Aging induces cardiac diastolic dysfunction ... - Wiley Online Library

Aging Cell (2005) 4, pp57–64

Doi: 10.1111/j.1474-9728.2005.00146.x

Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification

Blackwell Publishing, Ltd.

Shi-Yan Li,1 Min Du,2 E. Kurt Dolence,1 Cindy X. Fang,1 Gabriele E. Mayer,1 Asli F. Ceylan-Isik,1 Karissa H. LaCour,1 Xiaoping Yang,1 Christopher J. Wilbert,1 Nair Sreejayan1 and Jun Ren1 1

Division of Pharmaceutical Sciences & Center for Cardiovascular 2 Research and Alternative Medicine, and Department of Animal Science, University of Wyoming, Laramie, WY, USA

one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs. Key words: AGEs; aging; cardiac myocytes; contraction; oxidative stress.

Summary Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24 –26-month) mice using a MyoCam® system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening ± dL/dt). Oxidative stress and protein damage were (± evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, ± dL/ dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced phox phox p47 but not gp91 expression. In addition, level of cardiac AGEs was ∼2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4 –7 was distinctively modified in aged heart using

Correspondence Dr Jun Ren, Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071-3375, USA., Fax: + 1307 766 2953; e-mail: [email protected] Accepted for publication 6 January 2005 © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005

Introduction The prevalence of heart failure is 70 times higher in persons aged 65 or older than in those aged 20–34 [American Heart Association, 2005; calculated from the proportions of old (av. 11.225) versus young (av. 0.15) people suffering heart failure]. Approximately 80% of hospital admissions for heart failure in the US involve patients aged > 65 years (Rich, 1999). Cardiac functional reserve declines with age and cardiac aging is a continuous and irreversible process that accounts for the most common cause of death in elderly people (Fleg et al., 1995; Lakatta, 1999). The aging heart displays left ventricular wall thickening and myocardial enlargement (Lakatta, 1987). Although interactions among advanced age, occult disease and physical inactivity have been considered in interpreting age-associated changes in cardiovascular function, the ‘aging process’ itself has been proved to be independently related to change of cardiac structure and performance such as cardiac hypertrophy and prolonged myocardial contraction (Lakatta, 1987, 1999; Fleg et al., 1995). Several cellular mechanisms are thought to be involved in cardiac aging, including prolonged action potential duration, altered myosin heavy chain (MHC) isoform expression and sarcoplasmic reticulum (SR) function, all of which may lead to changes in cardiac excitation–contraction (E-C) coupling (Lakatta, 1987, 1999; Kass et al., 2001). Cardiac E-C coupling cycle or cardiac cycle has been demonstrated to be prolonged with increased 2+ age, probably due to cytosolic Ca overload-induced dysregu2+ lation of cytosolic Ca homeostasis (Zhou et al., 1998; Lakatta 2+ et al., 2001). The cytosolic Ca load is determined by multiple factors, including membrane structure and permeability, the regulatory proteins within the membrane, and reactive oxygen species (ROS), which affect both membrane structure and function. However, the link between advanced age and altered cardiac E-C coupling has not been fully understood. Therefore, the aim of the present study was to examine the effect of aging on cardiomyocyte contractile function and its causal relationship with cardiac protein oxidation, accumulation of advanced glycation endproducts (AGEs) and protein oxidative modification. 57

58 Cardiac diastolic dysfunction and AGE in aging, S.-Y. Li et al.

Table 1 General features of young (2–3 months) and old (24–26 months) FVB mice

Body weight (g) Heart weight (mg) Heart/ weight (mg g−1) Liver weight (mg) −1 Liver/ body weight (mg g ) Kidney weight (mg) −1 Kidney/ body weight (mg g ) −1 Fasting blood glucose (mg dL )

Young (n = 16)

Old (n = 17)

18.8 ± 1.4 120 ± 13 6.24 ± 0.33 906 ± 78 47.53 ± 1.69 249 ± 19 13.26 ± 0.26 93.3 ± 3.5

28.3 ± 0.8* 179 ± 12* 6.33 ± 0.40 1392 ± 48* 49.29 ± 1.51 422 ± 22* 14.87 ± 0.59 95.6 ± 6.4

Data are expressed as mean ± SEM, *P < 0.05 vs. young group.

