Effect of High Density Lipoproteins on Protein Expression in Myoblast Cell Lines Nikolina Babic,†,‡ Craig Beeson,†,§ and Norman J. Dovichi*,† University of Washington, Department of Chemistry, P.O. Box 351700, Seattle, Washington 98195 Received September 28, 2004
We have studied the effects of high-density lipoprotein (HDL) on rat heart (H9c2) and skeletal (L6) myoblasts using two-dimensional gel electrophoresis and tandem mass spectrometry. Gels generated from control cells and cells treated with 250 µg/mL HDL showed significant differences in the 7-10 pI region and the 30-50 kDa mass region. In particular, the membrane binding protein, annexin II, the voltage-dependent anion channel, glyceraldehyde-3-phosphate dehydrogenase and other glycolytic proteins are differentially expressed. Keywords: high-density lipoprotein • myoblast • 2-D electrophoresis
Introduction The majority of circulating high density lipoproteins (HDL) occurs as an ensemble of spherical particles that consist of approximately 50% lipids and 50% proteins, predominantly apolipoproteins. The lipoproteins are very important in helping tissues balance cholesterol levels. Cells make their own cholesterol and they scavenge cholesterol from the diet. An important role of HDL particles is to remove excess cholesterol in peripheral tissues and then deliver it to the liver for synthesis and excretion of bile salts. Insufficient levels of HDL, or excess levels of cholesterol, can cause cholesterol accumulation in blood vessels, leading to formation of plaques and preventing adequate oxygen supply to the tissue (ischemia). This condition, known as atherosclerosis, can lead to coronary heart disease and it is a major cause of heart disease in the world today. Recently, several reports have shown that HDL triggers certain signaling events, such as phosphorylation cascades and Ca2+ signals.1-3 This molecule has also been shown to act as a mitogen4 and to protect cells against environmentally induced stress.5,6 The mechanism by which HDL protects cells from stress remains unclear. It is likely that this complex molecule regulates several different pathways by modifying different proteins involved in these signaling events. Although the role of HDL in atherosclerosis is relatively well characterized, the effect of HDL on skeletal muscle protein expression has not been reported. We hypothesized that elevated HDL levels will modify the expression profiles of several proteins in rat myoblasts. These differences in the protein expression are likely to be related to the physiological changes associated with high * To whom correspondence should be addressed. E-mail: dovichi@ chem.washington.edu. † University of Washington. ‡ Present address: Mayo Clinic, Department of Laboratory Medicine and Pathology, 200 1st Street SW, Rochester, Minnesota 55905. § Present address: Medical University of South Carolina, Department of Pharmaceutical Sciences, 280 Calhoun Street, P.O. Box 250140, Charleston, South Carolina 29425.
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and low levels of serum HDL, which is correlated with atherosclerosis and metabolic syndromes. We recently employed two-dimensional electrophoresis (2-DE) to separate proteins expressed in rat myoblasts and ingel digestion, mass spectrometry, and database searching to identify those proteins.7 We have now extended our study to determine whether HDL has any effect on the protein expression in rat myoblasts. Lipoproteins regulate cholesterol synthesis in many tissues and one might expect to see changes in the synthesis of proteins correlated with increased levels of HDL. Rather, we were surprised to find significant changes in expression of proteins involved in energy metabolism.
