Alterations of Mitochondrial Enzymes Contribute to Cardiac Hypertrophy before Hypertension Development in Spontaneously Hypertensive Rats Chao Meng,†,¶ Xian Jin,†,¶ Li Xia,‡ Shao-Ming Shen,| Xiao-Ling Wang,‡ Jun Cai,§ Guo-Qiang Chen,‡,| Li-Shun Wang,*,‡ and Ning-Yuan Fang*,† The Department of Geriatrics, Ren-Ji Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200001, China, The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, SJTU-SM Shanghai 200001, China, Department of Pathology, SJTU-SM, Shanghai 200025, China, and Institute of Health Sciences, Shanghai Institutes for Biological Sciences-SJTU-SM, Shanghai 200025, China Received December 10, 2008
Mitochondrial dysfunction is recently thought to be tightly associated with the development of cardiac hypertrophy as well as hypertension. However, the detailed molecular events in mitochondria at early stages of hypertrophic pathogenesis are still unclear. Applying two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF tandem mass spectrometry, here we identified the changed mitochondrial proteins of left ventricular mitochondria in prehypertensive/ hypertensive stages of cardiac hypertrophy through comparing spontaneously hypertensive rats (SHR) and the age-matched normotensive Wistar Kyoto (WKY) rats. The results revealed that in the hypertrophic left ventricle of SHR as early as 4 weeks old with normal blood pressure, 33 mitochondrial protein spots presented significant alterations, with 17 down-regulated and 16 up-regulated. Such alterations were much greater than those in 20-week-old SHR with elevated blood pressure. Of the total alterations, the expression of two mitochondrial enzymes, trifunctional enzyme alpha subunit (Hadha) and NADH dehydrogenase 1 alpha subcomplex 10 (Ndufa10), were found to have special expression modification patterns in SHR strain. These data would provide new clues to investigate the potential contribution of mitochondrial dysfunction to the development of cardiac hypertrophy. Keywords: Cardiac hypertrophy • Hypertension • Mitochondrion • Proteomics • Spontaneously hypertensive rats
1. Introduction The risk of cardiovascular disease for patients at any level of high blood pressure increases markedly with damage to the heart, kidneys, brain, or large arteries.1 Cardiac hypertrophy, particular in the left ventricle (LV), is one manifestation of the major target-organ damages by hypertensive stress.2 During hypertension, the increased left ventricular wall stress caused by hypertension-induced pressure overload leads to myocardial hypertrophy. The prevalence of left ventricular hypertrophy (LVH) ranges from 20% in mildly hypertensive patients to almost 100% in those with severe or complicated hypertension.3 Although initially regarded as an adaptive reaction to withstand * To whom correspondence should be addressed. Dr. Ning-Yuan Fang, No. 145, Shan-Dong Middle Road, Shanghai 200001, P. R. China. E-mail:
[email protected]. Fax: 0086-21-58893771. Additional corresponding author: Dr. Li-Shun Wang. E-mail:
[email protected]. † The Department of Geriatrics, Shanghai Jiao-Tong University School of Medicine (SJTU-SM). ¶ These two authors equally contributed to this work. ‡ Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education. | Shanghai Institutes for Biological Sciences-SJTU-SM. § Department of Pathology, SJTU-SM. 10.1021/pr801059u CCC: $40.75
2009 American Chemical Society
an increased workload and maintain the constant cardiac function,4 the long-term development of ventricular hypertrophy will lead to functional deterioration and is associated with an increased risk of arrhythmia and cardiovascular mortality.5-7 However, clinically, the development and degree of LVH do not always parallel the level of blood pressure.8 The left ventricular mass index was reported to increase in normotensive children and adults with genetic risk of hypertension.9-13 It is suggested that in addition to elevated blood pressure (BP), other extrinsic factors, such as neurohormonal activation, oxidative stress and cytokinese, and intrinsic genetic predisposition may participate in pathogenesis of the LVH.14 The heart has perpetually high energy demands. The supply of ATP in heart is critical for myocardial contractility and electrophysiology.15 Over 90% of energy consumption of the heart is supplied by mitochondrion,16 which is one of the most complex organelles and plays key roles in many cellular functions including energy production, calcium homeostasis, and cell signaling.17,18 Recent investigations showed that the inefficient energy production due to mitochondrial dysfunction in brain and blood vessels would result in the elevation of blood pressure.19,20 Recently, the importance of the metabolic reJournal of Proteome Research 2009, 8, 2463–2475 2463 Published on Web 03/05/2009
