A Nitrate Ester of Sedative Alkyl Alcohol Improves Muscle Function and Structure in a Murine Model of Duchenne Muscular Dystrophy

Nitric oxide (NO) has major physiological and cellular effects on muscle growth, repair, and function. In most muscle biopsies from humans with myopathies, sarcolemma-localized neuronal nitric oxide synthase (nNOS) is either reduced or not detected, particularly in dystrophin-deficient Duchenne muscular dystrophy (DMD). Abnormal NO signaling at the sarcolemmal level is integrally involved in the pathogenesis and accounts, at least in part, for the muscle weakness of DMD. Dystrophic muscle fibers exhibit an increased susceptibility to contraction-induced membrane damage. Muscle relaxants function to prevent muscle wasting by decreasing nerve impulses and reducing calcium influx that regulates tensing or tightening of muscle fibers. We have recently developed a new class of nitric esters that combines the pharmacological functions of NO and muscle relaxation. Here, we report the synthesis and properties of the nitric ester (MMPN) of 2-methyl-2-<i>n</i>-propyl-1,3-propanediol (MPP) and its effect in mdx dystrophic mice, a murine model of DMD. MMPN produced significant improvements in biochemical, pathological, and functional phenotypes in the mouse model. The endurance of exercise was extended by 47% in time to exhaustion and 84% in running distance. Serum CK level was decreased by 30%. Additionally, MMPN decreased intracellular free calcium concentration without causing skeletal muscle weakness. No hepatic or renal toxicities were observed during the study. Our investigations unveil a potential new treatment for muscular diseases

Stable overexpression levels of Large on glycosylation of α-DG in the Lec15 cells.

<p>Pro-5, B421 clones overexpressing (+) or without (−) Large-MYC or Lec15.2 cells overexpressing Large-MYC at high +(H), modest +(M) low +(L) or without Large-MYC (−) were tested by immuno-blot assay. The blot of the Lec15.2 samples had 10 mins exposure, while all other blot had about 10 seconds' exposure time.</p

Stable overexpressing Large induces functional glycans in CHO cells.

<p>(A) Equal amount of lysates (60 µg proteins each lane) extracted from the cells stably expressing Large as indicated. The immuno-blotting procedure was described in methods section. The glycan antibody IIH6 and anti-MYC antibody (9E10) were used to detect the functional glycans and Large–MYC, respectively. In addition, an anti-β-DG (MANDAG2) antibody was used for detecting dystroglycan as the loading control. (B) The cells as indicated were seeded in 96-well plates and immuno-fluorescent staining was performed as described in the methods section. The images in top panel present the negative controls, while the images of bottom panel present the Large positive cells. A rabbit polyclonal anti-MYC antibody and the IIH6 antibody were used to stain the Large-MYC and glycosylated α-DG, respectively. The cells were also stained with DAPI to visualize the nucleus. The bar is 50 µm. (C) The cells as indicated were seeded in 96-well plate for 24 hour growth. The protocol of laminin clustering assay is described in the methods section. The laminins-DyLight488 was added to the cell culture for 6 hours incubation. A rabbit polyclonal anti-MYC antibody was used to stain Large-MYC protein followed by staining with secondary anti-rabbit antibodies conjugated with Alex594. In addition, DAPI was added to each well to stain the DNA for cells counting. The images were captured with a fluorescent microscope as described in methods section. The bar is 50 µm.</p

Effects of Large-induced pathway on N-linked glycans.

<p>(A) All cells were grown in the completed media as described in the methods section and the lysates harvested from the indicated cells with stably expressing LARGE were treated with pNGase F for 4 hours, while it is absent in the negative controls reactions as described in the methods section. IIH6 antibody was used to determine the LARGE induced glycans, and the anti-β-DG antibody was used to determine the β-DG proteins as described above. The blot of the Lec15.2 samples with the IIH6 antibody had 30 mins exposure, while all other blots had about 10 seconds' exposure time.</p

LARGE modifies both mucin type O-glycans and N-linked glycans on α-DG in ldlD cells while without galactosylation.

<p>(A) Predicted structures of the glycans in N-linked, Mucin and O-mannosyl pathway in the ldlD cells. (B)The ldlD-LG cells were maintained in F12 nutrition mix media with 3% lipoprotein deficient bovine serum for more than 48 hours prior to the experiment. The cells were seeded into 6-well plates one day before addition of indicated sugars (Gal at 10 µM and GalNAc at 200 µM). After being treated with the sugar(s) for 24 hour, the cell lysates were harvested and equal amount of the lysates were loaded. An immuno-blotting assay was performed to detect the functional glycans and β-DG with the IIH6 and β-DG antibody, respectively. (C) The quantitative data of the expression levels of the functional glycans were obtained with AlphaImage AIC software based on densitometers followed the manufacture instructions. The IIH6 expression levels were normalized with the expression levels of β-DG (N = 3). (D) The lysates harvested from the ldlD-LG cells growing in the conditions as indicated and the experimental procedure is the same as described in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016866#pone-0016866-g004" target="_blank">Fig. 4A</a>). (E) Quantitative analysis of the data of (D) (N = 3).</p

Illustration of the role of Large in the glycosylation of α-DG.

