Optimization of Culture Conditions for Determining Protein ...

The Professional Animal Scientist 22 (2006):283–291

Optimization of Culture Conditions for Determining Protein Degradation in Myoblasts Using Extracts of Adult Bovine Muscle Treated with Muscle Enhancing 1 Compounds J. L. MONTGOMERY,*2 W. M. HARPER, Jr.,* M. F. MILLER,* K. J. MORROW, Jr.,†3 K. W. BRADEN,*4 and J. R. BLANTON, Jr.*5 *Department of Animal Science, Texas Tech University, Lubbock, TX †Department of Cell Biology and Biochemistry, Texas Tech Health Sciences Center, Lubbock, TX

Abstract The objective of this study was to optimize a methodology in which muscle extracts from animals treated with growth agents are added to growth media and then applied to muscle cell cultures to determine effects of growth agents on indirect cellular protein degradation. Experiments were designed as a 2 × 2 × 2 × 2 factorial arrangement of cell type (C2C12 myoblasts or primary bovine muscle cell

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This research was partially supported by Texas Tech University and the Department of Animal and Food Sciences. This journal paper is no. T-5-447 from the Texas Tech University Agricultural Research Program. 2 Current address: Intervet Inc., 29160 Intervet Lane, PO Box 318, Millsboro, DE. 3 Current address: Meridian Biosciences, Inc., 3471 River Hills, Dr., Cincinnati, OH. 4 Current address: Auburn University, 142 Upchurch Hall, Auburn, AL. 5 To whom correspondence should be addressed: [email protected]

cultures), growth medium [(Dulbecco’s Modified Eagle’s Medium (DMEM)or skeletal muscle cell basal medium (SKBM)], muscle extract medium [potassium phosphate buffer (KPB) or prerigor extraction buffer], and β-adrenergic agonist treatments of the bovine (control or treated) to determine differences in cellular protein degradation. There was a treatment media × β-adrenergic agonist treatment × cell type interaction (P < 0.0001). C2C12 myoblasts with the β-adrenergic agonist in DMEM media had less protein degradation than controls in DMEM (P < 0.05). Primary bovine muscle cell cultures treated with the β-adrenergic agonist treatment in DMEM media had greater protein degradation than did controls in DMEM (P < 0.05). However, primary bovine muscle cell cultures treated with the β-adrenergic agonist treatment in SKBM media had reduced protein degradation than did controls in SKBM (P < 0.05). There was a treatment media × β-adrenergic agonist treatment × muscle cell extraction buffer interaction (P = 0.04). The β-agonist treatment de-

creased protein degradation when muscle samples were extracted with KPB or prerigor extraction buffer and made with SKBM culture media (P < 0.05). The results indicated SKBM and C2C12 myoblasts had the most consistent differences for showing relative β-adrenergic agonist treatment effects on indirect protein degradation. Key words: skeletal muscle, cell culture, protein degradation

Introduction Muscle protein turnover is a ratio dependent on both protein synthesis and protein degradation. Muscle protein degradation has been measured by constant infusion with radiolabeled tracers (Mulvaney et al., 1985; Zhang et al., 1996), flood administering or injection of radioactively-labeled compounds to live animals (Lorenzen et al., 2000), and perfusion of tissue sections with radiolabeled tracers (Ward and Buttery, 1979). Meth-

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ods employed to measure protein turnover in livestock are typically expensive and time consuming due to enormous isotope and animal costs (Skjaerlund et al., 1988). Also, a number of methods used to measure protein turnover are only able to measure either protein synthesis or degradation rates in a single study. Thus, a rapid method that allows researchers to determine treatment effects on relative indirect protein synthesis and degradation rates simultaneously in bovine would be highly beneficial. Another report from this laboratory discusses the measurement of protein synthesis using muscle extracts from bovine treated with pharmaceutical agents (Montgomery et al., 2006). Herein a similar method to measure protein degradation is discussed. Because both methods utilize the same muscle extracts, cell culture system, and supplies, experimental outcomes of treated animals can be determined for either protein synthesis, protein degradation or both simultaneously. Thus, an in vitro incubation system is employed using muscle extracts from treated animals that are added to growth media and applied to cell cultures to screen for relative changes that occur in vivo in response to hormonal, pharmaceutical, or physiological treatments. The results listed indicate how cell culture conditions can be optimized for determining relative treatment differences in protein degradation. However, the methods discussed demonstrated only indirect effects on protein degradation, and further studies will be needed to determine exactly how each individual compound being tested directly affects muscle protein degradation.

