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It is well established that heat stress (HS) negatively affects growth rate in swine. Although reduced feed intake undoubtedly plays a significant role in this reduction, studies in laboratory animals and other nonswine species indicate muscle growth also is affected by HS-related alterations in muscle physiology. Evidence is now emerging that heat shock proteins (Hsp), produced in response to HS and other types of cellular stress, may play an important role in regulating the rate and efficiency of muscle growth. Because muscle satellite cells play a crucial role in postnatal muscle growth, the effects of HS on rates of satellite cell proliferation, protein synthesis, and protein degradation play an important role in determining the rate and extent of muscle growth. Consequently, in the current study we have examined the effects of mild HS (40.5°C for 48 h) on the rates of proliferation, protein synthesis, and protein degradation and on quantities of Hsp90, Hsp70, and Hsp25/27mRNA and protein in cultured porcine muscle satellite cells (PSC). Mild HS of PSC cultures resulted in 2.5-, 1.4-, and 6.5-fold increases (P< 0.05) in the abundance of Hsp90, Hsp70, and Hsp25/27mRNA, respectively, relative to control cultures. Abundance of Hsp 90, 70, and 25/27 proteins was also increased in HS PSC cultures compared with those in control cultures. Proliferation rates in HS PSC cultures were 35% less (P< 0.05) than those in control cultures. Protein synthesis rates in HS-fused PSC cultures were 85% greater (P< 0.05) than those in control cultures, and protein degradation rates in HS-fused PSC were 23% less (P< 0.05) than those in control cultures. In light of the crucial role satellite cells play in postnatal muscle growth, the HS-induced changes we have observed in rates of proliferation, protein turnover, and abundance of HspmRNA and Hsp protein in PSC cultures indicate that mild HS affects the physiology of PSC in ways that could affect muscle growth in swine.

We previously showed that a combined trenbolone acetate (TBA)/estradiol-17β (E2) implant significantly increases IGF-I mRNA levels in the LM of feedlot steers by 28 d after implantation. Here we compare the effects of E2 (25.7 mg), TBA (120 mg), and combined TBA (120 mg)/E2 (24 mg) implants on IGF-I, IGF-I receptor (IGFR-1), estrogen receptor (ER)-α and androgen receptor (AR) mRNA levels in the LM of steers. Twenty yearling crossbred steers with an average initial BW of 421.1 ± 3.6 kg were stratified by BW and randomly assigned to 1 of 4 treatments: 1) nonimplanted, control; 2) implanted with TBA and E2; 3) implanted with E2; or 4) implanted with TBA. Steers were weighed weekly starting on d 0, and muscle biopsy samples were taken from each steer on d 0 (before implantation), 7, 14, and 28. Ribonucleic acid was prepared from each sample and real-time reverse transcription-PCR was used to determine the levels of IGF-I, IGFR-1, ER-α, and AR mRNA. Body weight of implanted steers, adjusted by using d-0 BW as a covariant, tended (P= 0.09) to be greater than that of control steers. On d 7 and 28, IGF-I mRNA levels were greater (58 and 78%, respectively; P< 0.009) in E2-implanted animals than in control steers. Similarly, on d 28 the LM IGF-I mRNA level was 65% greater (P= 0.017) in TBA/E2-implanted steers than in control animals. In contrast, the TBA implant did not increase (P= 0.99) LM IGF-I mRNA levels after 28 d of implantation. Muscle IGFR-1, AR, and ER-α mRNA levels were not different (P> 0.47) in any of the treated groups compared with the control group. These data suggest that E2 is responsible for the increased muscle IGF-I mRNA level observed in steers implanted with a combined TBA/E2 implant.

IGF-binding protein-5 (IGFBP-5) is produced by porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of this study was to produce recombinant porcine IGFBP-5 (rpIGFBP-5) and assess its IGF-I-dependent and IGF-I-independent actions on the proliferation of PEMCs. To accomplish this, we have expressed porcine IGFBP-5 in the baculovirus system, purified and characterized the expressed rpIGFBP-5 and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography, suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P>0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P>0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5, demonstrating the IGF-independent activity that rpIGFBP-5 possesses in PEMCs, since Long-R3-IGF-I is an IGF analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby can stimulate proliferation. The anti-rpIGFBP-5 IgY produced against rpIGFBP-5 specifically recognized native porcine IGFBP-5 in PEMC media that also contained porcine IGFBP-2, -3, and -4. This antibody is capable of neutralizing the effects of both rpIGFBP-5 and endogenously produced porcine IGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots. The production of rpIGFBP-5 and a neutralizing antibody to porcine IGFBP-5 provides a powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitromuscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo.

Myogenic cells isolated from lamb fetuses (approximately mid-gestation) exhibited a concentration-dependent decrease in myogenic cell proliferation in response to transforming growth factor (TGF) beta-1 (P< .001). Half-maximal inhibition of proliferation occurred at approximately .05 ng of TGF beta-1/mL and maximal inhibition of proliferation occurred at approximately .1 ng of TGF beta-1/mL. The specificity of this inhibition was confirmed by neutralization of the activity following exposure to a TGF beta antibody. The TGF beta-1 also suppressed proliferation of ovine satellite cells isolated from 5-d-old lambs (P< .0035), but to a lesser extent than observed for embryonic cells. In contrast, TGF beta-1 did not significantly suppress serum-stimulated proliferation of ovine satellite cells isolated from 30- or 150-d-old lambs. Similarly, TGF beta-1 did not suppress proliferation of skeletal muscle fibroblast-like cells isolated from either fetal lambs or 150-d-old lambs. In fact, proliferation of fibroblast-like cells derived from embryonic ovine muscle was enhanced by exposure to TGF beta-1 at all levels tested; however, a concentration-dependent response was not observed. Media transfer experiments showed that conditioning of culture media by postnatally derived cells did not render TGF beta-1 inactive. The studies described in this manuscript suggest that sensitivity of ovine myogenic cells to the antiproliferative effect of TGF-beta may vary with the stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)