HDAC Inhibitors as Epigenetic Regulators

The relaxing solution contained 4 mM MgATP, 1 mM free Mg2+, 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate, and sufficient KCl to adjust the ionic strength to 180. muscle fibers from the fast-twitch WAF1 extensor digitorum longus and the slow-twitch soleus muscle. A significant effect of the size of individual MNDs in hypertrophic muscle Boc-D-FMK fibers on both specific force and myosin content was observed. This effect was muscle cell type specific and suggested there is a critical volume individual myonuclei can support efficiently. The large MNDs found in fast muscles ofMstn/mice were correlated with the decrement in specific force and myosin content inMstn/muscles. Thus, myostatin inhibition may not be able to maintain the appropriate MND for optimal function.Qaisar, R., Renaud, G., Morine, K., Barton, E. R., Sweeney, H. L., Larsson, L. Is functional hypertrophy and specific force coupled with the addition of myonuclei at the single muscle fiber level? Keywords:IGF-1, myosin, myostatin Skeletal muscle plasticity is well documented, and muscle size is large in response to,e.g., functional overload, exercise, and hormonal influences. In general, muscle force is proportional to the cross-sectional area (CSA) of the contractile material (specific force), but a gain in muscle size need not result in maintained specific force. Muscle cells are postmitotically fixed multinuclear cells with hundreds of nuclei dispersed along the length of the cell. Each nucleus controls gene transcription in a finite volume of cytoplasm, the myonuclear domain (MND). The hypothesis that an increase in DNA content is a prerequisite for maintenance of metabolic and work demands and transport distances in hypertrophied fibers is supported in some (14), but not all, studies (5,6). In this context, two important signaling molecules that regulate muscle growth are insulin-like growth factor 1 (IGF-1) and myostatin (79), but they have strikingly different effects on specific force. IGF-1, a protein with anabolic effects on muscle and muscle hypertrophy, has been closely linked with an increased IGF-1 expression. A mouse model has been developed in which IGF-1 is expressed in skeletal muscleviaa tissue-restricted transgene (10). These mice show a larger muscle mass than wild-type mice, which are characterized by maintenance of specific force (1113), and satellite cell activation has been proposed to be the source of new myonuclei in these fibers (4). Myostatin, a member of the TGF- superfamily, is a negative regulator of skeletal muscle mass (14). Inhibition of myostatin or its gene results in larger muscle mass than in wild-type mice (1416). However, this hypertrophy is dissimilar from IGF-1-mediated hypertrophy in that the specific force has been reported to be less in myostatin-null mice (9,17). However, contractile measurements have typically been measured at the whole-muscle level, and it cannot be excluded that changes in specific force are secondary to changes in muscle fiber orientation rather than changes in the contractile properties at the single Boc-D-FMK muscle fiber level. Both IGF-1 and myostatin signaling pathways have been considered for the pharmacological treatment of different muscle-wasting conditions, and clinical trials have been initiated with the aim of improving muscle mass and function in patients with severe muscle wasting. Ultimately, the success of these trials will be evaluated with regard to muscle function and patient quality of life, Boc-D-FMK and not solely on the changes in muscle mass. Thus, there is, accordingly, a significant need for an improved mechanistic understanding of the effects of these interventions on skeletal muscle mass and function. We hypothesize that the myonuclear organization differs between hypertrophic muscle fibers from myostatin-knockout (Mstn/) and IGF-1-overexpressing (mIgf1+/+) muscle fibers. This, in turn, will have a significant effect on myofibrillar protein synthesis and turnover, affecting contractile proteins and regulation of muscle contraction at the cross-bridge level. Using a novel algorithm recently developed in our group to analyze the 3-dimensional (3D) organization of MNDs from confocal images (18), we compared the organization of myonuclei in single muscle fibers in parallel with function and myosin content from the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus from control,Mstn/, andmIgf1+/+mice. == MATERIALS AND METHODS == All animal experiments were conducted in accordance with the University of Pennsylvania Animal Care and Use Committee. C57 BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA),Mstn/were obtained from Dr. Se Jin Lee (Johns Hopkins University, Baltimore, MD, USA), and MyLC/IGF-I (mIgf1+/+) mice have been described in detail elsewhere (10). Four mice were included in each group (control,Mstn/,.