research articles modelling processes inherent in the hypertrophic growth response of heart (whether primary or secondary) have been recognized, as reviewed by Ritchie and Delbridge.21 On one hand, the decreased energy production in the hypertrophic growth response would lead to disturbances of intracellular Ca2+ handling, mitochondria calcium overload and reappearance of fetal isoforms of some ATPases involved in contractile function.21-23 On the other hand, the increased oxidative stress due to mitochondrial dysfunction and thus the decreased energy production also participates in the development of cardiac hypertrophy either in response to neurohumoral stimuli or chronic pressure overload.24 As a most widely used animal model of human genetic hypertension, the spontaneously hypertensive rats (SHR) also provides an useful experimental model to explore the development and progression of cardiac hypertrophy.25 LVH is observed early in prehypertensive SHR at the age of 30 days, followed by an active phase of hypertrophic growth between 16 and 20 weeks with enhanced cardiac function after blood pressure rises.26-29 As reported, the underlying mechanisms for cardiac hypertrophy in prehypertensive and sustained hypertensive phases are attributed to biochemical stress and mechanical stresses, respectively.25 In our previous work, many metabolic proteins were found to be significantly modified in hypertrophic LV during both phases of hypertension.30,31 Many of these proteins were known as mitochondrial enzymes. Mitochondrial enzymes are direct executors of energy production and controllers of mitochondria energy output, whose deregulation may probably result in mitochondrial dysfunction. Therefore, in consideration of the important role that mitochondria play in the pathogenesis of cardiac diseases, we speculated that both biochemical and mechanical stresses might exert influences on mitochondria to cause cardiac hypertrophy. In this study, aiming to take a close look at the molecular events that happened in hypertrophic LV mitochondria both in prehypertensive and sustained hypertensive phases, we isolated mitochondria from the left ventricle of SHR and age-matched normotensive Wistar Kyoto (WKY) rats at age of 4 and 20 weeks, and then used 2D-DIGE to illustrate the time course of altered metabolic protein expression in the normotensive and sustained hypertensive stage.
2. Materials and Methods 2.1. Animals and Blood Pressure Measurement. Male SHR and WKY rats at the age of 4 and 20 weeks were purchased from the SLAC Laboratory Animal Co. LTD (Shanghai, China) and housed in a temperature-controlled environment (22-24 °C) with a 12/12-h light-dark cycle. All procedures were approved by the local ethics committee of Shanghai Jiao-Tong University School of Medicine. After the rats had been brought to the laboratory, blood pressure was measured using tail cuff with a sphygmomanometer (BP-98A, softron, Japan). Blood pressure was measured three times and the three values were averaged. 2.2. Myocardial Diameter. The rats were anesthetized with an intraperitoneal injection of 5% chloral hydrate (1 mL/100 g body weight). The entire heart was rapidly excised, rinsed in ice-cold isotonic saline (0.9% w/v) and sectioned into right ventricular free wall, cardiac atrium, and left ventricular free wall. A piece of left ventricular tissue was fixed in formalin and embedded in paraffin blocks and stained with hematoxylin and eosin (HE). The sections were then examined under light microscopy and quantitated using the QWin Plus software 2464