Experimental Section Sample Preparation. Rat heart and skeletal muscle myoblasts, H9c2 and L6, were purchased from American Type Culture Collection (Rockville, MD). Heart myoblasts were passaged eight times while skeletal muscle myoblasts were passaged four times. Control cells were grown in media with 10% FCS that was low in HDL (140 µg/mL). The HDL(+) cells were grown in the same media supplemented with 250 µg/mL HDL. The cells were lifted from substrate with trypsin/EDTA and then they were extensively washed with a balanced salt solution prior to lysis. Cells were then lysed in the following buffer: 7 M urea, 2 M thiourea, 2% CHAPS, and 0.5% ASB-14. The amount of buffer used was 50 µL/106 cells. Once the buffer was added, the samples were homogenized by vortexing for 10 s. The samples were then incubated at 4 °C for approximately 1.5 h and ultracentrifuged for 30 min (at 12 000 rpm). Finally, 150 µL of cell lysate was combined with 190 µL of rehydration buffer (8 M urea, 0.5% CHAPS, 0.2% DTT and 0.2% Pharmalyte 3-10 NL or IPG buffer 6-11 NL). Two-Dimensional Electrophoresis. Unless otherwise noted, all the equipment used for 2-DE was purchased from Bio-Rad Laboratories Inc. (Hercules, CA). The first dimension separation was carried out using 18 cm Immobiline Dry Strips (Amersham Pharmacia, Piscataway, NJ) with appropriate pH gradients. 10.1021/pr049826g CCC: $30.25
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Effect of HDLs on Protein Expression
Figure 1. Scanned images of 2-DE gels of L6 myoblast cell line. An IPG 3-10 NL strip was used in the first dimension and an SDS-PAGE (12% T) gel was used in the second dimension. The L6 cells were cultured in DMEM/F-12 medium. Approximately 300 µg of protein was loaded and the gels were stained using a BioRad Silver Stain Kit.
Each strip was loaded with lysate obtained from 2.5 × 106 cells (approximately 300 µg of protein) and covered with Dry Strip Cover Fluid (Pharmacia Biotech, Piscataway, NJ). The IPGphor platform (Pharmacia Biotech, Piscataway, NJ) was used to carry out IEF. The running conditions for 2-DE were performed as per manufacturer’s directions. Once the 2-D separation was complete, the gels were stained and protein spots visualized using the Silver Stain Kit. Mass Spectrometry Sample Preparation. The samples for MS analysis were prepared by excising the spots of interest from the 2-D gel and carrying out an in-gel trypsin digestion. Collected spots were first destained by 8 min incubation with 500 µL of 30 mM K3Fe(CN)6 and 100 mM Na2S2O3 solution. The samples were then washed 4 times (8 min each wash) with 1 mL of Nanopure water. The gel pieces were cut and dehydrated in 100% ACN for 15 min. Dried gel spots were resuspended in 20 µg/mL of sequence-grade modified porcine trypsin in 50 mM NH4HCO3 and 5 mM CaCl2 and incubated on ice for 1 h. After the incubation period, the excess liquid was discarded and 30 µL 50 mM NH4HCO3 and 5 mM CaCl2 of was added. The samples were incubated overnight at 37 °C (approximately 12-15 h). The supernatant was then removed and saved while the remaining gel pieces were washed with 30 µL of 20 mM NH4HCO3 and sonicated for 15 min. The supernatant was removed again and combined with the previous fraction. The samples were washed three more times by sonicating for 10 min in 30 µL of 50%ACN/5% formic acid. The supernatants were combined, frozen, and evaporated to a few microliters. The evaporated samples were then reconstituted with 10 µL of 0.3% TFA and purified using zip tips with 0.6 µL C18 resin (Millipore Corporation, Billerica, MA). The purification procedure was performed following manufacturers instructions. All samples were stored at -80 °C until ready to use. Prior to use 1 µL of 2.5% of TFA was added. MS
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Figure 2. Scanned images of 2-DE gels of L6 myoblast cell line. An IPG 3-10 NL strip was used in the first dimension and an SDS-PAGE (12% T) gel was used in the second dimension. L6 cells were cultured in DMEM/F-12 growth medium, supplemented with 250 µg/mL HDL. Approximately 300 µg of protein was loaded and the gels were stained using a BioRad Silver Stain Kit.