Baseline mechanical properties of left ventricular myocytes Aged cardiac myocytes exhibited significantly larger crosssectional areas compared with those of the young counterparts (P < 0.05 between the two groups). Myocytes isolated from 24month-old FVB mice displayed a similar extent of contractile capacity as indicated by amplitude of peak shortening (PS) and maximal velocities of shortening/relengthening (± dL /dt), compared with those from 2-month-old young mice. Duration of shortening (TPS) was not different between the young and aged groups. However, the duration of relengthening (TR90) was significantly prolonged in the aged myocytes compared with the young myocytes (Fig. 1).

Results General features of young and old mice

Effect of increasing stimulation frequency on myocyte shortening

The impacts of aging on body, heart, liver and kidney weight, and blood glucose level are shown in Table 1. Advanced age increased the absolute weight of body, heart, liver and kidney but did not affect the size of these organs when normalized to body weight. Aging did not affect the fasting blood glucose level.

Rodent hearts normally contract at very high frequencies, whereas our mechanical evaluation was conducted at 0.5 Hz. To evaluate the impact of aging on cardiac contractile function under higher frequencies, we increased the stimulating frequency up −1 to 5.0 Hz (300 beats min ) and recorded the steady-state peak shortening. Cells were initially stimulated to contract at 0.5 Hz

Fig. 1 Contractile properties of ventricular myocytes from young and aged mouse hearts. (A) Cross-sectional area; (B) peak shortening (PS) as percentage of resting cell length; (C) maximal velocity of shortening (± dL/dt); (D) maximal velocity of relengthening (– dL/dt); (E) time-to-peak shortening (TPS); (F) time-to-90% relengthening (TR90). Mean ± SEM, n = 60 cells/group. *P < 0.05 vs. young group. © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005

Cardiac diastolic dysfunction and AGE in aging, S.-Y. Li et al. 59

Fig. 2 Peak shortening (PS) amplitude of ventricular myocytes isolated from young and aged mouse hearts at different stimulus frequencies (0.1, 0.5, 1.0, 3.0 and 5.0 Hz). PS at each stimulus frequency was normalized to that of 0.1 Hz of the same cell. Mean ± SEM, n = 20 and 22 cells for the young and aged myocyte group, respectively. *P < 0.05 vs. young group.

for 5 min to ensure steady state before commencing the frequency study. All the recordings were normalized to PS at 0.1 Hz of the same myocyte. Figure 2 shows a steeper negative staircase in PS in aged myocytes with increasing stimulating frequency compared with young myocytes. The percentage decrease of myocyte shortening from 0.1 Hz was significantly greater in aged myocytes than in young myocytes at all higher 2+ frequencies (0.5–5 Hz), suggesting that intracellular Ca cycling function may be reduced under aging. The baseline PS was not significantly different between young (4.84 ± 0.14%, n = 20) and aged (5.88 ± 0.52%, n = 22, P > 0.05 vs. young group) myocytes.

Effect of aging on oxidative stress, protein carbonyl formation and NAD(P)H oxidase levels Aging is often associated with enhanced oxidative stress leading to irreversible damage of membrane proteins or lipids (Forster et al., 1996; Cakatay et al., 2003; Ozbey et al., 2003). The glutathione (GSH) and glutathione disulfide (GSSG) levels are commonly used markers for oxidative stress. A low GSH/GSSG ratio suggests increased oxidative stress. Results in Fig. 3(A) indicate that advanced age significantly lowered the GSH/GSSG ratio. Consistently, protein carbonyl formation, which is indicative of protein oxidation and protein damage determined by both spectrometry and immunoblot, was significantly elevated in the aged group (Fig. 3). Because the NAD(P)H oxidases are predominant sources of ROS leading to oxidative stress (Cai & Harrison, 2000; Cai et al., 2003), the levels of two major subunits of NAD(P)H, phox phox gp91 and p47 , were evaluated by immunoblot. In agreement with elevated oxidative stress in the aged group, the level of p47, a cytosal component of NAD(P)H, increased significantly © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005

