Tatiana Y. Kostrominova

Number of contractions to maintain mass and force of a denervated rat muscle

Within 5 weeks, denervated extensor digitorum longus (EDL) muscles of rats lose 66% of mass, 91% of force, and 76% of fiber cross‐sectional area (CSA). We previously determined the parameters of electrical stimulation for denervated rat EDL muscles to generate tetanic contractions sufficient to maintain mass and force close to control values. Using these parameters, we tested the hypothesis that a range exists for number of contractions per day, below and above which values for mass, maximum force, and fiber CSA are lower than values for innervated control muscles. For 5 weeks, denervated EDL muscles were stimulated to generate between 25 and 5000 contractions daily with contractions separated by constant intervals of rest, repeated 24 h per day. Force was not maintained with fewer than 200 or more than 800 contractions daily, whereas mass and fiber CSA were not maintained with fewer than 50 contractions daily. Protocols of stimulation that maintain muscle mass and force during prolonged periods of denervation may minimize problems clinically associated with denervation atrophy. Muscle Nerve 30: 77–86, 2004

Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats

Non‐technical summary  The force developed by a single fibre in frog muscles is transmitted laterally to the muscle surface with little or no loss. To demonstrate this phenomenon in mammals, a ‘yoke’ apparatus was developed that attached to the surface of whole, parallel‐fibred muscles and permitted measurements of the lateral transmission of forces. We then demonstrated that for wild‐type mice and rats longitudinal and lateral transmission of forces in muscles were not different. In contrast, for skeletal muscles of dystrophic mice and very old rats, in which the dystrophin‐associated glycoprotein complex (DGC) of fibres was disrupted, the forces transmitted laterally were impaired severely. We conclude that during contractions of skeletal muscles, an intact DGC is essential for the lateral transmission of force and disruptions of the DGC lead to sarcomere instability and contraction‐induced injury.

Reparative myogenesis in long-term denervated skeletal muscles of adult rats results in a reduction of the satellite cell population

This study, conducted on 25‐month denervated rat hindlimb muscles, was directed toward elucidating the basis for the poor regeneration that is observed in long‐term denervated muscles. Despite a ∼97.6% loss in mean cross‐sectional area of muscle fibers, the muscles retained their fascicular arrangement, with the fascicles containing ∼1.5 times more fibers than age‐matched control muscles. At least three distinct types of muscle fibers were observed: degenerating, persisting (original), and newly formed (regenerated) fibers. A majority of newly formed fibers did not appear to undergo complete maturation, and morphologically they resembled myotubes. Sites of former motor end‐plates remained identifiable in persisting muscle fibers. Nuclear death was seen in all types of muscle fibers, especially in degenerating fibers. Nevertheless, the severely atrophic skeletal muscles continued to express developmentally and functionally important proteins, such as MyoD, myogenin, adult and embryonic subunits of the nicotinic acetylcholine receptor, and neural‐cell adhesion molecule. Despite the prolonged period of denervation, slow and fast types of myosin were found in surviving muscle fibers. The number of satellite cells was significantly reduced in long‐term denervated muscles, as compared with age‐matched control muscles. In 25‐month denervated muscle, satellite cells were only attached to persisting muscle fibers, but were never seen on newly formed fibers. Our data suggest that the absence of satellite cells in a population of immature newly formed muscle fibers that has arisen as a result of continuous reparative myogenesis may be a crucial, although not necessarily the only, factor underlying the poor regenerative ability of long‐term denervated muscle. Anat Rec 263:139–154, 2001.

Survival of Schwann cells in chronically denervated skeletal muscles.

Abstract. It is well established that over time Schwann cells disappear from the endoneurial space of the distal stump of a chronically transected sciatic nerve trunk. Nevertheless, the status of the Schwann cells within terminal branches of the transected sciatic nerve remains poorly understood. To elucidate this issue we examined the endoneurial space of the intramuscular nerves in rat hindlimb skeletal muscles, which had been denervated for a 25-month period. Based on specific ultrastructural characteristics, we identified a small population of viable Schwann cells within the intramuscular nerve trunks. The surviving Schwann cells continued to be immunopositive for both S-100 protein and neural cell adhesion molecule. In addition, reverse transcription-polymerase chain reaction and/or Western blot analyses have shown that at least two molecules, brain-derived neurotrophic factor and a non-catalytic truncated form of tyrosine protein kinase receptor B, which could potentially participate in the process of nerve repair, were detectable in chronically denervated skeletal muscle. Our results demonstrate that Schwann cells can survive inside the intramuscular nerve trunks of denervated skeletal muscle for a 25-month period without axonal contact.