experiments were carried out using a Finnigan LCQ instrument (Thermo Electron Corporation, Woburn, MA), coupled to Magic 2000 HPLC (Michrom Bioresources, Inc., Auburn, CA). Western Blotting. All of the equipment used for Western blots was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA) unless stated otherwise. The samples were prepared in the following manner: 4 × 106 L6 or H9c2 cells were mixed with 200 µL of hot reducing SDS loading buffer (50 mM Tris, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). The suspension was homogenized by pippetting and vortexing for 10 s. Samples were then placed into a boiling water bath for 10 min and cellular debris was removed by ultracentrifugation (10 000 rpm, 10 min). The pellet was discarded and a 25 µL sample was loaded into each well of the pre-cast 12% tris-glycine gels (Cambrex Bio Science Rockland, Inc., Rockland, ME). Separated proteins were blotted onto the Immun-Blot PVDF Membrane. Prior to placing the gels into the transfer cassette, the gels and PVDF membranes were equilibrated in transfer buffer (39 mM glycine, 48 mM Tris, 0.037% SDS and 20% methanol) for 15-20 min. Transfer conditions were 150 mA for 80 min. Blocking primary and secondary antibody incubations and detection were carried out following the protocol outlined in the manual for ECL Plus Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ). Membranes were scanned using the Storm 840 Imager purchased from Molecular Dynamics (Sunnyvale, CA). Primary antibodies were used as follows: porin (Ab-5) or VDAC was used at concentration of 1 µg/mL (Calbiochem, San Diego, CA); annexin II (H-50) antibody was used at 1:200 dilution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and finally, GAPDH antibody was used at 1:2000 dilution (Novus Biological, Littleton, CO). All of the primary antibodies were rabbit polyclonal antibodies. The secondary antibody used was Journal of Proteome Research • Vol. 4, No. 2, 2005 345
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Table 1. Summary of Protein Identification in HDL-treated and Untreated Cells spot no.
1 2 3 4 5 6 7 8 9 10 11 12
protein ID
accession no.
theoretical pI/MW
Aldehyde dehydrogenase class 3 Acidic ribosomal protein P0 Annexin A1 Aldose reductase-like protein Annexin II Annexin II GAPDH Lactate dehydrogenase A GAPDH VDAC 1 Mitochondrial malate dehydrogenase* Aldose reductase-like protein Mitochondrial malate dehydrogenase* Lactate dehydrogenase A VDAC 1 Mitochondrial malate dehydrogenase* Aldehyde reductase 1 Aldehyde reductase 1
NM_031972 gi|71138 NM_012904 AJ277957 NM_019905 NM_019905 gi|120707 NM_017025 gi|120707 NM_031353 NM_031151 AJ277957 NM_031151 NM_017025 NM_031353 NM_031151 NM_012498 NM_012498
6.8/50 6.2/34 7.3/39 7.5/36 7.7/39 7.7/39 8.3/36 8.3/36 8.3/36 8.5/31 8.7/36 7.5/36 8.7/36 8.3/36 8.5/31 8.7/36 6.7/36 6.7/36
donkey anti-rabbit IgG-HRP purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 2-D Gel Analysis. The 2-D gel analysis software package PDQuest (Bio-Rad Laboratories Inc., Hercules, CA) was used to quantify spots of interest. Since gels vary in spot intensity, clarity and streaking, this software allows the user to define certain parameters on the one gel and then apply those settings to all the other gels, to eliminate these interferences. Prior to the spot detection, the faintest spot on a gel was defined. This set the sensitivity and minimum peak value parameters. Following this, the largest spot was defined by drawing a box around the largest spot cluster on the image. This step is necessary to define the parameters used in the Floating Ball background subtraction. Upon the spot detection in PDQuest, the original gel scan was automatically filtered, smoothed and fit to a Gaussian curve resulting in 3-D Gaussian spots. All quantitation analysis was performed on the Gaussian image. Spot quantity is the total intensity of a defined spot in 2-D gel image and it corresponds to the amount of protein present. The formula used for calculating the quantity of Gaussian spots is as follows: spot height*π*σx*σy, where: spot height is the peak of the Gaussian representation of the spot; σx and σy are standard deviations of the Gaussian distribution of the spot in the x and y-axis direction, respectively.
