Metabolic Enzymes, Antioxidants, and Cytoskeletal Proteins Are Significantly Altered in Vastus Lateralis Muscle of K-Depleted Cadaveric Subjects Ratree Tavichakorntrakool,† Pote Sriboonlue,† Vitoon Prasongwattana,† Anucha Puapairoj,‡ Pa-thai Yenchitsomanus,§,| Supachok Sinchaikul,⊥ Shui-Tein Chen,⊥,# Chaisiri Wongkham,† and Visith Thongboonkerd*,∇ Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok, Thailand, Institute of Biological Chemistry and Genomic Research Center, Academia Sinica, Taipei, Taiwan, Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan, and Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Received November 3, 2008
Abstract: Molecular mechanisms underlying myopathy caused by prolonged potassium (K) depletion remain poorly understood. In the present study, we examined proteome profile of vastus lateralis muscle obtained from cadaveric subjects who had K depletion (KD) (muscle K < 80 µmol/g wet weight) compared to those who had no KD (NKD) (muscle K g 80 µmol/g wet weight) (n ) 6 per group). Muscle proteins were extracted, resolved by 2-DE, and visualized with CBB-R250 stain. Spot matching and intensity analysis revealed significant changes in levels of 11 (6 increased and 5 decreased) protein spots in the KD group. Q-TOF MS and MS/MS analyses identified these altered proteins as metabolic enzymes (aldehyde dehydrogenase 1A1, uridine diphosphoglucose pyrophosphorylase, enolase 1, cytosolic malate dehydrogenase, and carbonic anhydrase III), antioxidants (peroxiredoxin-3 isoform b), cytoskeletal proteins (slow-twitch skeletal troponin I and myosin light chain 2), and others. These altered proteins are involved in many cellular functions, including bioenergetics, acid-base regulation, oxidative stress response, and muscle contractility. Vali* To whom correspondence should be addressed. Visith Thongboonkerd, MD, FRCPT, Medical Proteomics Unit, Office for Research and Development, 12th Floor Adulyadej Vikrom Building, 2 Prannok Road, Siriraj Hospital, Bangkoknoi, Bangkok 10700, Thailand. Phone/Fax: +66-2-4184793. E-mail:
[email protected] (or)
[email protected]. † Department of Biochemistry, Faculty of Medicine, Khon Kaen University. ‡ Department of Pathology, Faculty of Medicine, Khon Kaen University. § Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University. | Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC). ⊥ Institute of Biological Chemistry and Genomic Research Center, Academia Sinica. # Institute of Biochemical Sciences, College of Life Science, National Taiwan University. ∇ Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University.
2586 Journal of Proteome Research 2009, 8, 2586–2593 Published on Web 02/26/2009
dation was done by Western blot analysis, which confirmed the increased level of peroxiredoxin-3 and decreased level of troponin-I in the KD muscle. Linear regression analysis also revealed a significant negative correlation between peroxiredoxin-3 level and muscle K content (r ) -0.887; p < 0.001), as well as a significant positive correlation between troponin-I level and muscle K content (r ) 0.618; p < 0.05). Our results implicate the important roles these altered proteins play in the development of KD-associated myopathy. Keywords: Antioxidants • Cytoskeletal proteins • Metabolic enzymes • Pathophysiology • Potassium depletion • Proteome • Proteomics • Skeletal muscle
Introduction Maintenance of homeostasis in normal cells requires a normal range of potassium (K) level in blood circulation. Skeletal muscle is the main organ responsible for K storage in human body. Therefore, the most acceptable method for determination of total body K status is the measurement of muscle K content and K depletion (or deficiency) is widely defined when muscle K content is 69 or ions scores >37 for PMF and MS/MS analyses, respectively. Western Blot Analysis. Equally, 20 µg of total proteins extracted from vastus lateralis muscle (as described above) was resolved in 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were incubated at 4 °C overnight with primary antibodies to peroxiredoxin-3 (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA), troponin-I (Santacruz) or to β-actin (Abcam, Inc.; Cambridge, U.K.); all with a dilution of 1:4000 in 5% skimmed milk/PBS. After washing, the membrane was incubated further with respective secondary antibodies conjugated with horseradish peroxidase with a dilution of 1:8000. Reactive protein bands were then visualized with ECL Plus Western blot detection system (GE Healthcare). Band intensity was measured by ImageQuant TL software (GE Healthcare). Statistical Analysis. All data are reported as Mean ( SD. Statistical analysis was performed with SPSS software (version 11.0) (SPSS Corporation, Chicago, IL). To test for differences between 2 sample groups, an unpaired t-test was used. To test for correlations between two parameters, a linear regression analysis was performed. P-values