Materials and Methods Chemicals. Amino acid mixture L[14C(U)] (NEC-445E) was obtained from Perkin Elmer Life and Analytical Sciences, Inc. (Wellesley, MA). Dulbecco’s Modified Eagle’s Medium (DMEM) and gentamycin sulfate were purchased from Invitrogen Corp. (Carlsbad, CA). Fetal bovine se-

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rum (FBS) was purchased from Atlanta Biologicals (Nocross, GA). Skeletal muscle cell basal medium (SKBM) was purchased from BioWhittaker (Walkersville, MD). RPMI-1640 cell culture media and antibiotic and antimycotic (penicillin, 100 U/mL; streptomycin, 100 µg/mL) were purchased from Sigma Chemical Corp. (Saint Louis, MO). Protein concentration determination was made using a protein assay kit from Bio-Rad Laboratories (Hercules, CA). All other chemicals and reagents were of analytical grade and were readily available from commercial sources. Animals and Muscle Sample Preparations. Steers from a control group of cattle (n = 10) and from a treated group (n = 10) fed a diet with a commercially available β-adrenergic agonist compound for 30 d were harvested at a commercial processing plant after a 5-d withdrawal; muscle samples were collected and prepared as described elsewhere (Montgomery et al., 2006). From each animal, two 10-g muscle samples were collected: 1 sample was extracted in 30 mL of icecold 0.01 M potassium phosphate buffer (KPB, pH 7.4), and the other sample was extracted in 30 mL of icecold prerigor extraction buffer (50 mM Tris, 10 mM EDTA, pH 8.3). Tissue samples were homogenized for 45 sec and centrifuged for 30 min at 40,000 × g, prepared as elsewhere described (Montgomery et al., 2006), and the protein concentration determined according to Layne (1957). Treatment Media. Two different cell culture media preparations were made for each of the 20 animals and extraction buffers. In a 15-mL conical vial, muscle extract was added at a level of 400 µg protein/mL into treatment media consisting of SKBM with 15% FBS, 1% penicillin and streptomycin, and 0.1% gentamycin (vol/ vol) The muscle extract averaged approximately 10% of the final level in media. In a second 15-mL conical vial, muscle extract was added at a level of 400 µg protein/mL into treatment media consisting of DMEM with 15% FBS, 1% penicillin and

streptomycin, and 0.1% gentamycin (vol/vol) The treatment media were then filter sterilized through a 0.22 µM filter and stored at 4°C until application to cell cultures. Isolation and Culture of Primary Bovine Muscle Cell Cultures. Primary bovine muscle cell cultures were isolated and cultured as outlined in detail by Kerth (1999) and Pollard et al. (2001). Primary bovine muscle cell cultures were prepared following procedures outlined by Hembree et al. (1991) as adapted by Montgomery et al. (2002). To test for fibroblast contamination, primary bovine muscle cultures were grown in DMEM in 6-cm petri dishes at a density of 15,000 cells/ cm2. At approximately 80% confluency the DMEM media was replaced with fusion media, which consisted of the DMEM and antibiotics as described above and 2% (vol/vol) horse serum. Fibroblast contamination was presumed low because 60 to 80% of the nuclei fused to form myotubes when incubated in fusion media for 8 d. Cell Cultures. C2C12 myoblasts (CRL-1772) were purchased from American Type Culture Collection (ATCC; Manassas, VA). C2C12 myoblasts and primary bovine muscle cell cultures were thawed from liquid nitrogen storage and transferred to DMEM growth media (media containing 15% FBS, 1% penicillin and streptomycin, and 0.1% gentamycin; vol/vol) and placed into 75-cm2 canted neck tissue cell culture flasks and incubated in a culture incubator (37°C, humidified environment, 5% CO2, 95% air). Cell cultures were grown to approximately 80% confluence in the cell culture flasks and subcultured. Cells were added to 24-well culture plates at a density of 2,500 cells/cm2 in 1 mL of growth media and incubated in a culture incubator for approximately 48 h until they reached 60 to 70% confluence. Protein Degradation Assay. The procedure for determining the rate of protein degradation as measured by a pulse-chase uptake of labeled amino