Results Effect of HDL on Protein Expression in L6 Myoblasts. The effect of HDL on protein expression levels in L6 cells was determined by comparison of 2-D gels obtained using control cells and L6 cells supplemented with 250 µg/mL HDL. These gels are shown in Figures 1 and 2, respectively. The horizontal axis shows the first dimension separation, based on protein isoelectric point, while the vertical axis shows separation according to protein molecular weight. The proteins we have identified are shown in Table 1. The major differences between the control cells and those treated with HDL seem to be in the 30-40 kDa and pI in 7-10 region. A comparison of this region for gels run with replicate samples is shown in Figure 3. Spots of high density that were consistent among the replicate gels were identified by mass spectrometric analysis of tryptic peptides. Criteria used to accept identifications included the extent of sequence coverage, the number of 346
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estimated pI/MW (Gel 1)
estimated pI/MW (Gel 2)
7.6/52 8.0/34
7.6/52
estimated pI/MW (Gel 3)
estimated pI/MW (Gel 4)
8.0/31 8.1/34 8.1/32 8.3/36 8.5/30
8.0/34 8.0/33 8.2/34 8.2/32 8.3/36 8.5/30
8.0/34 8.0/32 8.2/35
8.2/34 8.2/33 8.5/35
8.5/36 8.6/31
8.6/37 8.7/30
9.2/30
9.2/30
9.3/31
9.5/30
9.1/30
9.1/31
9.2/30
8.8/30
9.0/29
7.4/36 7.4/34
peptides matched, and the correlation factors. The correlation factor (Xcorr) indicates how well the measured product ion spectrum matches theoretical spectra created from the database sequences.8 Within a given set of data, higher Xcorr values generally indicate better match. In our analyses, we disregarded all the matches that produced Xcorr values lower than 2.5 for doubly charged peptides and gave less than two peptides matched. Several peptides that were unique to protein in question were also considered, even though the sequence coverage was relatively poor. The most significant differences between the cells treated with HDL and control cells appear to be in the expression of the membrane binding protein, annexin II and the glycolytic protein glyceraldehydes-3-posphate dehydrogenase (GAPDH). Lactate dehydrogenase A (LDH A) and/or NAD(P)H dehydrogenase are also apparently upregulated in cells grown with additional HDL. The voltage-dependent anion channel (VDAC), which is a member of the mitochondrial transition pore, occurs at different pI values, probably due to different post-translational modifications. Effect of HDL on Protein Expression in H9c2 Myoblasts. The protein expression profile of rat heart myoblasts, H9c2 cells, show great similarity to skeletal muscle cells. Protein fingerprints of nontreated (control) cells and H9c2 cells treated with 250 µg/mL HDL are shown in Figures 4 and 5, respectively. The protein identifications for gels shown in these figures are shown in Table 1. It appears that HDL affects the same proteins as in L6 cells. The differences in post-translational modifications of VDAC are pronounced in H9c2 cells grown in additional HDL. There also seems to be more mitochondrial malate dehydrogenase in these cells than in L6 cells. Western Blot Identification of Proteins Affected by HDL Presence. Annexin II, GAPDH and VDAC expression was examined using Western blots of lysate from L6 cells. This was done to ensure that the correct proteins are identified as upregulated, since comigration is a common problem in 2-DE. One spot on a 2D gel may contain several proteins, and it is difficult to determine which protein affects the spot intensity in observed gels. The 2-DE spots corresponding to annexin II, GAPDH, and VDAC were more intense in gels from cells grown with added HDL. VDAC, however, appeared to be present in different phosphorylation states. The data obtained with
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Effect of HDLs on Protein Expression
Figure 3. Scanned images of 2-DE gels as shown in Figures 1 and 2. Replicate gels were generated from different samples. Circles represent spots identified as having changed in intensity by greater than 1.5-fold.
Figure 4. Scanned images of 2-DE gels of H9c2 myoblast cell line. An IPG 3-10 NL strip was used in the first dimension and an SDS-PAGE (12% T) gel was used in the second dimension. The H9c2 cells were cultured in DMEM/F-12 medium. Approximately 300 µg of protein was loaded and the gels were stained using a BioRad Silver Stain Kit.
