Expression of Becker-like and full-length human dystrophins was stable for at least 6 months in m... more Expression of Becker-like and full-length human dystrophins was stable for at least 6 months in mdx mouse muscle following intramuscular plasmid DNA injection. Intramuscular injection of a single plasmid DNA encoding both luciferase and dystrophin resulted in stable luciferase expression for at least 2 months in mdx muscle, whereas injection of plasmid DNA encoding only luciferase did not result in stable luciferase expression. These results suggest that expression of either full-length or Becker-like dystrophins protects mdx mouse myofibers from degeneration.
European Journal of Applied Physiology and Occupational Physiology, Aug 1, 1998
Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cel... more Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P < 0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P < 0.05) BrdU labeling in uninjured soleus muscles compared to weight-bearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P > 0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers.
The ability of satellite cells to survive the ischemic conditions at the core of orthotopically f... more The ability of satellite cells to survive the ischemic conditions at the core of orthotopically free grafted rat extensor digitorum longus muscles was examined. Cell cultures of isolated core and peripheral regions of whole muscle grafts maintained in vivo for more than 24 hours indicated that no viable cells were present in the core, whereas the number of cells from the peripheral region was greatly increased. Muscles were examined with the electron microscope to determine the fate of satellite cells of the core at various times after transplantation. The population of satellite cells in the core was reduced beginning at 18 hours and had virtually disappeared by 24-28 hours. This reduction did not appear to be the result of satellite cell death. Although there was abundant morphological evidence that myonuclei as well as myofiber cytoplasmic organelles were degenerating, there was little indication of satellite cell death in situ any time period studied. These studies suggest that satellite cells cannot survive, but migrate from the ischemic core to more peripheral regions of whole muscle transplants. In addition, they suggest that migration is an important aspect of the regeneration response in the free graft system and permits the myogenic population to contribute en masse to the centripetal wave of regeneration from the time it is initiated at the muscle periphery.
The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogen... more The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.
Fiber type classification of p-phenylene-diamine-stained transverse sections of rat skeletal musc... more Fiber type classification of p-phenylene-diamine-stained transverse sections of rat skeletal muscle correlated with electron microscopic observations demonstrate that satellite cells are not equally distributed among the various fiber types in either the EDL or soleus at 1 and 12 months of age. Of the three fiber types in the EDL, satellite cells are observed most frequently on Type IIA fibers, and at approximately equal frequencies on Type IIB and Type I fibers. Satellite cells in the soleus, in contrast to the EDL, are not often encountered on Type IIA fibers, but are preferentially associated with Type I fibers in that muscle. The frequency with which satellite cells are found on specific fiber types is distinct for the soleus and EDL and appears to correspond with their growth characteristics. The distribution of satellite cells according to fiber type does not provide an explanation for the differences in the percentage of satellite cell nuclei between the EDL and soleus.
Normal adult mouse tibialis anterior muscles were perfused continuously with 3H-thymidine for nin... more Normal adult mouse tibialis anterior muscles were perfused continuously with 3H-thymidine for nine days. Quantitation of the satellite cell population in these muscles reveals that not only is the frequency of satellite cell nuclei low, but that those present are mitotically quiescent.
The effect of the local anaesthetic Marcaine on muscle and non-muscle cell types was examined usi... more The effect of the local anaesthetic Marcaine on muscle and non-muscle cell types was examined using an in vitro assay. Each of the cell types examined (myotubes, myoblasts, fibroblasts and liver parenchyma) expressed morphological alterations when incubated in Marcaine-medium. Myotubes were the most sensitive of the cells studied and exhibited several pronounced membrane structural changes after short incubation periods in Marcaine-medium. The toxic effects of Marcaine were irreversible and the myotubes continued to degenerate despite being placed in fresh medium. Myoblasts and non-muscle cells, however, demonstrated a rapid recovery when removed from the Marcaine-medium. Since Marcaine is thought to complete with Ca++ for specific sites on cell membranes, it is proposed that the differential effects which were observed are dependent upon the level of calcium related activities being carried out by the cells.
5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a ... more 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.
Regenerative properties of age-associated changes in the intrinsic laryngeal muscles following in... more Regenerative properties of age-associated changes in the intrinsic laryngeal muscles following injury are unclear. The purpose of this study was to investigate the regenerative properties of the thyroarytenoid (TA) muscle in an aging rat model. The hypothesis was that following myotoxic injury, old animals would exhibit a decrease in mitotic activities of muscle satellite cells when compared with younger rats, suggesting reduced regenerative potential in the aging rat TA muscle. Animal group comparison. Regeneration responses following injury to the TA muscle were examined in 18 young adult, middle-aged, and old Fischer 344/Brown Norway rats. TA muscle fiber cross-sectional area (CSA), satellite cell mitosis (number/fiber), and regeneration index (CSA injured side/CSA noninjured side) were measured and compared across age groups. Young adult animals had a significantly higher regeneration index than the middle-aged and old groups. Within the lateral region of the TA muscle (LTA), the regeneration index was significantly higher in the young adult animals than in the middle-aged and old animals. The regeneration index of the medial TA was significantly higher than the LTA across all age groups. The regenerative capacity of the TA muscle is impaired with increasing age.