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acids was conducted as described by Ballard et al. (1986) and described in full detail by Montgomery et al. (2002). Briefly, C2C12 myoblasts and primary bovine muscle cultures were subcultured and grown in 24-well plates as described above. The DMEM growth media was aspirated and replaced with 1.0 mL of either of the DMEM or SKBM growth media. For all plates, 1 µCi of 14C-labeled amino acid mixture was added to label each well. Plates were incubated in the cell culture incubator for 24 h. After the 24-h labeling period, media from each well was removed and discarded. Each well was gently rinsed twice with 1 mL of fresh respective growth media. The rinse media was removed and discarded and treatment media added. For each of the different treatment media and different muscle extracts per animal, 3 wells were treated for each of the cell culture types. Cells were incubated in treatment media at 37°C for 4 h, after which treatment media was removed and discarded. Each well was gently rinsed with 1 mL of fresh respective growth media twice. Rinse media was removed and discarded. For each well, 1.0 mL of the previously incubated treatment media was added. Plates were incubated once more at 37°C for 2 h. From each well, 0.4 mL of treatment media was removed and placed in a 1.8-mL cryovial. To this cryovial, 0.4 mL of ice-cold 20% trichloroacetic acid (TCA; wt/vol) was added; vials were capped and vortexed, producing a TCA-precipitated media, which was stored at 4°C overnight. Additionally, 0.4 mL of medium from each of the 24 wells was removed and placed in a scintillation vial. Five mL of scintillation fluid was added and radioactivity in disintegrations per minute (DPM) determined using a scintillation counter. The remaining medium from each well was then removed and stored in a cryovial for protein determination (media sample; Bradford, 1976). Each well was gently rinsed twice with 1 mL of fresh growth media. Rinse media was re-

moved and discarded. To each well, 0.5 mL of NaOH was added and the plates were incubated at 37°C for 2 h to lyse the cells, after which 0.5 mL of 20% (wt/vol) cold TCA was added to each well to precipitate cellular proteins. Plates were stored at 4°C for at least 12 h. Each well was gently agitated and the cell layer of precipitated proteins were scraped from the culture wells with a rubber policeman. For each well, 0.2 mL of precipitated protein solution of the cell layer was removed and the protein concentration of each well was determined in quadruplicate according to Bradford (1976). The wells were rinsed twice with TCA. Pooled TCA rinses for each well were collected on a glass fiber filter using a vacuum collection manifold to retain the precipitated proteins. Plates and filters were rinsed with 5% (wt/vol) cold TCA. From each well of TCA-precipitated protein sample, 0.1 mL of solution was removed and the protein concentration of the TCA-precipitated protein samples was determined in quadruplicate according to Bradford (1976). The remaining contents of the TCA-precipitated protein sample vials were collected on glass fiber filters. The vials and filters were rinsed with 5% (wt/ vol) cold TCA. Filters were placed in scintillation vials, and the DPM for each filter were determined using a scintillation counter. The protein concentration of the media samples also was determined according to Bradford (1976). Percent protein degradation was calculated as [DPM TCA precipitate/(µg protein per 700 µl)] × 100]/[(DPM media/µg protein per 400 µl) + (DPM cell layer/µg protein per 800 µl) + (DPM TCA precipitate/µg protein per 700 µl)]. Percent protein degradation per well was determined and the triplicates were averaged to determine percent protein degradation for each individual treatment replicate. Assay Validation. A number of procedures were implemented to validate the assay prior to conducting these experiments. The effect of reaction