Western blots is shown in Figure 6 and they are in agreement with the MS data. Quantification of Differential Protein Spots. Several 2-D gels of L6 cells grown with and without HDL were analyzed using PDQuest to measure spot intensity. The intensity variations between HDL-treated and untreated gels were used to calculate standard errors of the mean and the data are tabulated in Table 2. We have included only spots whose
Figure 5. Scanned images of 2-DE gels of H9c2 myoblast cell line. An IPG 3-10 NL strip was used in the first dimension and an SDS-PAGE (12% T) gel was used in the second dimension. The H9c2 cells were grown in DMEM/F-12 medium, supplemented with additional 250 µg/mL HDL. Approximately 300 µg of protein was loaded and the gels were stained using a BioRad Silver Stain Kit.
intensities changed by more than 1.5-fold with a confidence level of p < 0.05.
Discussion We have identified several proteins that are most affected by presence of additional HDL in the culture medium in two different myoblast cell lines. These are VDAC, GAPDH, LDH Journal of Proteome Research • Vol. 4, No. 2, 2005 347
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Figure 6. Western blots of proteins showing differences in protein expression due to additional HDL present in the media: a. VDAC; b. GAPDH; c. annexin II; d. β-Actin loading control. All the blots were obtained using L6 myoblasts. Table 2. Quantitative Analyses of Differential Expression spot no.a
NoHDL (quantityb ×103)
HDL (quantity ×103)
2 3 4 5 6 7
57 ( 8 55 ( 3 117 ( 12 52 ( 2 58 ( 9 100 ( 15
189 ( 9 147 ( 15 246 ( 37 79 ( 5 137 ( 21 167 ( 21
a Identified in Figure 2. b Units of OD*IU2, where IU ) image units ) 0.1 mm.
A/NAPD(H) dehydrogenase, and annexin II. Interestingly, these proteins are all involved in regulation of cellular energy metabolism.9-11 It has been shown recently that only the HDLtreated cells significantly increase glycolysis when exposed to mitochondrial inhibitors such as sodium azide (N. Babic & C. Beeson, unpublished results). The VDAC is a part of the mitochondrial permeabilitytransition pore and it is involved in trafficking of ATP and ADP between the cytosol and mitochondria matrix. It has been reported that VDAC can be phosphorylated in vitro by the catalytic subunit of protein kinase A (PKA).12 A different study implicated VDAC 1 as a direct binding partner with protein kinase C (PKC).13 The phosphorylation of VDAC reduces the probability of pore opening. Some conditions that induce pore opening are high Ca2+ levels, low glucose and ATP and high inorganic phosphate levels, conditions caused by inhibition of mitochondrial respiration. Another protein apparently upregulated in the cells grown with additional HDL is LDH-A, which is also regulated by PKC. It has been shown that the activation of PKC stabilizes LDH-A mRNA complex.14 LDH catalyzes the reversible reaction of lactate to pyruvate, with LDH-A isozyme favoring lactate production. Lactate is important source of oxidative fuel for the heart in vivo. During the conditions of oxygen deficiency, the acid accumulates in the cell and the lactate production is favored.
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The metabolic enzyme, GAPDH, is a highly versatile molecule that can recognize many different molecules. Besides its primary role in the glycolytic pathway (tetrameric form), it is also involved in other processes, such as membrane fusion, microtubule binding, nuclear RNA transport and DNA replication and repair, to name a few.15 Recently, it has been discovered that the activity of GAPDH is diminished under ischemic conditions.16 Annexins are a family of proteins that bind to membranes and anionic phospholipids in Ca2+ dependent manner.17 It was suggested that annexins might be universal modulators of membrane trafficking.9,10 Annexin II, in particular, is involved in plethora of cellular processes. Some examples are DNA replication,18-20 inhibition of blood coagulation, and signal transduction in mitogenesis and differentiation.21 It has been recently reported that annexin II binds to the adenine nucleotide transporter (ANT) protein in stressed cells.22 The ANT protein is the partner of the VDAC protein, which our results show to be also modulated in response to HDL. Among the many activities associated with HDL, modulation of glucose utilization is not one of them. Our results have shown that exposure of myoblasts to HDL alters the expression and post-transitional state of proteins involved in glycolysis and mitochondrial respiration. Evidently, in muscle and perhaps in other tissues, HDL also appears to have a role in the regulation of energy metabolism.
