The role of Pitx2 in maintaining the phenotype of myogenic precursor cells in the extraocular muscles.

Many differences exist between extraocular muscles (EOM) and non-cranial skeletal muscles. One striking difference is the sparing of EOM in various muscular dystrophies compared to non-cranial skeletal muscles. EOM undergo continuous myonuclear remodeling in normal, uninjured adults, and distinct transcription factors are required for the early determination, development, and maintenance of EOM compared to limb skeletal muscle. Pitx2, a bicoid-like homeobox transcription factor, is required for the development of EOM and the maintenance of characteristic properties of the adult EOM phenotype, but is not required for the development of limb muscle. We hypothesize that these unique properties of EOM contribute to the constitutive differences between EOM and non-craniofacial skeletal muscles. Using flow cytometry, CD34(+)/Sca1(-/)CD45(-/)CD31(-) cells (EECD34 cells) were isolated from extraocular and limb skeletal muscle and in vitro, EOM EECD34 cells proliferated faster than limb muscle EECD34 cells. To further define these myogenic precursor cells from EOM and limb skeletal muscle, they were analyzed for their expression of Pitx2. Western blotting and immunohistochemical data demonstrated that EOM express higher levels of Pitx2 than limb muscle, and 80% of the EECD34 cells expressed Pitx2. siRNA knockdown of Pitx2 expression in EECD34 cells in vitro decreased proliferation rates and impaired the ability of EECD34 cells to fuse into multinucleated myotubes. High levels of Pitx2 were retained in dystrophic and aging mouse EOM and the EOM EECD34 cells compared to limb muscle. The differential expression of Pitx2 between EOM and limb skeletal muscle along with the functional changes in response to lower levels of Pitx2 expression in the myogenic precursor cells suggest a role for Pitx2 in the maintenance of constitutive differences between EOM and limb skeletal muscle that may contribute to the sparing of EOM in muscular dystrophies

Loss of Synaptic Vesicles from Neuromuscular Junctions in Aged MRF4-Null Mice

MRF4 belongs to the basic helix-loop-helix class of transcription factors and this and other members of its family profoundly influence skeletal muscle development. Less is known about the role of these factors in aging. As MRF4 is preferentially expressed in sub-synaptic nuclei, we postulated it might play a role in maintenance of the neuromuscular junction. To test this hypothesis, we examined the junctional regions of 19-20-month-old mice and found decreased levels of SV2B, a marker of synaptic vesicles, in MRF4-null mice relative to controls. There was a corresponding decrease in grip strength in MRF4-null mice. Taken together, these data suggest that the intrinsic muscle factor, MRF4 plays an important role in maintenance of neuromuscular junctions

Loss of Synaptic Vesicles from Neuromuscular Junctions in Aged MRF4-Null Mice

MRF4 belongs to the basic helix-loop-helix class of transcription factors and this and other members of its family profoundly influence skeletal muscle development. Less is known about the role of these factors in aging. As MRF4 is preferentially expressed in sub-synaptic nuclei, we postulated it might play a role in maintenance of the neuromuscular junction. To test this hypothesis, we examined the junctional regions of 19-20-month-old mice and found decreased levels of SV2B, a marker of synaptic vesicles, in MRF4-null mice relative to controls. There was a corresponding decrease in grip strength in MRF4-null mice. Taken together, these data suggest that the intrinsic muscle factor, MRF4 plays an important role in maintenance of neuromuscular junctions

Pitx2-positive nuclei are located within myofibers and outside of myofibers in the satellite cell position.

<p>(A, B) Pitx2 (green) and dystrophin (red) localization in wild-type mouse EOM. Arrows point down to Pitx2-positive myonuclei. Arrowheads point up to Pitx2-positive nuclei outside of the dystrophin ring. (C) Pitx2 (green) and laminin (red) localization in wild-type mouse EOM. (D) Pitx2 (green) and dystrophin (red) localization in wild-type mouse TA. Arrow points to a rare Pitx2-positive cell. (E) Pitx2 (green) and dystrophin (red) localization in human EOM. (F) Pitx2 (green) and dystrophin (red) localization in aged (19 months old) wild-type mouse EOM. (G) Percentages of Pitx2-positive nuclei in relation to dystrophin in wild-type mouse EOM and TA. (H) Percentages of Pitx2-positive nuclei in relation to laminin in wild-type mouse EOM and TA. orb, orbital layer of EOM. glob, global layer of EOM. Scale bars are 50 µm. * Indicates significant difference from EOM orbital inside (P≤0.05). # Indicates significant difference from EOM global inside (P≤0.05).</p

Pitx2-positive cells co-express MyoD, but not Pax7.

<p>(A) Pitx2 (red) and Pax7 (green) localization in human EOM. (B) Pitx2 and (C) MyoD localization in wild-type mouse EOM. (D) Co-localization of Pitx2 (red) and MyoD (green) in wild-type mouse EOM. Scale bars are 50 µm.</p

The Ca\u3csub\u3ev\u3c/sub\u3e 1.2 Ca\u3csup\u3e2+\u3c/sup\u3e Channel is Expressed in Sarcolemma of Type I and IIA Myofibers of Adult Skeletal Muscle

Although Ca2+-dependent signaling pathways are important for skeletal muscle plasticity, the sources of Ca2+ that activate these signaling pathways are not completely understood. Influx of Ca2+ through surface membrane Ca2+ channels may activate these pathways. We examined expression of two L-type Ca2+ channels in adult skeletal muscle, the CaV 1.1 and CaV 1.2, with isoform-specific antibodies in Western blots and immunocytochemistry assays. Consistent with a large body of work, expression of the CaV 1.1 was restricted to skeletal muscle where it was expressed in T-tubules. CaV 1.2 was also expressed in skeletal muscle, in the sarcolemma of type I and IIa myofibers. Exercise-induced alterations in muscle fiber types cause a concomitant increase in the number of both CaV 1.2 and type IIa–positive fibers. Taken together, these data suggest that the CaV 1.2 Ca2+ channel is expressed in adult skeletal muscle in a fiber type–specific manner, which may help to maintain oxidative muscle phenotype

Phage-Displayed Random Peptide Libraries in Mice: Toxicity After Serial Panning

Purpose: In vivo screening of phage-displayed random peptide libraries (RPLs) has been used to identify peptide ligands to targets found on endothelial cells of blood vessels supplying specific tissues such as brain, kidney, and tumor tissue. Peptides that bind specifically to blood vessels supplying tumor tissue have been conjugated to cytotoxic agents and used to successfully eradicate tumors in a mouse model. With the ultimate goal of developing similar methods for treating human cancer, we describe an in vivo RPL screening process that, unlike previous in vivo experiments, does not harm the animal being screened. Methods: RPLs were administered to FVB, BalbC, and tumor-bearing MRL/MpJ-fasLPR mice in a variety of dosing formats. Tumor nodules were excised 10 min following infusion and phage were amplified from the specimens. Phage were reinjected into the same animal within 48 h. This process was repeated twice for a total of three in vivo screens of mouse tumor tissue within the same animal. Mice were observed for systemic side effects, histopathologic damage, and presence of phage in organs. Peptide sequences were determined from several third-pan phage clones. Results: Overall there was minimal toxicity from administration of single or repeat doses of RPLs. Amino acid consensus sequences were identified and some of the sequences were similar to those of peptide ligands that bind matrix metalloproteinases. Conclusions: Serial administration of an RPL is well tolerated and serial panning in individual mice leading to consensus sequence motifs is possible. Based on these preclinical data the Food and Drug Administration has approved the implementation of human clinical trials with this technique