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time was tested for linearity and has been previously published: degradation rates were found to decrease linearly over a 36-h reaction time (Montgomery et al., 2002). Additionally, the effect of protein concentration of the muscle extract was tested to determine effects on degradation rates. In the current study, 400 µg protein/mL into treatment media was used. In assay validation tests, protein concentration of 100 to 600 µg protein/ mL into treatment media was tested. The protein concentration was found to linearly increase (r2 = 0.93) degradation rates (data not shown). Although 400 µg of protein concentration of the muscle extract per milliliter media used by Haugk et al. (1995) was used in a different assay method, the same concentration appeared to be appropriate for the current indirect protein degradation methodology. Thus, the reaction time and muscle extract concentration used in these protein degradation assays seemed to be appropriate. Data analysis. The experiment was designed as a 2 × 2 × 2 × 2 arrangement of cell type (C2C12 myoblasts or bovine muscle cell cultures), growth medium (DMEM or SKBM), muscle extract medium (KPB or prerigor extraction buffer), and β-agonist feeding treatment of the bovine (control or treated). Percent protein degradation was determined in triplicate from each animal and individual treatment and expressed as mean ± standard error of the mean. Treatment interactions, calculation of means, and standard error of the means were determined by analysis of variance procedures on PC SAS (SAS Inst., Inc., Cary, NC) and mean separation was conducted with Fisher’s protected Least Significance Difference. In all cases, the criterion for statistical significance was P < 0.05.

Results and Discussion There was a main effect of cell type on percent protein degradation (P < 0.005). Primary bovine muscle cell cultures had a greater rate of protein

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Figure 1. Effect of cell type on percent protein degradation. C2C12 myoblasts and primary bovine muscle cell cultures were treated with muscle extracts from control bovine or bovine treated with a β-adrenergic agonist. a,bMeans with different letters differ (P < 0.05).

degradation (P < 0.05) than C2C12 myoblasts (Figure 1). There was a cell type × muscle extraction buffer interaction (P = 0.001). Protein degradation rates were greater (P < 0.05) in primary bovine muscle cell cultures than in C2C12 myoblasts when muscle samples were extracted in KPB (Figure 2). However, there were no differences (P > 0.05) in degradation rates between cell types when muscle samples were extracted in prerigor extraction buffer. There was not a cell type × cell culture media interaction

(P = 0.81; data not shown), a cell type × cell culture media × muscle extraction buffer interaction (P = 0.85; data not shown), a cell type × β-adrenergic agonist treatment interaction (P = 0.07; data not shown), or a cell type × muscle extraction buffer × β-adrenergic agonist treatment interaction (P = 0.51; data not shown). There was a main effect of β-adrenergic agonist treatment (P = 0.001) on protein degradation. β-Adrenergic agonist treatment decreased percent protein degradation in both primary bo-

Figure 2. Effect of cell type and muscle extraction buffer on percent protein degradation. C2C12 myoblasts and primary bovine muscle cell cultures were treated with muscle extracts from control bovine or bovine treated with a β-adrenergic agonist, extracted in potassium phosphate buffer (KPB) or prerigor extraction buffer. Cell type and muscle extraction buffer interacted (P = 0.001). a,bMeans with different letters differ (P < 0.05).

