Shannon N. Bremner

Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle

Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, thin filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die within 8–11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle thin filament lengths were up to 25% shorter compared with wild type, and thin filaments were uniform in length both within and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.

Quantitative analysis of neonatal skeletal muscle functional improvement in the mouse.

SUMMARY Postnatal skeletal muscle growth is classically attributed to fiber hypertrophy and myogenic differentiation, but these processes do not account for the size-independent increase of muscle mechanical performance that occurs during postnatal growth. There is also little knowledge about the precise time-course of contractile function or the underlying factors that affect it. The present study investigated morphological factors (muscle fiber size and myofibrillar packing), biochemical factors (myosin heavy chain isoform and desmin intermediate filament protein expression), and muscle architecture during postnatal development in mice. Physiological testing of the mouse tibialis anterior revealed that maximum isometric stress increased from 27±3 kPa at postnatal day 1 to 169±10 kPa by postnatal day 28, roughly a sixfold increase. Morphological measurements revealed a robust increase in the size-independent packing of myofibrillar matrix material occurring with the functional improvement, with just 48.1±5.5% of the cross-sectional area filled with myofibrils at postnatal day 1 whereas 92.5±0.9% was filled by day 28. Expression of four myosin heavy chain isoforms (embryonic, neonatal, IIX and IIB), as well as desmin, correlated significantly with muscle mechanical function. Stepwise multiple regression showed that, of the variables measured, percentage content of neonatal myosin heavy chain was the best predictor of mechanical function during the postnatal time-course. These data provide the first specific structural basis for increases in muscle tension development during growth. Therefore, models of muscle growth must be modified to include an intrinsic quality enhancement component.

Quantitative analysis of muscle fibre type and myosin heavy chain distribution in the frog hindlimb: implications for locomotory design.

To investigate the design of the frog muscular system for jumping, fibre type distribution and myosin heavy chain (MHC) isoform composition were quantified in the hindlimb muscles of Rana pipiens. Muscles were divided into two groups: five large extensor muscles which were predicted to shorten and produce mechanical power during jumping (JP), and four much smaller muscles commonly used in muscle physiology studies, but that do not shorten or produce power during jumping (NJP). Fibres were classified as one of four different types (type 1, 2, 3 or tonic) or an intermediate type (type 1–2) based on␣their relative myosin-ATPase reactivity and MHC immunoreactivity in muscle cross-sections according to previous nomenclature established for amphibian skeletal muscle. Type 1 fibres correspond to the fastest and most powerful of the twitch fibres, and type 3 fibres are the slowest and least powerful. Myosin-ATPase histochemistry revealed that the JP muscles were co mposed primarily of type 1 fibres (89%) with a small percentage of type 2 (7%) and intermediate type 1–2 fibres (4%). The fibre type composition of NJP muscles was more evenly distributed between type 1 (29%), type 2 (46%) and type 1–2 (24%) fibres. Tonic fibres comprised less than 2% of the muscle cross-section in both JP and NJP groups. Similarly, MHC composition determined by quantitative SDS–PAGE revealed that JP muscles were composed predominantly of type 1 MHC (86%), with a balance of type 2 MHC (14%). The opposite pattern was found for MHC composition in the NJP muscles: type 1 (28%), type 2 (66%) and type 3 (6%). These results demonstrate that the large extensor muscles that produce the power required for jumping have a fibre type distribution that enables them to generate high levels of mechanical power, with the type 1 isoform accounting for 85–90% of the total M HC content.

Increased efficacy and decreased systemic-effects of botulinum toxin A injection after active or passive muscle manipulation

The effect of physical manipulation on the outcome of neurotoxin (NT) injection was studied in a rat tibialis anterior (TA) model system where dorsiflexion torque could be measured precisely. After determination of initial torque, all rats received a one‐time botulinum toxin A (BTX‐A) injection (dose 6.0 units/kg in a volume of 100μL) into the TA midbelly. Four experimental groups were studied: one group was subjected to BTX‐A injection alone (BTX‐A only, n=8), one was subjected to BTX‐A injection followed immediately by 10 isometric contractions (ISO; n=9), and the third was subjected to BTX‐A followed immediately by 10 muscle passive stretch/release cycles (PS; n=10). After 1 month, maximum dorsiflexion torque of the injected and contralateral legs was determined followed by quantification of TA fiber area. Post‐injection torque was significantly reduced by around 80% in all NT‐treated extremities 1 month after injection (p<0.05). While all NT‐treated extremities demonstrated a significant torque decrease relative to their pre‐injection levels, ISO and PS groups demonstrated significantly lower torques compared with the BTX‐A only group which received no physical manipulation (p<0.05) indicating greater efficacy. Perhaps even more surprising was that the ISO and PS groups both demonstrated a significantly smaller contralateral effect compared with the BTX‐A only group that received no manipulation (p<0.05) indicating a decreased systemic‐effect. Muscle fiber size generally correlated with dorsiflexion torque. These data demonstrate that both neuromuscular activity (seen in the ISO group) and muscle movement (seen in the PS group) increased the efficacy of BTX‐A and decreased the systemic side effects.

PASSIVE MECHANICAL PROPERTIES AND RELATED PROTEINS CHANGE WITH BOTULINUM NEUROTOXIN A INJECTION OF NORMAL SKELETAL MUSCLE

The effects of botulinum neurotoxin A on the passive mechanical properties of skeletal muscle have not been investigated, but may have significant impact in the treatment of neuromuscular disorders including spasticity. Single fiber and fiber bundle passive mechanical testing was performed on rat muscles treated with botulinum neurotoxin A. Myosin heavy chain and titin composition of single fibers was determined by gel electrophoresis. Muscle collagen content was determined using a hydroxyproline assay. Neurotoxin‐treated single fiber passive elastic modulus was reduced compared to control fibers (53.00 kPa vs. 63.43 kPa). Fiber stiffness and slack sarcomere length were also reduced compared to control fibers and myosin heavy chain composition shifted from faster to slower isoforms. Average titin molecular weight increased 1.77% after treatment. Fiber bundle passive elastic modulus increased following treatment (168.83  kPa vs. 75.14 kPa). Bundle stiffness also increased while collagen content per mass of muscle tissue increased 38%. Injection of botulinum neurotoxin A produces an effect on the passive mechanical properties of normal muscle that is opposite to the changes observed in spastic muscles.