References (1) O’Connell, B. J.; Genest, J. Circulation 2001, 104, 1978-1983. (2) Nofer, J.-R.; Fobker, M.; Hobbel, G.; Voss, R.; Wolinska, I.; Tepel, M.; Zidek, W.; Junker, R.; Seedorf, U.; von Eckardstien, A.; Assmann, G.; Walter, M. Biochemistry 2000, 39, 15199-15207. (3) Xia, P.; Vadas, M. A.; Rye, K.-A.; Barter, P. J.; Gamble, J. R. J. Biol. Chem. 1999, 274, 33143-33147. (4) Nofer, J.-R.; Junker, R.; Pulawski, E.; Fobker, M.; Levkau, B.; von Eckardstein, A.; Seedorf, U.; Assmann, G.; Walter, M. Thromb. Haemost. 2001, 85, 730-735. (5) Calabresi, L.; Rossoni, G.; Gomaraschi, M.; Sisto, F.; Berti, F.; Frencesschini, G. Circ. Res. 2003, 92, 330-337. (6) Sugano, M.; Tsuchida, K.; Makino, N. Biochem. Biophys. Res. Comm. 2000, 272, 872-876. (7) Babic, N.; Beeson, C. C.; Dovichi, N. J., unpublished results. (8) Eng, J.; McCormack, A.; Yates, J., III. J. Am. Soc. Mass. Spectrom. 1994, 5, 976-989. (9) Emans, N.; Grovel, J. P.; Walter, C.; Gerke, V.; Kellner, R.; Griffiths, G.; Gruenberg, J. J. Cell Biol. 1993, 120, 1357-1369. (10) Gruenberg, J.; Emans, N. Trends Cell Biol. 1993, 3, 224-227. (11) Mazurek, S.; Hugo, F.; Failing, K.; Eigenbrodt, E. J. Cell Physiol. 1996, 167, 238-250. (12) Bera, A. K.; Ghosh, S. J. Struct. Biol. 2001, 135, 67-72. (13) Baines, C. P.; Song, C. X.; Zheng, Y. T.; Wang, G. W.; Zhang, J.; Wang, O. L.; Guo, Y.; Bolli, R.; Cardwell, E. M.; Ping, P. Circ. Res. 2003, 92, 873-880. (14) Short, S.; Tian, D.; Short, M. L.; Jungmann, R. A. J. Biol. Chem. 2000, 275, 12963-12969. (15) Sirover, M. A. J. Cell Biol. 1997, 66, 133-140. (16) Eaton, P.; Wright, N.; Hearse, D. J.; Shattock, M. J. J. Mol. Cell Cardiol. 2002, 34, 1549-1560. (17) Liu, J.; Rothermund, C. A.; Ayala-Sanmartin, J.; Vishwanatha, J. K. BMC Biochem. 2003, 4, 10-25. (18) Jindal, H. K.; Chaney, W. G.; Anderson, C. W.; Davis, R. G.; Vishwanatha, J. K. J. Biol. Chem. 1991, 266, 5169-5176. (19) Hitesh, K.; Jindal, H. K.; Jamboor, K.; Vishwanatha, J. K. Biochemistry 1990, 29, 4767-4773. (20) Vishwanatha, J. K.; Kumble, S. J. Cell Sci. 1993, 105, 533-540. (21) Roda, O.; Valero, M. L.; Peiro, S.; Andreu, D.; Real, F. X.; Navarro, P. J. Biol. Chem. 2003, 278, 5702-5709. (22) Verrier F.; D. A.; Lebras M.; Metivier D.; Kroemer G.; Mignotte B.; Jan G.; Brenner C. Oncogene 2004, 23, 8049-8064.
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