vine muscle cell cultures and C2C12 myoblasts (Figure 3) when compared to cultures treated with muscle samples from control cattle. However, there was not a main effect of muscle extraction buffer (P = 0.29; data not shown) or cell culture media (P = 0.85; data not shown). Also, there was not a cell culture media × muscle extraction buffer interaction (P = 0.79) or β-adrenergic agonist treatment × muscle extraction buffer interaction (P = 0.30; data not shown). There was a cell culture treatment media × β-adrenergic agonist treatment interaction (P < 0.0001) on percent protein degradation of both culture types. When treatment media was made with SKBM, protein degradation percentage was dramatically decreased (P < 0.05) by β-adrenergic agonist treatment when compared to control muscle samples made with SKBM (Figure 4). Treatment media with DMEM resulted in no differences in protein degradation rates (P > 0.05) between β-adrenergic agonist and control treatments. There was a treatment media × β-adrenergic agonist treatment × cell type interaction (P < 0.0001) on protein degradation rates. C2C12 myoblasts treated with DMEM β-adrenergic agonist treatment media had a lesser protein degradation rate than controls in DMEM (P < 0.05; Figure 5a), indicating that bovine cells were more sensitive. However, there was no difference in protein degradation rates in C2C12 myoblasts between control and β-adrenergic agonist treatments when samples were placed in SKBM. Primary bovine muscle cell cultures treated with DMEM β-adrenergic agonist treatment media had a greater protein degradation rate than controls in DMEM (P < 0.05; Figure 5b). However, primary bovine muscle cell cultures treated with SKBM β-adrenergic agonist treatment media had a lower protein degradation rates than controls in SKBM (P < 0.05; Figure 5b). There was a treatment media × βadrenergic agonist treatment < muscle cell extraction buffer interaction (P = 0.04) on protein degradation.


Figure 3. Effect of β-adrenergic agonist treatment on percent protein degradation. Muscle cells were treated with muscle extracts from control bovine or bovine treated with a β-adrenergic agonist. a,bMeans with different letters differ (P < 0.05).

Protein degradation was increased by β-adrenergic agonist treatment when muscle samples were extracted with KPB and placed in DMEM culture treatment media in comparison to control samples in KPB and DMEM (P < 0.05; Figure 6a). However, β-ad-

renergic agonist treatment decreased protein degradation when muscle samples were extracted with KPB and made with SKBM culture treatment media when compared with control samples in SKBM (P < 0.05; Figure 6a). Protein degradation was de-

Figure 4. Effect of cell culture media type and β-adrenergic agonist treatment on percent protein degradation. Bovine and C2C12 myoblast muscle cells were treated with muscle extracts from control bovine or bovine treated with a β-adrenergic agonist in Dulbecco’s Modified Eagle’s Medium (DMEM) or skeletal muscle cell basal medium (SKBM). Cell culture media type and β-adrenergic agonist treatment interacted (P = 0.0001).a–cMeans with different letters differ (P < 0.05).

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creased by β-adrenergic agonist treatment when muscle samples were extracted with prerigor extraction buffer and placed in SKBM culture treatment media in comparison to control samples in prerigor extraction buffer and SKBM (P < 0.05; Figure 6b). However, protein degradation was not affected (P > 0.05) by β-adrenergic agonist treatment when muscle samples were extracted with prerigor extraction buffer and made in DMEM culture treatment media (Figure 6b). Thus, β-adrenergic agonist treatment differences indicating decreased protein degradation appeared to be typical of the literature when SKBM cell culture media was employed for both extraction buffers and cell types (Mersmann, 1995, 1998). We have discussed an optimization of in vitro muscle cell culture conditions for determining relative indirect protein degradation levels using extracts of muscle from animals. In vivo studies that measure muscle protein degradation in livestock typically require the use of radiolabeled compounds (Ward and Buttery, 1979; Skjaerlund et al., 1988). These types of in vivo experiments are expensive, time consuming, and labor intensive. A consequence of the expense is that muscle protein degradation experiments rarely allow for protein degradation determinations on large numbers of animals, especially in bovine, whereas the current method allows for large numbers due to its economy. β-Adrenergic receptors have been shown to be present on the surface of almost every type of mammalian cell (Mersmann, 1998). Norepinephrine and epinephrine are endogenous physiologically active β-adrenergic agonists. A typical physiological response of a β-adrenergic agonist binding to a β-adrenergic receptor in livestock is a modification of growth with increased accretion of skeletal muscle and decreased accretion of fat (Mersmann, 1998). A number of researchers have shown that β-adrenergic agonists increase muscle protein synthesis and decrease muscle protein