Fig. 3 Oxidative stress and protein oxidative damage in cardiac tissue. (A) Ratio of GSH to GSSG; (B) protein carbonyl contents; (C) a representative gel depicting the patterns of total cardiac protein oxidation from both young and aged groups by immunostaining using anti-dinitrophenylhydrazine (DNPH) Western blot. Mean ± SEM, n = 8. *P < 0.05 vs. young group.

in aging, whereas no significant variation of gp91 detected from both groups (Fig. 4).

phox

level was

Effect of aging on cardiac methylglyoxal level and formation of AGEs A close link between oxidative stress and formation of AGEs has been demonstrated, in which AGEs are considered as one of the important factors in age-related cardiovascular disease (Barouch et al., 2003; Jerums et al., 2003; Chang et al., 2004). As a highly reactive dicarbonyl compound produced during cell metabolism and from degradation reactions, such as glucose autoxidation and lipid peroxidation, methylglyoxal is considered to be one of the potent precursors of AGEs contributing to protein aging. In the present study, content of AGEs was evaluated by ELISA and methylglyoxal level was measured by an o phenylenediamine-based assay (Chaplen et al., 1996). Cardiac AGE content was significantly increased in the aged group, whereas no difference in methylglyoxal level was observed between the


Fig. 4 NAD(P)H oxidase expression in cardiac tissue. (A) Representative gels depicting immunostaining using anti-NAD(P)H oxidase subunits of gp91phox and p47phox. (B) NAD(P)H oxidase subunit levels of gp91phox and p47phox. Mean ± SEM, n = 8 per data point. *P < 0.05 vs. young group.

aged and young groups (Fig. 5). To reveal the particular proteins modified by AGEs under aging, one- and two-dimensional SDS gel electrophoreses and Western blottings (Fig. 6) demonstrated that a group of proteins with a molecular weight range of 50 –75 kDa, isoelectric point (pI) 4 –7, were distinctively modified by AGEs.

Discussion The present study demonstrated that aging directly leads to cardiac contractile dysfunction represented as prolonged relaxation duration and impaired tolerance to increased stress (higher stimulus frequency). In addition, we found elevated oxidative stress, NAD(P)H oxidase expression, protein carbonyl formation, protein oxidation and protein modification as well as enhanced AGE level in aged hearts. Our results favor the idea that oxidative stress and AGE accumulation may be related to aging-induced cardiomyocyte dysfunction. Prolonged relaxation duration has been indicated in the aging myocardium (Lim et al., 1999, 2000). Our current study demonstrated a prolonged duration of relengthening (TR90) associated with normal peak shortening (PS) amplitude and maximal rate of shortening /relengthening (± dL /dt) and duration of shortening (TPS). This mechanical dysfunction is consistent with the data indicating cardiac oxidative stress, protein oxidative damage and protein modification by AGEs. Several mechanisms have been

Fig. 5 Cardiac tissue methylglyoxal (MG) levels and AGE contents. (A) MG was measured by high-performance liquid chromatography. (B) AGEs were determined by ELISA using anti-AGE monoclonal antibody (6D12). Mean ± SEM, n = 8 per data point. *P < 0.05 vs. young group.

postulated for such aging-associated mechanical defects. It has been reported that the depressed rate of shortening may be associated with aging-induced shifts in contractile protein isoforms, such as the shift of MHC isoform from the fast type (V1) to the slow type (V3). Aging has also been demonstrated to reduce myofilament Ca2+ sensitivity significantly. Finally, prolonged duration of relaxation may simply be a consequence of 2+ impaired sarco(endo)plasmic reticulum Ca -ATPase (SERCA) + 2+ and Na /Ca exchanger function with advanced age (Chiamvimonvat, 2002; Janczewski et al., 2002; Kass et al., 2004). Our study favors a role of oxidative stress and protein oxidation/ modification in aging-associated cardiac mechanical dysfunction. Interestingly, our data revealed prolonged relaxation duration (TR90) but normal maximal velocity of relaxation (± dL /dt). This discrepancy may indicate that protein(s) responsible for rapid relaxation (rapid phase of ventricular filling during diastole) such as MHC isozyme may be normal whereas cellular machineries responsible for reduced phase ventricular filling 2+ + 2+ or slow cytosolic Ca extrusion (e.g. Na –Ca exchanger, mito2+ chondrial or sarcolemmal Ca pumps) are at fault. Further study is warranted to elucidate the expression and function of these myocardial proteins. Increased oxidative stress has been hypothesized to play an important role in the aging process (Sohal, 2002; Mutlu-Turkoglu © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005