<p>LGIFG: Large-induced functional glycans.</p

Overexpressing DPM2 restores the Large function in the Lec15.2 cells.

<p>(A) Transient transfection was conducted to introduce mouse <i>DPM2</i> cDNA into the Lec 15.2-LG cells. 48 hours after transfection, the cell lysates were harvested and immuno-blotting assay and laminins overlay assay were conducted as described above. (B) A transient transfection was conducted to introduce DPM-GFP cDNA into the cells in 96-well plate. 48 hours the after transfection, cells were fixed and an indirect immuno-fluorescent staining with IIH6 antibody was conducted with the protocol as described in the methods section. The images were captured with a fluorescent microscope as described in methods section. The bar is 50 µm.</p

Additional file 1 of Triazine-cored polymeric vectors for antisense oligonucleotide delivery in vitro and in vivo

Additional file 1. 1) Polymer’s structure and corresponding code; 2) Comparison of transduction and cytotoxicity between TAP (10 μg) and LF-2k (4 μg) mediated 2′-OMePSE50

Tris[2-(acryloyloxy)ethyl]isocyanurate Cross-Linked Low-Molecular-Weight Polyethylenimine as Gene Delivery Carriers in Cell Culture and Dystrophic <i>mdx</i> Mice

Hyperbranched poly­(ester amine)­s (PEAs) were successfully synthesized by Michael addition reaction between tris­[2-(acryloyloxy)­ethyl]­isocyanurate (TAEI) and low-molecular-weight polyethylenimine (LPEI, <i>M</i>_w 0.8k, 1.2k, and 2.0k) and evaluated <i>in vitro</i> and <i>in vivo</i> as gene carriers. PEAs effectively condensed plasmid DNA with particle sizes below 200 nm and surface charges between 11.5 and 33.5 mV under tested doses [at the ratios 2–10:1 of polymer/pDNA­(w/w)]. The PEAs showed significantly lower cytotoxicities when compared with PEI 25k in two different cell lines. The PEAs (C series) composed of PEI 2k showed higher transgene expression compared to PEAs of PEI 0.8k (A series) or 1.2k (B series). Highest gene transfection efficiency in CHO, C2C12 myoblast, and human skeletal muscle (HSK) cell lines was obtained with TAEI/PEI-2K (C12) at a ratio of 1:2. Both C12, C14­(TAEI/PEI-2K at a ratio of 1:4) demonstrated 5–8-fold higher gene expression as compared with PEI 25k in <i>mdx</i> mice <i>in vivo</i> through intramuscular administration. No obvious muscle damage was observed with these new polymers. Higher transfection efficiency and lower toxicity indicate the potential of the biodegradable PEAs as safe and efficient transgene delivery vectors

Development of a Nitric Oxide-Releasing Analogue of the Muscle Relaxant Guaifenesin for Skeletal Muscle Satellite Cell Myogenesis

Nitric oxide (NO) mediates activation of satellite precursor cells to enter the cell cycle. This provides new precursor cells for skeletal muscle growth and muscle repair from injury or disease. Targeting a new drug that specifically delivers NO to muscle has the potential to promote normal function and treat neuromuscular disease, and would also help to avoid side effects of NO from other treatment modalities. In this research, we examined the effectiveness of the NO donor, iosorbide dinitrate (ISDN), and a muscle relaxant, methocarbamol, in promoting satellite cell activation assayed by muscle cell DNA synthesis in normal adult mice. The work led to the development of guaifenesin dinitrate (GDN) as a new NO donor for delivering nitric oxide to muscle. The results revealed that there was a strong increase in muscle satellite cell activation and proliferation, demonstrated by a significant 38% rise in DNA synthesis after a single transdermal treatment with the new compound for 24 h. Western blot and immunohistochemistry analyses showed that the markers of satellite cell myogenesis, expression of myf5, myogenin, and follistatin, were increased after 24 h oral administration of the compound in adult mice. This research extends our understanding of the outcomes of NO-based treatments aimed at promoting muscle regeneration in normal tissue. The potential use of such treatment for conditions such as muscle atrophy in disuse and aging, and for the promotion of muscle tissue repair as required after injury or in neuromuscular diseases such as muscular dystrophy, is highlighted