Advances in research on the prenatal development of skeletal muscle in animals in relation to the quality of muscle-based food. I. Regulation of myogenesis and environmental impact

Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only slightly affected. Initial studies on low and high maternal protein feeding are published. Although there are only a few studies, first results also reveal an influence of nutrition on skeletal muscle development in fish and poultry. Finally, environmental temperature has been identified as a critical factor for growth and development of skeletal muscle in both fish and poultry.     

Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter.     

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0–0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.

     

Quinolones as HCV NS5B polymerase inhibitors

Hepatitis C virus (HCV) infection is treated with a combination of peginterferon alfa-2a/b and ribavirin. To address the limitations of this therapy, numerous small molecule agents are in development, which act by directly affecting key steps in the viral life-cycle. Herein we describe our discovery of quinolone derivatives, novel small-molecules that inhibit NS5b polymerase, a key enzyme of the viral life-cycle. A crystal structure of a quinoline analog bound to NS5B reveals that this class of compounds binds to allosteric site-II (non-nucleoside inhibitor-site 2, NNI-2) of this protein.     

3-heterocyclyl quinolone inhibitors of the HCV NS5B polymerase

The discovery and optimization of a novel class of quinolone small-molecules that inhibit NS5B polymerase, a key enzyme of the HCV viral life-cycle, is described. Our research led to the replacement of a hydrolytically labile ester functionality with bio-isosteric heterocycles. An X-ray crystal structure of a key analog bound to NS5B facilitated the optimization of this series of compounds to afford increased activity against the target enzyme and in the cell-based replicon assay system.     

Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.     

Expression of calpastatin isoforms in muscle and functionality of multiple calpastatin promoters

Calpastatin is the specific endogenous inhibitor of calpain proteinase that is encoded by a single gene. Transient transfection assays in both a non-fusing skeletal muscle and non-muscle cell-line demonstrated that the putative porcine calpastatin promoter regions 5' to exons 1xa, 1xb, and 1u were functional. Both real-time quantitative and semi-quantitative RT-PCR on porcine skeletal muscle total RNA indicated that steady-state expression of Type I and III mRNAs containing exons 1xa and 1u, respectively, was at equivalent levels whilst the expression of Type II mRNA containing exon 1xb was significantly less (p<0.001). Immunoprobing of Western blotted muscle extracts with an antibody raised against a peptide sequence encoded by exon 1xa indicated that Type I protein was expressed and that there was significantly more Type I protein in cardiac than skeletal muscle (p<0.001). The results suggest that the expression of the single calpastatin gene was differentially controlled at several levels.     

Response of the porcine MYH4-promoter and MYH4-expressing myotubes to known anabolic and catabolic agents in vitro

Myosin heavy chain-IIB (MyHC-IIB; encoded by MYH4 or Myh4) expression is often associated with muscle hypertrophic growth. Unlike other large mammals, domestic pig breeds express MyHC-IIB at both the mRNA and protein level.AimTo utilise a fluorescence-based promoter-reporter system to test the influence of anabolic and catabolic agents on increasing porcine MYH4-promoter activity and determine whether cell hypertrophy was subsequently induced.MethodsC2C12 myoblasts were co-transfected with porcine MYH4-promoter-driven ZsGreen and CMV-driven DsRed expression plasmids. At the onset of differentiation, treatments (dibutyryl cyclic-AMP (dbcAMP), Des(1–3) Insulin-Like Growth Factor-1 (IGF-I), triiodo-l-thyronine (T3) and dexamethasone (Dex)) or appropriate vehicle controls were added and cells maintained for up to four days. At day 4 of differentiation, measurements were collected for total fluorescence and average myotube diameter, as indicators of MYH4-promoter activity and cell hypertrophy respectively.ResultsPorcine MYH4-promoter activity increased during C2C12 myogenic differentiation, with a marked increase between days 3 and 4. MYH4-promoter activity was further increased following four days of dbcAMP treatment and average myotube diameter was significantly increased by dbcAMP. Porcine MYH4-promoter activity also tended to be increased by T3 treatment, but there were no effects of Des(1–3) IGF-I or Dex treatment, whereas average myotube diameter was increased by Des(1–3) IGF-I, but not T3 or Dex.ConclusionPorcine MYH4-promoter activity responded to dbcAMP, Des(1–3) IGF-I and T3 treatment in vitro as observed previously in reported in vivo studies. However, we report that increased MYH4-promoter activity was not always associated with muscle cell hypertrophy. The fluorescence-based reporter system offers a useful tool to study muscle cell hypertrophic growth.