Fig. 6 Representative gels depicting cardiac protein non-enzymatic glycation immunostaining using anti-AGE monoclonal antibody (6D12). (A) One-dimensional SDS gel electrophoresis analysis showed the cardiac proteins modified by AGEs. Two-dimensional SDS gel electrophoresis analysis demonstrated the proteins modified by AGEs from the young group (B) and old group (C).

et al., 2003). It is suggested that oxidative damage to proteins plays a crucial role in age-associated cardiovascular disease because oxidation of proteins leads to loss of catalytic function, and thus constitutes one of the mechanisms linking oxidative stress/damage and age-associated losses in physiological function. Protein carbonyl detection as a measure of oxidative damage revealed extensive oxidative damage in cardiac proteins of old mice compared with young mice. The NAD(P)H oxidases serve as a predominant source of ROS generation and a hallmark of oxidative stress. In the determination of two major NAD(P)H oxidase subunits, gp91phox and p47phox, the p47phox level was found to be elevated significantly in the aged group, whereas no overt phox difference in gp91 level was observed. As documented elsephox where (Cai et al., 2003; Li & Shah, 2002), p47 is an important cytosolic subunit component, the expression and translation of which determines the activation of the oxidase. The activation of NAD(P)H oxidase and the production of ROS by these enzyme systems are regarded as one of the fundamental mechanisms in driving oxidative stress and contributing to the pathogenesis of numerous cardiovascular diseases, including hypertension, cardiac hypertrophy, heart failure, restenosis and atherosclerosis (Cai & Harrison, 2000; Cai et al., 2003). AGEs, by contrast, are a heterogeneous group of non-enzymatic glycation products of proteins that accumulate in the circulation and various tissues (Brownlee, 1995). It is suggested that accumulation of AGEs may account for the pathogenesis of cardiovascular dysfunction associated with aging (Pirola et al., 2003; Yan et al., 2003; Li et al., 2005). In agreement with those ideas, our results showed that AGE content in the heart was significantly elevated in the aged group. In particular, a group of proteins with a molecular range of 50–75 kDa, pI 4 –7, was found to be distinctively modified by AGEs. Methylglyoxal, a reactive dicarbonyl © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005

compound endogenously produced mainly from glycolytic intermediates, is known as one of the potent precursors of AGEs. However, cardiac content of methylglyoxal measured in this study from both young and aged groups exhibited a non-significant difference. It is possible that the methyglyoxal-detoxifying enzyme glyoxalase system was altered ineffectively in senescence. It has been suggested that methylglyoxal is detoxified by the glyoxalase system through which the over-expression of AGE formation is prevented (Shinohara et al., 1998). Enzymatic analysis is warranted to clarify this question. In summary, our study revealed impaired cardiac relaxation and depressed tolerance to increased stress (stimulus frequency) in aged cardiac myocytes. Our data suggest a causal association between these mechanical dysfunctions and cardiac oxidative stress, protein oxidation/modification, suggesting that these oxidative events may play a role in the deteriorated cardiac mechanical function under aging. Given what we know about the cardiac E-C coupling in aging, the in-depth mechanism of action and clinical value of employing antioxidants, AGE production inhibitors or cross-linking breakers in delaying cardiac aging may have some promising future.

Experimental procedures Experimental animals The experimental protocol described in this study was approved by the Institutional Animal Care and Use Committee of the University of Wyoming (Laramie). Male young (2 months of age) and old (∼24 –26 months of age) mice were from the inbred albino FVB line housed within the School of Pharmacy Animal Facility, University of Wyoming. The mice were maintained on


a 12 : 12-h light–dark illumination cycle and allowed food and water ad libitum. Blood glucose levels were measured using a glucose monitor (Accu-ChekII, model 792, Boehringer Mannheim Diagnostics, Indianapolis, IN, USA) and body weights were measured with a standard laboratory scale.