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Meng et al. (Leica, Wetzlar, Germany). The average diameter of cardiac myocytes in each rat was measured from g50 randomly selected cells. The final results were shown as means and SD of average diameters of three rats each in SHR or WKY rats, respectively. 2.3. Isolation of Left Ventricular Mitochondria and Heart Protein Extraction. Isolation of mitochondria was performed as preciously described by Yu et al.32 Briefly, most of left ventricular tissues were harvested and cut into small pieces in isolation buffer (0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 and 1% protease inhibitor cocktail from Roche), followed by differential centrifugation and density gradient centrifugation (Optiprep, Axis Shield/Cedarlane, Hornby, ON, CA). Protein concentration was determined using the Bio-Rad RC DC protein assay kit (Bio-Rad, Hercules, CA). Simultaneously, part of left ventricular, right ventricular, cardiac atrium, brain, liver, and kidney tissues were pulverized under liquid nitrogen and resuspended with same lysis buffer and method for further analysis. 2.4. Protein Labeling and Two-Dimensional Gel Electrophoresis. 2-D DIGE was performed according to the manufacturer’s instructions CyDyes (GE Healthcare) with minor modifications.33 Shortly, 50 µg of left ventricular protein in SHR or WKY rats was minimally labeled with 400 pmol of either Cy3 or Cy5 for comparison on the same two-dimensional (2-D) gel. Internal standard aliquots of each sample were pooled and labeled with Cy2. The labeled samples were incubated for 30 min on ice in the dark and then 1 µL of 10 mM lysine was added to stop the reaction. The samples were diluted in rehydration solution (8 M urea, 2% (w/v) CHAPS, 65 mM DTT and 0.5% (v/v) Bio-lyte 3/10 ampholyte) and applied to 17 cm IPG strips (17 cm, pH 3-10 NL, Bio-Rad, Hercules, CA) until 80 kVh were reached. Once the IEF was completed, the IPG strips were equilibrated for 30 min with 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, followed by reduction with 1% of DTT and alkylation with 2.5% iodoacetamide. The second-dimensional separations were carried out on 12% SDS-polyacrylamide gels, and gels were run at 16 mA/gel for 30 min, and then 24 mA/gel until the dye reached the bottom of the gels. Three replicas from each sample were made following this procedure. 2.5. Image Analysis and In-Gel Digestion. Fluorescence images of the gels were acquired on a Typhoon TRIO scanner (GE Healthcare, Freiburg, Germany) with a resolution of 100 µm. Sensitivity was adjusted in a prescan with 1000 µm resolution so that no channel had saturated spots. Spot detection was performed on the gel images using the DeCyder software (Version 6.5). Differential in-gel analysis (DIA) module was done by setting the target spot number to 2000. All six gels were added to the appropriate workspace and group and matched to a master gel sequentially using the DeCyder module BVA (version 6.5.14). For grouped comparison, significance was calculated via a one-way ANOVA (p < 0.05) and differentially expressed proteins with an absolute ratio of at least 1.3-fold were selected. The statistical analysis was calculated by the DeCyder software (version 6.5). The preparative gel was done and stained with Coomassie Brilliant Blue. Proteins of interest were excised, transferred into the ZipPlate micro-SPE Plate wells (Millipore, Billerica, MA). The proteins were digested according to the manufacturer’s protocol (Millipore), as described previously.34 2.6. Mass Spectrometry and Protein Identification. Protein identification was processed and analyzed by searching the Swiss-Prot protein database for Rattus norvegicus using the
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Mitochondrial Proteomics of Cardiac Hypertrophy in SHR
Figure 1. Hemodynamics and cardiac hypertrophy in SHR at selected age. (A-C) The SBP (A), DBP (B) and myocyte diameters (C) in WKY rats and SHR at the age of 4 and 20 weeks. The data represent the mean with bar as SD of three rats each group. The symbols (*) and (**) indicate p-values of less than 0.05 and 0.01 compared with WKY rats, respectively. (D) HE staining under light microscopy (40×) for transverse sections of left ventricular myocytes of WKY (left) and SHR (right) at the age of 4 (upper) and 20 (lower) weeks. (E) Enrichment of mitochondria was detected by anti-Cox IV antibody. The fractions of nuclei, endoplasmic reticulum and cytosol were monitered with Western blots. “Total” or “Mito” represents proteins from the whole left ventricle or, left ventricle mitochondria of these rats labeled 1-3, respectively.