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Figure 5. Effect of cell culture media type, β-adrenergic agonist treatment, and cell type on percent protein synthesis. (a) C2C12 myoblasts or (b) bovine muscle cell cultures were treated with muscle extracts from control bovine or bovine treated with a β-adrenergic agonist in Dulbecco’s Modified Eagle’s Medium (DMEM) or skeletal muscle cell basal medium (SKBM). a,b Means with different letters differ (P < 0.05). There was a treatment media × β-adrenergic agonist treatment × cell type interaction (P = 0.0001).

degradation (Kim and Sainz, 1992; Mersmann, 1995; Yang and McElligott, 1989). The anabolic effects of βadrenergic agonists have been observed in ruminant muscle in vivo, and are thought to be primarily mediated via an inhibition of protein degradation (Bohorov et al., 1987). β-Adrenergic agonists have been shown to decrease protein degradation in rat L8 muscle cells by regulating concentra-

tions of some proteases in muscle (Hong and Forsberg, 1995). The calcium-requiring proteases µ- and m-calpain have been found to be involved in myofibrillar protein turnover and protein degradation in muscle cell cultures (Huang and Forsberg, 1998). βAdrenergic agonist treatments have been shown to decrease protein degradation by reducing calpain activity and increasing the calpain inhibitor

calpastatin (Wang and Beermann, 1988; Kretchmar et al., 1990; Sainz et al., 1993). Thus, a β-adrenergic agonist treatment was used as the ideal testing model for optimizing culture conditions for measuring muscle protein degradation in the present study. In the present study we found that relative indirect protein degradation was greater in the primary bovine muscle cultures than C2C12 myoblasts. Additionally, we reported that the β-adrenergic agonist treatment decreased protein degradation regardless of cell type. C2C12 myoblasts are a cloned line of murine myoblasts. Symonds et al. (1990) reported that the β2-adrenergic agonist cimaterol had no effect on protein degradation of ovine primary muscle cell cultures. In contrast, McMillan et al. (1992) reported clenbuterol (a β2adrenergic agonist) administration to primary rat muscle cell cultures resulted in decreased protein degradation. Harper et al. (1987) reported that cloned myoblasts were more responsive for protein degradation than primary muscle cultures from livestock when treated with growth promoting hormones. However, in the present study C2C12 myoblasts appeared to be just as responsive to the treatment as the primary bovine muscle cell cultures if samples were placed in SKBM, although more variable when placed in DMEM. A number of treatments affect protein degradation. Bukoski et al. (1989) showed that the active form of vitamin D increased protein degradation. Harper et al. (1987) and Roe et al. (1989) found that adding insulin-like growth factor type I, insulin, epidermal growth factor, and other hormones decreased protein degradation in primary ovine muscle cell cultures. Also, the anabolic steroid trenbolone acetate has been shown to decrease muscle cellular protein degradation (Hayden et al., 1992). Cell culture media proved to be an important factor in determining differences in protein degradation levels. When the extracted muscle was placed in SKBM cell culture media,


Figure 6. Effect of cell culture media type, β-adrenergic agonist treatment, and muscle extraction buffer on percent protein degradation of muscle cells. Muscle cells were treated with muscle extracts in (a) potassium phosphate buffer (KPB) or (b) prerigor extraction buffer from control bovine or bovine treated with a β-adrenergic agonist in Dulbecco’s Modified Eagle’s Medium (DMEM) or skeletal muscle cell basal medium (SKBM).a,bMeans with different letters differ (P < 0.05). There was a treatment media × β-adrenergic agonist treatment × muscle cell extraction buffer interaction (P = 0.04).

