Changes in skeletal muscle and tendon structure and function following genetic inactivation of myostatin in rats

Abstract

Key pointsr Myostatin is an important regulator of muscle mass and a potential therapeutic target for the treatment of diseases and injuries that result in muscle atrophy.r Targeted genetic mutations of myostatin have been generated in mice, and spontaneous loss-of-function mutations have been reported in several species. The impact of myostatin deficiency on the structure and function of muscles has been well described for mice, but not for other species.r We report the creation of a genetic model of myostatin deficiency in rats using zinc finger nuclease technology.r The main findings of the study are that genetic inactivation of myostatin in rats results in increases in muscle mass without a deleterious impact on the specific force production and tendon mechanical properties. The increases in mass occur through a combination of fibre hypertrophy, hyperplasia and activation of the insulin-like growth factor-1 pathway, with no substantial changes in atrophy-related pathways.r This large rodent model has enabled us to identify that the chronic loss of myostatin is void of the negative consequences to muscle fibres and extracellular matrix observed in mouse models. Furthermore, the greatest impact of myostatin in the regulation of muscle mass may not be to induce atrophy directly, but rather to block hypertrophy signalling.Abstract Myostatin is a negative regulator of skeletal muscle and tendon mass. Myostatin deficiency has been well studied in mice, but limited data are available on how myostatin regulates the structure and function of muscles and tendons of larger animals. We hypothesized that, in comparison to wild-type (MSTN +/+ ) rats, rats in which zinc finger nucleases were used to genetically inactivate myostatin (MSTN / ) would exhibit an increase in muscle mass and total force production, a reduction in specific force, an accumulation of type II fibres and a decrease and stiffening of connective tissue. Overall, the muscle and tendon phenotype of myostatin-deficient rats was markedly different from that of myostatin-deficient mice, which have impaired contractility and pathological changes to fibres and their extracellular matrix. Extensor digitorum longus and soleus muscles of MSTN / rats demonstrated 20-33% increases in mass, 35-45% increases in fibre number, 20-57% increases in isometric force and no differences in specific force. The insulin-like growth factor-1 pathway was activated to a greater extent in MSTN / muscles, but no substantial differences in atrophy-related genes were observed. Tendons of MSTN / rats had a 20% reduction in peak strain, with no differences in mass, peak stress or stiffness. The general morphology and gene expression patterns were similar between tendons of both genotypes. This large rodent model of myostatin deficiency did not have the negative consequences to muscle fibres and extracellular matrix observed in mouse models, and suggests that the greatest impact of myostatin in the regulation of muscle mass may not be to induce atrophy directly, but rather to block hype...

Inhibition of p38 mitogen-activated protein kinase signaling reduces fibrosis and lipid accumulation after rotator cuff repair

“…16, 25 The general techniques and methodologies in this study have been previously published and are provided in brevity in the current manuscript. 16, 17, 27, 30 …”

“…27, 33 Briefly, 100 mg of muscle was homogenized in Tissue Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Scientific), homogenized, spun at 12,000 × g for 10 min, and the supernatant was collected and stored at −80°C until use. A BCA protein assay (Thermo Scientific) was used to determine protein content, and the relative abundance of phosphorylated p38 MAPK (phosphorylation at Thr 180 and Tyr 182 residues) from 50 μg of total protein was analyzed using a Milliplex-p38 phospho protein magnetic bead assay (EMD Millipore Corporation, Billerica, MA, USA).…”

“…16, 17, 27, 30 Briefly, fibers were placed in a chamber filled with relaxing solution, and secured at one end to a servomotor (model 322C, Aurora Scientific, Aurora, ON, Canada) and at the other end to a force transducer (model 403A, Aurora Scientific). The length of the fiber was adjusted until an average sarcomere length of 2.5μm was achieved.…”

Tissue Engineering of Tendons

“…[27][28][29] For example, the secretion of GDF-8 from myogenic cells regulates the structure and function of tendon tissues in both prenatal and postnatal development. 18,30,31 Actually, our recent study revealed that the mechanical strength of in vivo engineered tendons could be significantly reduced when myocytes and fibroblasts were isolated by differential adhesion rates (data not shown), and this thus becomes our future direction for exploring the detailed mechanism in muscle-derived cell engineered tendon regeneration and maturation.…”

Analysis of polymorphisms in the equine MSTN gene in Polish populations of horse breeds

“…Myostatin has the larger impact on muscle mass than any other single signaling molecule, and it appears to regulate muscle fiber size by interacting with both the protein synthesis and degradation pathways in muscle fibers (Mendias et al, 2015). It has been demonstrated that increased levels of myostatin is due to upregulation of the muscle-specific E3 ligases, Atrogin-1 and muscle ring finger protein 1 (MuRF1) through Smad 3 mediated up regulation of Atrogin-1 and forkhead box O1 (Lokireddy et al, 2011).…”