Expression of Becker-like and full-length human dystrophins was stable for at least 6 months in m... more Expression of Becker-like and full-length human dystrophins was stable for at least 6 months in mdx mouse muscle following intramuscular plasmid DNA injection. Intramuscular injection of a single plasmid DNA encoding both luciferase and dystrophin resulted in stable luciferase expression for at least 2 months in mdx muscle, whereas injection of plasmid DNA encoding only luciferase did not result in stable luciferase expression. These results suggest that expression of either full-length or Becker-like dystrophins protects mdx mouse myofibers from degeneration.
European Journal of Applied Physiology and Occupational Physiology, Aug 1, 1998
Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cel... more Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P < 0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P < 0.05) BrdU labeling in uninjured soleus muscles compared to weight-bearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P > 0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers.
The ability of satellite cells to survive the ischemic conditions at the core of orthotopically f... more The ability of satellite cells to survive the ischemic conditions at the core of orthotopically free grafted rat extensor digitorum longus muscles was examined. Cell cultures of isolated core and peripheral regions of whole muscle grafts maintained in vivo for more than 24 hours indicated that no viable cells were present in the core, whereas the number of cells from the peripheral region was greatly increased. Muscles were examined with the electron microscope to determine the fate of satellite cells of the core at various times after transplantation. The population of satellite cells in the core was reduced beginning at 18 hours and had virtually disappeared by 24-28 hours. This reduction did not appear to be the result of satellite cell death. Although there was abundant morphological evidence that myonuclei as well as myofiber cytoplasmic organelles were degenerating, there was little indication of satellite cell death in situ any time period studied. These studies suggest that satellite cells cannot survive, but migrate from the ischemic core to more peripheral regions of whole muscle transplants. In addition, they suggest that migration is an important aspect of the regeneration response in the free graft system and permits the myogenic population to contribute en masse to the centripetal wave of regeneration from the time it is initiated at the muscle periphery.
The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogen... more The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.
Fiber type classification of p-phenylene-diamine-stained transverse sections of rat skeletal musc... more Fiber type classification of p-phenylene-diamine-stained transverse sections of rat skeletal muscle correlated with electron microscopic observations demonstrate that satellite cells are not equally distributed among the various fiber types in either the EDL or soleus at 1 and 12 months of age. Of the three fiber types in the EDL, satellite cells are observed most frequently on Type IIA fibers, and at approximately equal frequencies on Type IIB and Type I fibers. Satellite cells in the soleus, in contrast to the EDL, are not often encountered on Type IIA fibers, but are preferentially associated with Type I fibers in that muscle. The frequency with which satellite cells are found on specific fiber types is distinct for the soleus and EDL and appears to correspond with their growth characteristics. The distribution of satellite cells according to fiber type does not provide an explanation for the differences in the percentage of satellite cell nuclei between the EDL and soleus.
Normal adult mouse tibialis anterior muscles were perfused continuously with 3H-thymidine for nin... more Normal adult mouse tibialis anterior muscles were perfused continuously with 3H-thymidine for nine days. Quantitation of the satellite cell population in these muscles reveals that not only is the frequency of satellite cell nuclei low, but that those present are mitotically quiescent.
The effect of the local anaesthetic Marcaine on muscle and non-muscle cell types was examined usi... more The effect of the local anaesthetic Marcaine on muscle and non-muscle cell types was examined using an in vitro assay. Each of the cell types examined (myotubes, myoblasts, fibroblasts and liver parenchyma) expressed morphological alterations when incubated in Marcaine-medium. Myotubes were the most sensitive of the cells studied and exhibited several pronounced membrane structural changes after short incubation periods in Marcaine-medium. The toxic effects of Marcaine were irreversible and the myotubes continued to degenerate despite being placed in fresh medium. Myoblasts and non-muscle cells, however, demonstrated a rapid recovery when removed from the Marcaine-medium. Since Marcaine is thought to complete with Ca++ for specific sites on cell membranes, it is proposed that the differential effects which were observed are dependent upon the level of calcium related activities being carried out by the cells.
5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a ... more 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.
Regenerative properties of age-associated changes in the intrinsic laryngeal muscles following in... more Regenerative properties of age-associated changes in the intrinsic laryngeal muscles following injury are unclear. The purpose of this study was to investigate the regenerative properties of the thyroarytenoid (TA) muscle in an aging rat model. The hypothesis was that following myotoxic injury, old animals would exhibit a decrease in mitotic activities of muscle satellite cells when compared with younger rats, suggesting reduced regenerative potential in the aging rat TA muscle. Animal group comparison. Regeneration responses following injury to the TA muscle were examined in 18 young adult, middle-aged, and old Fischer 344/Brown Norway rats. TA muscle fiber cross-sectional area (CSA), satellite cell mitosis (number/fiber), and regeneration index (CSA injured side/CSA noninjured side) were measured and compared across age groups. Young adult animals had a significantly higher regeneration index than the middle-aged and old groups. Within the lateral region of the TA muscle (LTA), the regeneration index was significantly higher in the young adult animals than in the middle-aged and old animals. The regeneration index of the medial TA was significantly higher than the LTA across all age groups. The regenerative capacity of the TA muscle is impaired with increasing age.
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