Skeletal muscle growth is accomplished chiefly through the actions of satellite cells, a heterogeneous population that includes the adult muscle stem cell. Located adjacent to a mature muscle fiber, satellite cells typically reside in a quiescent state. Little information exists detailing satellite cell regulation of reversible G(0). One member of the mitosin family of centromere proteins, LEK1 (leucine/glutamic acid/lysine protein 1), is present in the nucleus of nondividing mouse satellite cells. The objective of this study was to evaluate LEK1 as a marker of quiescent bovine satellite cells (BSC) in vitro and in vivo. The BSC were isolated from young bull calves (< or =7 d) and cultured in vitro for up to 9 d before fixation and immunostaining for LEK1. Results demonstrated that all myogenic cells contain the protein, with immunostaining primarily within the nucleus and immediate perinuclear region. Immunocytochemical detection of LEK1 in cryosections of mature cows revealed that the protein was present in a fraction of satellite cells and muscle fiber nuclei. Approximately 20% of Pax7-expressing satellite cells contained LEK1. An equivalent percentage of myonuclei, as defined by nuclei within a dystrophin boundary, contained nuclear LEK1. To gain insight into the functional role of LEK1, BSC were transiently transfected with plasmids coding for putative dominant inhibitory LEK1 proteins [DeltaLEK1(991) and DeltaLEK1(911)] and evaluated for cell proliferation. Both forms of DeltaLEK1 inhibited (P < 0.05) BSC proliferation, as indicated by a decrease in Ki67 immunopositive cells. In C2C12 myoblasts, DeltaLEK1(911) inhibited (P < 0.05) myoblast determination protein 1 (MyoD)-directed muscle gene transcriptional activity; DeltaLEK1(991) had no effect on TnI-Luc transcription. By contrast, both DeltaLEK1 fusion proteins inhibited myogenin expression in BSC without disrupting myoblast fusion. These results provide evidence that LEK1 serves to coordinate proliferation and differentiation in myogenic cells. Coupling the immunostaining pattern and functional data, we propose that LEK1 may serve as a useful marker for satellite cells that are preparing to fuse into adjacent fibers as well as an indicator of recently added myonuclei.

The goal of this study was to evaluate the effects of ractopamine-hydrochloride (RAC) supplementation on the myosin heavy chain isoform distribution and shelf-life properties of muscles from beef top round, knuckle, and loin. Thirty-four steer carcasses were selected from 4 separate slaughter groups. Within each slaughter group (3 groups, n = 8; 1 group, n = 10), steers were separated into pens (n = 8) and fed 0 or 200 mg x animal(-1) x d(-1) of RAC for the final 28 d of feeding. Seventy-two hours postmortem, the longissimus lumborum, semimembranosus (SM), adductor, gracilis, vastus lateralis (VL), and rectus femoris were removed from each carcass. A subsample of each muscle was collected for immunohistochemical analysis. Whole muscles were vacuum packaged and wet aged at 1 +/- 2 degrees C for 13 d before processing into steaks for a 5-d simulated retail display study. Daily, steaks were analyzed for reduction of nitric oxide metmyoglobin, lean color, fat color, and surface discoloration. Objective measures of metmyoglobin, oxymyoglobin, L*, a*, and b* values were recorded daily. Ractopamine significantly (P < 0.05) changed the fiber type isoform distribution in all muscles except the SM. The VL and gracilis presented the greatest fiber type switch with approximately 21% of type I fibers switching to type IIA fibers. However, the fiber type shifts induced by RAC supplementation had little to no effect on subjective and objective color measurements during the 5-d retail display period. Metmyoglobin and oxymyoglobin accumulation, L*, a*, and b*-values were not affected (P > 0.05) by RAC supplementation. Percent nitric oxide metmyoglobin reduction data indicate that reducing ability of RAC-treated steaks from the adductor and longissimus lumborum were significantly affected (P < 0.05). Visual panel data suggest that RAC tended (P < 0.10) to have the most detrimental effect on the lean color and surface discoloration scores of steaks from the VL during the last 3 d of display. Ractopamine significantly (P < 0.05) increased the surface discoloration of the rectus femoris and SM on d 5. Although RAC supplementation had no effect on objective color measurements, subjective measurements indicate that it may have some effect on surface discoloration of some muscles.

The objective of this study was to evaluate the effects of coadministration of ractopamine-HCl (RAC) and trenbolone acetate plus estradiol (TBA) on LM fiber cross-sectional area (CSA), diameter, fiber-associated myonuclei, and satellite cell number. Culled crossbred beef cows (n = 98; 11 +/- 1.8 yr old; BCS 4.3 +/- 0.03) from a single ranch in south Florida were fed a concentrate diet for 92 d in a 2 x 2, randomized block design. Cows were blocked by BW on arrival into light (initial BW = 369.75 +/- 2.68 kg and end BW = 501.96 +/- 6.90 kg) and heavy (initial BW = 418.31 +/- 2.75 kg and end BW = 522.15 +/- 7.09 kg) groups before assignment to treatment. Factors included dietary treatment (0 or 15 ppm) and implant status (0 or 80 mg of trenbolone acetate + 16 mg of estradiol). Ractopamine was provided in the diet to 2 pens or half the treatments during the final 35 d of feeding. Cows were slaughtered on d 92. Forty-eight hours postmortem, the 6th-rib portions of the LM were obtained from 10 randomly selected carcasses from each treatment group (n = 40). Cryosections (12 mum) were immunostained for dystrophin and myosin heavy chain I or II for the measurement of fiber CSA and type, respectively. Fiber-associated nuclei and satellite cell numbers were measured in serial cryosections. There was a RAC x TBA interaction (P < 0.05). Type I fiber CSA and diameter were increased (P < 0.05) by TBA and RAC. Type I CSA and diameter were larger (P < 0.05) in TBA + RAC than RAC only. Type II fiber CSA and diameter were not affected by TBA (P = 0.48), RAC (P = 0.15), or TBA + RAC (P = 0.60). Satellite cell numbers and fiber-associated nuclei were not affected (P > 0.05) by implant status or ractopamine supplementation. These results indicate that TBA and RAC preferentially increase the size of type I fibers in cull cows.