1 repetition maximum [1RM]: 200.11 ± 58.91 kg) performed 1 repetition of squats and box squats using 60, 70, and 80% of their 1RM in a randomized fashion. Subjects completed the movement while standing on a force plate and with 2 linear position transducers attached to the bar. Force and velocity were used to calculate power. Peak force and peak power were determined from the force-time and power-time curves during the concentric phase of the lift. Muscle activity (electromyography) was recorded from the vastus lateralis, vastus medialis, biceps femoris, and longissimus. Results indicate that peak force and peak power are similar between the squat and box squat. However, during the 70% of 1RM trials, the squat resulted in a significantly lower peak force in comparison to the box squat (squat = 3,269 ± 573 N, box squat = 3,364 ± 575 N). In addition, during the 80% of 1RM trials, the squat resulted in significantly lower peak power in comparison to the box squat (squat = 2,050 ± 486 W, box squat = 2,197 ± 544 W). Muscle activity was generally higher during the squat in comparison to the box squat. In conclusion, minimal differences were observed in kinetic variables and muscle activity between the squat and box squat. Removing the stretch-shortening cycle during the squat (using a box) appears to have limited negative consequences on performance.

Relationship between maximal squat strength and five, ten, and forty yard sprint times.

The purpose of this investigation was to examine the relationship between maximal squat strength and sprinting times. Seventeen Division I-AA male football athletes (height = 1.78 +/- 0.04 m, body mass [BM] = 85.9 +/- 8.8 kg, body mass index [BMI] = 27.0 +/- 2.6 kg/m2, 1 repetition maximum [1RM] = 166.5 +/- 34.1 kg, 1RM/BM = 1.94 +/- 0.33) participated in this investigation. Height, weight, and squat strength (1RM) were assessed on day 1. Within 1 week, 5, 10, and 40 yard sprint times were assessed. Squats were performed to a 70 degree knee angle and values expressed relative to each subject's BM. Sprints were performed on a standard outdoor track surface with timing gates placed at the previously mentioned distances. Statistically significant (p < or = 0.05) correlations were found between squat 1RM/BM and 40 yard sprint times (r = -0.605, p = 0.010, power = 0.747) and 10 yard sprint times (r = -0.544, p = 0.024, power = 0.626). The correlation approached significance between 5 yard sprint times and 1RM/BM (r = -0.4502, p = 0.0698, power = 0.4421). Subjects were then divided into those above 1RM/BM of 2.10 and below 1RM/BM of 1.90. Subjects with a 1RM/BM above 2.10 had statistically significantly lower sprint times at 10 and 40 yards in comparison with those subjects with a 1RM/BM ratio below 1.90. This investigation provides additional evidence of the possible importance of maximal squat strength relative to BM concerning sprinting capabilities in competitive athletes.

Mechanosensitive pathways controlling translation regulatory processes in skeletal muscle and implications for adaptation.

The ability of myofibers to sense and respond appropriately to mechanical signals is one of the primary determinants of the skeletal muscle phenotype. In response to a change in mechanical load, muscle cells alter their protein metabolism, primarily through the regulation of protein synthesis rate. Protein synthesis rates are determined by both translation efficiency and translational capacity within the muscle. Translational capacity is strongly determined by the ribosome content of the muscle; thus the regulation of ribosomal biogenesis by mechanical inputs has been an area of recent interest. Despite the clear association between mechanical signals and changes in protein metabolism, the molecular pathways that link these events are still not fully elucidated. This review focuses on recent studies looking at how mechanosignaling impacts translational events. The role of impaired mechanotransduction in aging is discussed, as is the connection between age-dependent signaling defects and compromised ribosomal biogenesis during mechanical overload. Finally, emerging evidence suggests that the nucleus can act as a mechanosensitive element and that this mode of mechanotransduction may have an important role in skeletal muscle physiology and adaptation.