Edward Schultz

Skeletal muscle satellite cells: Changes in proliferation potential as a function of age

Skeletal muscle satellite cells were isolated from soleus and extensor digitorum longus muscles of Sprague-Dawley rats at selected ages between 6 days and 30 months and grown in cell culture. Cells at each donor age were monitored individually to determine the number of progeny they were capable of producing. Under identical in vitro conditions, the average number of progeny produced was inversely proportional to donor age. Hence, the proliferation potential of satellite cells decreases with age.

Absence of exogenous satellite cell contribution to regeneration of frozen skeletal muscle

SummaryRat extensor digitorum longus (EDL) muscles were repeatedly frozen and thawed to kill completely all cellular constituents. Within four days, blood vessels and phagocytic cells invaded the muscles. Migrating or circulating myoblasts were not among the invading cells, and could be induced to invade the frozen muscle only when a physical bridge was created with an adjacent intact muscle. A further requirement for migration was that the connective tissue investments of both the frozen EDL and adjacent muscle had to be disrupted. This study demonstrates that regeneration of a muscle is primarily dependent upon the intrinsic satellite cell population, although under some circumstances recruitment of extrinsic cells is possible.

Effects of skeletal muscle regeneration on the proliferation potential of satellite cells

Skeletal muscle satellite cells are myogenic stem cells that function to repair damaged muscle fibers. Participation of satellite cells in a regeneration response following muscle injury results in a significant reduction in their cumulative proliferation potential. The magnitude of the reduction is proportional to the number of regeneration responses in which the cells participate.

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin.

SummaryMyofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against α- and γ-actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the “sarcomeric” proteins α-actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of “sarcomeric” proteins in regard to myofibrillogenesis.

AN ANATOMIC STUDY OF THE RAT LARYNX : ESTABLISHING THE RAT MODEL FOR NEUROMUSCULAR FUNCTION

The gross and microscopic anatomy of the rat larynx was studied with particular attention to myology and neuromuscular structures to further validate it as a model to evaluate morphologic and functional changes induced by botulinum injection. A laryngeal alar cartilage (LAlC), alar cricoarytenoid (ACA) muscle, and a superior cricoarytenoid muscle (SCA) were identified as anatomic structures not previously described. Two portions (medial and lateral) of the thyroarytenoid muscle (TA) were distinguished. The function of the ACA was suggested to be similar to the aryepiglottis muscle in humans and the function of the SCA was suggested to be similar to the human interarytenoid muscle. The predominant pattern of motor endplate (MEP) distribution in rat laryngeal muscles (posterior cricoarytenoid, lateral cricoarytenoid, cricothyroid, and SCA) was to have MEPs concentrated mostly at the midbelly of muscle where they were distributed throughout the cross-sectional area of the midbelly. The TA and ACA differed from this pattern. The lateral TA had MEPs concentrated at the anterior third of its belly and those of the medial TA were located at the midbelly. Motor endplates in the ACA were located mostly at the posterior portion of muscle. Muscle fiber-typing showed subtle differences between the intrinsic laryngeal muscles. Fast fibers were predominant in the rat laryngeal muscles. This study supports the expanded use of rats in studies of laryngeal neuromuscular function and disease in humans.

Hyperplasia of jejunal smooth muscle in the myenterically denervated rat

SummaryApplication of a cationic surfactant, benzalkonium chloride, to the serosa of rat jejunum results in an increase in thickness of both longitudinal and circular smooth muscle layers. The increase in thickness is due primarily to an increase in the number of smooth muscle cells (hyperplasia). Little cellular hypertrophy was observed. The time sequence of surfactant-induced effects on the muscle layers was determined. Within 24 h, total destruction of the longitudinal muscle and partial destruction of the circular muscle was evident. The myenteric plexus was also necrotic; however, the submucosal plexus remained intact. By 48 h after surfactant treatment, the smooth muscle cells remaining in the circular muscle layer had begun to divide, as indicated by the presence of mitotic figures and incorporation of 3H-thymidine. A repopulation of the longitudinal muscle layer began at this time, apparently the result of migration of cells arising in the circular muscle layer. By 5 days post-treatment, both muscle layers had regenerated to their original states. The myenteric plexus was totally absent. The denervated smooth muscle cells proceeded to divide until approximately day 15, resulting in hyperplasia of both muscle layers. Between 15 and 105 days, the number of muscle cells in the circular layer progressively declined, eventually returned to the value seen in control tissue. In contrast, the number of smooth muscle cells in the longitudinal layer remained elevated through the period of study (165 days). We hypothesize that the smooth muscle hyperplasia observed after serosal benzalkonium chloride application results from loss of the myenteric nerves.

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the α- and γ-isoforms of actin☆

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.

NITRIC OXIDE DONORS, SODIUM NITROPRUSSIDE AND S-NITROSO-N- ACETYLPENICILLAMINE, STIMULATE MYOBLAST PROLIFERATION IN VITRO

SummaryNitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 µM) of donor concentrations. However, 50 µM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 µM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.

Satellite cells express distinct patterns of myogenic proteins in immature skeletal muscle

Satellite cells are the myogenic cells lying between the myofiber sarcolemma and basal lamina. The objective of this study was to determine the expression patterns of MyoD, myogenin, and Pax7 within the satellite cell population in the growing rat soleus and extensor digitorum longus (EDL) muscles. Secondly, the expression of the myogenic markers was also studied within the interstitial cell compartment and myonuclei. It was discovered that the soleus contained a higher number of Pax7, MyoD, or myogenin‐positive nuclei compared with the EDL. Similarly, myogenin was expressed at a lower level in the myonuclei of the soleus compared with the EDL, and myogenin was expressed at a higher level in the interstitial compartment of the soleus compared with the EDL. When interstitial nuclei, myonuclei, and double‐labeled nuclei were used in the estimate of the satellite cell population, it was discovered that approximately of 13% of the myofibers in a transverse section of the soleus muscle and 4.1% of EDL myofibers exhibit a labeled satellite cell nucleus. Overall, results from this study suggest that expression patterns of these markers vary predictably among muscles with different growth dynamics and phenotypic characteristics. Developmental Dynamics 235:3230–3239, 2006.

Muscle fiber-type changes induced by botulinum toxin injection in the rat larynx.

This study examined muscle fiber-type alterations after single or multiple botulinum toxin (BT) injections to better understand possible morphologic changes induced by therapeutic BT injections in patients with spasmodic dysphonia. Muscle fiber staining was accomplished in rat intrinsic laryngeal muscles with antibodies to specific myosin heavy chains. Results indicated that the typical baseline distributions of type II muscle fibers (ie, types IIa, IIb, IIx, and IIL) were altered by BT injection, while no change was observed in type I fibers. Embryonic fibers were observed only along the needle insertion site at 7 days post BT injection. Although inferences from these animal data to human neuromuscular function must be made with caution, our findings provide insight into the possible cellular and molecular changes characterizing BT-injected muscles.