Morphological and Functional Characteristics of Three- Dimensional Engineered Bone-Ligament-Bone Constructs Following Implantation

The incidence of ligament injury has recently been estimated at 400,000/year. The preferred treatment is reconstruction using an allograft, but outcomes are limited by donor availability, biomechanical incompatibility, and immune rejection. The creation of an engineered ligament in vitro solely from patient bone marrow stromal cells (has the potential to greatly enhance outcomes in knee reconstructions. Our laboratory has developed a scaffoldless method to engineer three-dimensional (3D) ligament and bone constructs from rat bone marrow stem cells in vitro. Coculture of these two engineered constructs results in a 3D bone-ligament-bone (BLB) construct with viable entheses, which was successfully used for medial collateral ligament (MCL) replacement in a rat model. 1 month and 2 month implantations were applied to the engineered BLBs. Implantation of 3D BLBs in a MCL replacement application demonstrated that our in vitro engineered tissues grew and remodeled quickly in vivo to an advanced phenotype and partially restored function of the knee. The explanted 3D BLB ligament region stained positively for type I collagen and elastin and was well vascularized after 1 and 2 months in vivo. Tangent moduli of the ligament portion of the 3D BLB 1 month explants increased by a factor of 2.4 over in vitro controls, to a value equivalent to those observed in 14-day-old neonatal rat MCLs. The 3D BLB 1 month explants also exhibited a functionally graded response that closely matched native MCL inhomogeneity, indicating the constructs functionally adapted in vivo.

Skeletal muscle weakness due to deficiency of CuZn-superoxide dismutase is associated with loss of functional innervation

An association between oxidative stress and muscle atrophy and weakness in vivo is supported by elevated oxidative damage and accelerated loss of muscle mass and force with aging in CuZn-superoxide dismutase-deficient (Sod1(-/-)) mice. The purpose was to determine the basis for low specific force (N/cm(2)) of gastrocnemius muscles in Sod1(-/-) mice and establish the extent to which structural and functional changes in muscles of Sod1(-/-) mice resemble those associated with normal aging. We tested the hypothesis that muscle weakness in Sod1(-/-) mice is due to functionally denervated fibers by comparing forces during nerve and direct muscle stimulation. No differences were observed for wild-type mice at any age in the forces generated in response to nerve and muscle stimulation. Nerve- and muscle-stimulated forces were also not different for 4-wk-old Sod1(-/-) mice, whereas, for 8- and 20-mo-old mice, forces during muscle stimulation were 16 and 30% greater, respectively, than those obtained using nerve stimulation. In addition to functional evidence of denervation with aging, fiber number was not different for Sod1(-/-) and wild-type mice at 4 wk, but 50% lower for Sod1(-/-) mice by 20 mo, and denervated motor end plates were prevalent in Sod1(-/-) mice at both 8 and 20 mo and in WT mice by 28 mo. The data suggest ongoing denervation in muscles of Sod1(-/-) mice that results in fiber loss and muscle atrophy. Moreover, the findings support using Sod1(-/-) mice to explore mechanistic links between oxidative stress and the progression of deficits in muscle structure and function.

Application of WGA lectin staining for visualization of the connective tissue in skeletal muscle, bone, and ligament/tendon studies

During immunostaining of specific proteins in tissue sections using monoclonal and polyclonal antibodies, visualization of general tissue staining/background or major structural features is helpful to pinpoint precise localization of the protein of interest. Often in skeletal muscle research, immunostaining with antibodies against connective tissue or plasma membrane proteins (collagen 1, laminin, and caveolin 3) are used for this purpose. Although immunostaining for these proteins works well, it is time consuming, costly, limits the number of antibodies against protein of interest that can be used on a single section, and is not applicable to some staining techniques. Lectins were frequently used in earlier publications for skeletal muscle fiber boundaries and connective tissue visualization, but are not common in the current research studies. This work investigates costaining of muscle, bone, ligament, and tendon tissue sections with fluorescently tagged wheat germ agglutinin (WGA) lectin as a tool for the visualization of connective tissue. The results of this study show that fluorescent WGA lectin costaining is a cost‐effective, fast, and convenient method for connective tissue visualization, especially in the studies where extensive washes reduce staining of the structures that are the primary interest of the investigation. Microsc. Res. Tech. 74:18‐22, 2011.