MASCOT search engine of Matrix Science that integrated in the Global Protein Server Workstation. The mass tolerance, the most important parameter, was limited to 50 ppm. The results from both the MS and MS/MS spectra were accepted as a good identification when the GPS score confidence was higher than 95%. 2.7. Western Blot. For conventional SDS-PAGE, protein lysates were separated by DIGE as mentioned above and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blotted with antibodies against β-actin (Oncogene, San Diego, CA), β-tubulin (Oncogene, San Diego, CA), Calnexin (Stressgen, Canada), Lamin B (Santa Cruz, CA), Cox IV (Cell signaling, CA), Ndufa10 (Abcam, CA) and Hadha (a gift from Dr. Arnold W. Strauss35), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Dako Cytomation, Denmark). The protein signal was detected by luminol-enhanced chemiluminescence reagent (Pierce, Rockford, IL). The quantitative Cox IV bands were analyzed with Quantity One 4.4.0 software (Bio-Rad, Hercules, CA). 2.8. RT-PCR and Gene Sequence. Total cellular mRNAs were extracted by TRIzol reagent (Invitrogen, Carlsbad, CA) from left ventricular tissues in 3 SHR and 3 WKY rats at the ages of 4 and 20 weeks, and were treated with RNase-free DNase (Promega, Madison, WI). Then, reverse transcription (RT) was performed by TaKaRa RNA PCR kit (Takara, Dalian, China) following manufacturer’s instructions. The primers used were Ndufa10-F: 5′-ATAGGACCCTGTCGCGTTCACGTC-3′ and Ndufa10-R: 5′-CTGGCAATTTCTTGATCATTTGCT-3′ (refGene_ NM _199495). The 18S primers used as internal control which
were F, 5′-AGGCCCTGTAATTGGAATGA GTC-3′ and R, 5′GCTCCCAAGATCCAACTACGAG-3′. PCR products were purified from 1% agarose gels with TIANgel midi purification kit (Beijing, China) and sequenced in the DNA sequencing department of Biosune systems Biology, Shanghai. 2.9. PCR Verification and DNA Sequencing. Genomic DNA was extracted from blood leukocytes in 3 SHR and 3 WKY rats at the age of 20 weeks. DNA sequence of exon 3 of Ndufa10 gene was amplified and sequenced. The used primers were 5′TGGTTGTAGAGAACGGAGAAGCTC-3′ for forward and 5′-CTGCATCAACAGGGATTTTGAG G-3′ for reverse primer. 2.10. Statistical Analysis. The data were expressed as means ( SD. Blood pressure and myocardial cell diameters were analyzed with the Student’s t test between two groups. It was considered statistically significant if the p-value was less than 0.05.
3. Results 3.1. Blood Pressure Detection, Histological Analysis, and Mitochondrial Isolation of Left Ventricle at Selected Age. As shown in Figure 1A,B, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHR at the age of 4 weeks were 133.67 ( 7.26 mmHg and 90.89 ( 5.74 mmHg, respectively, which showed no significant differences compared with those of WKY rats (SBP 114 ( 22.73 and DBP 75.50 ( 20.57 mmHg). As the blood pressure of SHR steadily increased after 8 weeks,30 at 20 weeks’ age, the SBP and DBP in SHR (199.17 ( 18.77 and 156.61 ( 10.90 mmHg, respectively) became significantly higher than those of WKY rats (146.78 ( 7.82 and 114.33 ( 1.73 mmHg, respectively). Meanwhile, myocyte Journal of Proteome Research • Vol. 8, No. 5, 2009 2465
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Figure 2. Experimental design of proteomic analysis and representative 2D-DIGE images of protein expression profiles at age of 4 weeks. (A) The experimental workflow of the comparative proteomic analysis. (B) Representative overlaid images at age of 4 weeks. (C) The significantly down-regulated protein spots in 4-week-old SHR (left panel) and the significantly up-regulated protein spots in 4-week-old SHR (right panel).