the β-adrenergic agonist treatment decreased indirect protein degradation rates regardless of cell type. When the extracted muscle was placed in DMEM, treatment effects were highly variable, especially when primary bovine muscle cultures were treated. Thus, the cloned C2C12 myoblasts appeared to be less variable and more re-

sponsive to the β-adrenergic agonist treatment than the primary bovine myoblasts in DMEM. Harper et al. (1987) previously reported that cloned myoblasts were more responsive for protein degradation than primary muscle cultures. Treatment of the muscle cells with the β-adrenergic agonist samples decreased protein deg-

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radation rates when muscle samples were extracted in either KPB or prerigor extraction buffer as long as muscle extracts were placed in SKBM cell culture media and not in DMEM. The authors found this quite surprising because DMEM is the American Type Culture Collection-recommended growth media for C2C12 myoblasts. Furthermore, in our protein synthesis techniques, DMEM proved to be much more responsive for showing β-adrenergic agonist treatment effects than SKBM cell culture media (Montgomery et al., 2006). These experiments indicated that extracting muscle from treated animals in either KPB or prerigor extraction buffer was equally effective in showing β-adrenergic agonist responses in protein degradation if placed in SKBM cell culture media. For maximal consistency of determining relative protein degradation differences by applying muscle extracts from livestock treated with muscle-enhancing compounds, SKBM medium should be used with C2C12 myoblasts. We have described an optimization of a technique for rapidly determining protein degradation differences due to pharmaceutical and growth promoting treatments to livestock using a muscle cell in vitro system. The procedures are much less expensive in terms of isotope and animal costs than other invasive in vivo methods. However, animals would have to be treated with radiolabeled compounds to determine direct myofibrillar protein degradation differences in different muscles. These procedures are not intended to elucidate the mechanisms behind pharmaceutical enhancement of muscle cell protein degradation, although they prove to be very helpful in determining relative and in direct effects. However, caution should be exercised when interpreting in vitro results. The methods employed here can use either clonal or primary cells. However, primary cells will have greater genetic and animal variations in sensitivity compared to clonal C2C12 cells, and clonal cells may be more adapted to

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an in vitro environment. Also, the methods employed are intended to reflect relative changes that occur in vivo in response to hormonal, pharmaceutical, or physiological treatments. For instance, the effect of the β-adrenergic agonist in these experiments to decrease protein degradation rates is most likely not attributable to β-adrenergic agonist residues as the withdrawal time would have substantially reduced residues in muscle. However, the effect of the β-adrenergic agonist in these experiments to decrease protein degradation rates is most likely due to an increase in hepatocyte growth factor having a positive growth on cellular activation (Bischoff, 1986; Tatsumi et al., 1998), or to increases in muscle residues of insuline-like growth factor-1 or other growth factors (Awede et al., 2002). Additional studies would be needed to determine exactly how each individual compound affects myofibrillar protein degradation, as well as to determine direct effects of compounds on muscle protein degradation rates. The procedures described only provide evidence of general and indirect effects on protein degradation and additional in vivo studies will be needed to determine direct effects on myofibrillar protein degradation rates.

Implications Overall, the techniques described are an optimization for a procedure that aids in rapid determination of pharmaceutical and growth-promoting treatments to livestock on general and indirect muscle cell protein degradation in vitro. β-adrenergic agonist treatment differences indicating decreased protein degradation appeared to be typical of the literature when SKBM cell culture media was employed for both extraction buffers and cell types. However, for the most consistent and least variable results of determining protein degradation differences of β-adrenergic agonist treatment affects by applying muscle extracts from livestock with the de-

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scribed procedures, a cell culture media of SKBM should be used with clonal C2C12 myoblasts. These procedures are not intended to elucidate the mechanisms behind pharmaceutical enhancement of muscle cell protein degradation or to measure direct effects on protein degradation.

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