Skeletal muscle intermediate filaments form a stress-transmitting and stress-signaling network

ABSTRACT A fundamental requirement of cells is their ability to transduce and interpret their mechanical environment. This ability contributes to regulation of growth, differentiation and adaptation in many cell types. The intermediate filament (IF) system not only provides passive structural support to the cell, but recent evidence points to IF involvement in active biological processes such as signaling, mechanotransduction and gene regulation. However, the mechanisms that underlie these processes are not well known. Skeletal muscle cells provide a convenient system to understand IF function because the major muscle-specific IF, desmin, is expressed in high abundance and is highly organized. Here, we show that desmin plays both structural and regulatory roles in muscle cells by demonstrating that desmin is required for the maintenance of myofibrillar alignment, nuclear deformation, stress production and JNK-mediated stress sensing. Finite element modeling of the muscle IF system suggests that desmin immediately below the sarcolemma is the most functionally significant. This demonstration of biomechanical integration by the desmin IF system suggests that it plays an active biological role in muscle in addition to its accepted structural role.

Dramatic changes in muscle contractile and structural properties after 2 botulinum toxin injections

Introduction: Botulinum toxin is frequently administered serially to maintain therapeutic muscle paralysis, but the effect of repeated doses on muscle function are largely unknown. This study characterized the muscle response to 2 onabotulinum toxin (BoNT) injections separated by 3 months. Methods: Animal subjects received a single toxin injection (n = 8), 2 BoNT injections separated by 3 months (n = 14), or 1 BoNT and 1 saline injection separated by 3 months (n = 8). Results: The functional effect of 2 serial injections was exponentially greater than the effect of a single injection. While both groups treated with a single BoNT injection had decreased torque in the injected leg by approximately 50% relative to contralateral legs, the double BoNT injected group had decreased torque by over 95% relative to the preinjection level. Both single and double BoNT injections produced clear signs of fiber‐type grouping. Conclusions: These experiments demonstrate a disproportionately greater effect of repeated BoNT injections. Muscle Nerve 52: 649–657, 2015

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging

Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity.

mTOR regulates peripheral nerve response to tensile strain

While excessive tensile strain can be detrimental to nerve function, strain can be a positive regulator of neuronal outgrowth. We used an in vivo rat model of sciatic nerve strain to investigate signaling mechanisms underlying peripheral nerve response to deformation. Nerves were deformed by 11% and did not demonstrate deficits in compound action potential latency or amplitude during or after 6 h of strain. As revealed by Western blotting, application of strain resulted in significant upregulation of mammalian target of rapamycin (mTOR) and S6 signaling in nerves, increased myelin basic protein (MBP) and β-actin levels, and increased phosphorylation of neurofilament subunit H (NF-H) compared with unstrained (sham) contralateral nerves (P < 0.05 for all comparisons, paired two-tailed t-test). Strain did not alter neuron-specific β3-tubulin or overall nerve tubulin levels compared with unstrained controls. Systemic rapamycin treatment, thought to selectively target mTOR complex 1 (mTORC1), suppressed mTOR/S6 signaling, reduced levels of MBP and overall tubulin, and decreased NF-H phosphorylation in nerves strained for 6 h, revealing a role for mTOR in increasing MBP expression and NF-H phosphorylation, and maintaining tubulin levels. Consistent with stretch-induced increases in MBP, immunolabeling revealed increased S6 signaling in Schwann cells of stretched nerves compared with unstretched nerves. In addition, application of strain to cultured adult dorsal root ganglion neurons showed an increase in axonal protein synthesis based on a puromycin incorporation assay, suggesting that neuronal translational pathways also respond to strain. This work has important implications for understanding mechanisms underlying nerve response to strain during development and regeneration.NEW & NOTEWORTHY Peripheral nerves experience tensile strain (stretch) during development and movement. Excessive strain impairs neuronal function, but moderate strains are accommodated by nerves and can promote neuronal growth; mechanisms underlying these phenomena are not well understood. We demonstrated that levels of several structural proteins increase following physiological levels of nerve strain and that expression of a subset of these proteins is regulated by mTOR. Our work has important implications for understanding nerve development and strain-based regenerative strategies.

Systematic Test of Neurotoxin Dose and Volume on Muscle Function in a Rat Model

Introduction: Onabotulinum toxin serotype A (BT‐A) is used for a variety of motor and sensory disorders related to abnormal muscle activity. Methods: We developed a high‐resolution rodent model to allow precise determination of the effect of BT‐A dose (measured in units) and injectate volume (measured in μl) on the efficacy of the injection and systemic side effects. Dorsiflexion is the best indicator of injected and contralateral muscle function. Results: One month after injection, dorsiflexion torque of BT‐A‐injected limbs was decreased significantly in all experimental groups compared with saline controls (P < 0.05). Torque was also compared among the BT‐A groups, which demonstrated a significant effect of dose (P < 0.001), but no effect of volume (P > 0.2) and no dose × volume interaction (P > 0.3). Similar results were observed for other parameters measured. Conclusions: These data demonstrate that injection dose and not volume or concentration is the primary determinant of neurotoxin efficacy in a rodent model. Muscle Nerve 49: 709–715, 2014