diameter of the left ventricle in SHR at 4 weeks’ age was 15.00 ( 0.41 µm, which was significantly larger compared with that in WKY rats (10.87 ( 0.16 µm, p < 0.01). This disparity was enlarged to 25.60 ( 0.33 µm in SHR against 16.91 ( 0.59 µm in WKY rats (p < 0.01) at the age of 20 weeks (Figure 1C). Enlargement and disorder arrangement of myocytes were observed in the left ventricles of SHR (Figure 1D). It was suggested that LVH existed in SHR at the age of 4 weeks despite the normal blood pressure, and became severer at the age of 20 weeks. The mitochondria were isolated from left ventricle, the most active, heart energy consuming part of SHR and WKY rats. As shown in Figure 1E, the marker of mitochondrial inner membrane, Cox IV, was significantly enriched by 3.59 ( 0.26and 3.30 ( 0.09-folds compared with total heart protein extracts (using Quantity One 4.4.0 software). No significant contamination from other organelles was observed, which was verified by the corresponding subcellular resident proteins, including 2466
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nuclear protein lamin B, endoplasmic reticulum(ER) resident protein calnexin, and cytosolic protein β-actin. 3.2. Comparison of Protein Expressions in the Left Ventricular Mitochondria of SHR and WKY Rats at the Age of 4 Weeks. To investigate mitochondrial protein expressions in the myocardium before BP elevation, mitochondrial proteins from both SHR and WKY rats at the age of 4 weeks were extracted and applied to comparative analysis by DIGE (Figure 2A). Typical gels are shown in Figure 2B and C. From the approximate 2000 spots detected in each gel, 33 protein spots were found to be modified in SHR by the DeCyder software analysis with an absolute ratio of more than 1.3-folds with statistical significance (p < 0.05). Among these modified protein spots, 17 were down-regulated, and 16 were up-regulated. These deferential spots were cut from preparative gel, digested and applied to MALDI-TOF-TOF tandem MS analysis. All the 33 spots were successfully identified. Several spots differently located on 2D gel were identified as the same protein, indicating potential diversity of protein
Mitochondrial Proteomics of Cardiac Hypertrophy in SHR
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Figure 3. Distribution of deregulated mitochondrial proteins identified by MALDI-TOF/TOF. (A) Pie chart of protein classification. (B) Distribution of protein expression of mitochondria at different ages of WKY rats and SHR. The spot volume ratio was normalized with the value of related 4-week-old WKY rats. One way ANOVA using Decyder software (version 6.5) was used to determine the changes of the spots we found. Changes of spots in 4-week-old SHR comparing with WKY rats were significant. The significant changes of related ages were shown. (*p < 0.05, **p < 0.01).
forms including post-translational modification (PTM) change, mutation and isoform. Totally, these identified differential spots account for 17 nonredundant proteins (Table 1) related to metabolic energy pathways such as fatty acid oxidation (29.41%), glycolysis (5.88%), pyruvate oxidation (5.88%), tricarboxylic acid cycle (11.76%), electron transport chain oxidative phosphorylation (35.29%) and proteins with other functions (11.76%) (Figure 3A). 3.3. Comparison of Protein Expressions in the Left Ventricular Mitochondria of SHR and WKY Rats at the Age of 20 Weeks. To reveal the effect of hypertension development on the mitochondrial protein expression, the mitochondrial protein expression profiling of 20-week-old SHR and WKY rats were compared following the same procedure as described above. Spot volume of all four groups was normalized against that of 4-week-old WKY rats and expressed as ratio to it. Surprisingly, no new differential spots were found in addition to those of 4-week-old rats. Only 13 out of 33 spots continued to change significantly by more than 1.30-fold in SHR at the age of 20 weeks (marked in Figure 3B). The others no longer changed at 20 weeks. 3.4. Full-Length Hadha Is down-Regulated in Left Ventricle of SHR. On the 2D DIGE gel, five spots (1921, 996, 983, 1190 and 1192) located in different position were all identified to be mitochondrial trifunctional enzyme R subunit (Hadha). Hadha was composed of two domains with different functions: long-chain enoyl-CoA hydratase at N-terminal and long-chain 3-hydroxyacyl-CoA dehydrogenase at C-terminal (Figure 4A). Spot 1921, with a molecular weight of about 83 kDa, was observed to undergo a significant down-regulation in 4-week-old SHR (change fold -2.15, p < 0.05) and a slight down-regulation in 20-week-old SHR (change fold -1.3, p