Effect of implantation on engineered skeletal muscle constructs

The development of engineered skeletal muscle would provide a viable tissue for replacement and repair of muscle damaged by disease or injury. Our current tissue‐engineering methods result in three‐dimensional (3D) muscle constructs that generate tension but do not advance phenotypically beyond neonatal characteristics. To develop to an adult phenotype, innervation and vascularization of the construct must occur. In this study, 3D muscle constructs were implanted into the hindlimb of a rat, along the sciatic nerve, with the sural nerve isolated, transected and sutured to the construct to encourage innervation. Aortic ring anchors were sutured to the tendons of the biceps femoris muscle so that the construct would move dynamically with the endogenous muscle. After 1 week in vivo, the constructs were explanted, evaluated for force production and stained for muscle, nerve and collagen markers. Implanted muscle constructs showed a developing capillary system, an epimysium‐like outer layer of connective tissue and an increase in myofibre content. The beginning of α‐bungarotoxin clustering suggests that neuromuscular junctions (NMJs) could form on the implanted muscle, given more time in vivo. Additionally, the constructs increased maximum isometric force from 192 ± 41 μN to 549 ± 103 μN (245% increase) compared to in vitro controls, which increased from 276 ± 23 μN to 329 ± 27μN (25% increase). These findings suggest that engineered muscle tissue survives 1 week of implantation and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. However, in terms of force production, the muscle constructs need longer implantation times to fully develop an adult phenotype. Copyright

Adaptive changes in structure of skeletal muscles from adult Sod1 homozygous knockout mice

Cu/Zn superoxide dismutase (SOD1), which is localized cytoplasmically and in the mitochondrial intermembrane space, is an enzyme that is critically important for superoxide free-radical elimination. Compared with age-matched wild-type littermates (Sod1+/+), SOD1 homozygous knockout (Sod1-/-) mice have smaller body masses, heart and skeletal muscle masses, and muscle cross-sectional areas. At the light-microscopic level, cross sections of skeletal muscles from Sod1-/- mice show no gross structural abnormalities. Following the staining of muscles of Sod1-/- mice for succinate dehydrogenase (SDH) enzymatic activity, a grouping of SDH-positive fibers has been observed. Immunostaining for neural cell adhesion marker in the gastrocnemius muscle of Sod1-/- mice has revealed a small number of atrophic denervated muscle fibers. No denervated fibers are observed in extensor digitorum longus (EDL), tibialis anterior, or plantaris muscles. An increase in mRNA expression levels of myogenin and acetylcholine receptor alpha has been detected in muscles in Sod1-/- mice, but no changes in MyoD expression occur. Compared with fast oxidative fibers in EDL muscles of Sod1+/+ mice, those of Sod1-/- mice show increased accumulations of sub-sarcolemmal mitochondria. We conclude that the lack of SOD1 in adult Sod1-/- mice does not result in extensive denervation of skeletal muscle fibers, although the distribution of fiber types is modified, and that fast oxidative fibers develop alterations in the amount and spatial distribution of sub-sarcolemmal mitochondria.

TGF-β1 enhances contractility in engineered skeletal muscle

Scaffoldless engineered 3D skeletal muscle tissue created from satellite cells offers the potential to replace muscle tissue that is lost due to severe trauma or disease. Transforming growth factor‐beta 1 (TGF‐β1) plays a vital role in mediating migration and differentiation of satellite cells during the early stages of muscle development. Additionally, TGF‐β1 promotes collagen type I synthesis in the extracellular matrix (ECM) of skeletal muscle, which provides a passive elastic substrate to support myofibres and facilitate the transmission of force. To determine the role of TGF‐β1 in skeletal muscle construct formation and contractile function in vitro, we created tissue‐engineered 3D skeletal muscle constructs with varying levels of recombinant TGF‐β1 added to the cell culture medium. Prior to the addition of TGF‐β1, the primary cell population was composed of 75% Pax7‐positive cells. The peak force for twitch, tetanus and spontaneous force were significantly increased in the presence of 2.0 ng/ml TGF‐β1 when compared to 0, 0.5 and 1.0 ng/ml TGF‐β1. Visualization of the cellular structure with H&E and with immunofluorescence staining for sarcomeric myosin heavy chains and collagen type I showed denser regions of better organized myofibres in the presence of 2.0 ng/ml TGF‐β1 versus 0, 0.5 and 1.0 ng/ml. The addition of 2.0 ng/ml TGF‐β1 to the culture medium of engineered 3D skeletal muscle constructs enhanced contractility and extracellular matrix organization. Copyright