Cell isolation procedures Mouse hearts were removed and perfused with Krebs–Henseleit bicarbonate buffer containing (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 HEPES and 11.1 glucose, with 5% CO2−95% O2. Hearts were subsequently digested with −1 223 U mL collagenase D (Boehringer Mannheim) for 20 min at 37 °C. After perfusion, left ventricles were removed and minced 2+ before being filtered. Extracellular Ca was slowly added back to 1.25 mM. Functional study was conducted within 6 h of cell isolation and myocytes with obvious sarcolemmal blebs or spontaneous contractions were not used (Hintz et al., 2003).

Cell shortening/relengthening Mechanical properties of ventricular myocytes were assessed using a SoftEdge MyoCam® system (IonOptix Corp., Milton, MA, USA) (Hintz et al., 2003). In brief, left ventricular myocytes were placed in a Warner chamber mounted on the stage of an inverted −1 microscope (Olympus, IX-70) and superfused (∼1 mL min at 25 °C) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. The cells were field stimulated with a suprathreshold voltage at a frequency of 0.5 Hz (unless otherwise stated), 3-ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to an FHC stimulator (Brunswick, NE, USA). The myocyte being studied was displayed on a computer monitor using an IonOptix MyoCam camera. An IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), the amplitude myocytes shortened upon electrical stimulation – an indication of peak ventricular contractility; time-to-PS (TPS), the duration of myocyte shortening – an indication of systolic duration; time-to-90% relengthening (TR90), the duration to reach 90% relengthening – an indication of diastolic duration (90% rather 100% relengthening was used to avoid noisy signal at baseline level); and maximal velocities of shortening/relengthening (± dL/ dt), maximal slope (derivative) of shortening and relengthening phases – indication of maximal velocities of ventricular pressure increase/decrease. In the case of altering stimulus frequency, the steady-state contraction of myocytes was achieved (usually after the first five to six beats) before PS amplitude was recorded.

Glutathione and glutathione disulfide (GSH/GSSG) assay Glutathione levels were determined in cardiac tissue as an indication of oxidative stress. For measurement of GSH, frozen

tissue samples were homogenized in four volumes (w/v) of 1% picric acid. Acid homogenates were centrifuged at 16 000 g (30 min) and supernatant fractions collected. Supernatant fractions were assayed for total GSH and GSSG by the standard recycling method and GSH content was determined using a standard curve generated from known concentrations of GSH. The procedure consisted of using one-half of each sample for GSSG determination and the other half for GSH. Samples for GSSG determination were incubated at room temperature with 2 µL of 4-vinyl pyridine (4-VP) per 100-µL sample for 1 h after vigorous vortexing. Incubation with 4-VP conjugates any GSH present in the sample so that only GSSG is recycled to GSH without interference by GSH. The GSSG (as GSH × 2) was then subtracted from the total GSH to determine actual GSH level (Ren et al., 2003).

Quantification of protein carbonyl Protein carbonyl content of total protein lysate from cardiac tissue was determined as described (Hintz et al., 2003). Briefly, proteins were extracted and lysed to prevent proteolytic degradation. Protein was precipitated by adding an equal volume of 20% TCA to protein (0.5 mg) and centrifuged for 1 min. The TCA solution was removed and the sample resuspended in 10 mM 2,4-dinitrophenylhydrazine (2,4-DNPH) solution. Samples were incubated at room temperature for 15 –30 min, 500 µL of 20% TCA was added and samples were centrifuged for 3 min. The resultant supernatant was discarded, the pellet washed in ethanol/ethyl acetate (1 : 1, v/v) and allowed to incubate at room temperature for 10 min. The samples were centrifuged again for 3 min and the ethanol/ethyl acetate steps were repeated twice more. The precipitate was resuspended in 6 M guanidine solution, centrifuged for 3 min and insoluble debris removed. The maximum absorbance (360 –390 nm) was read vs. the appropriate blanks (2 M HCl) and the carbonyl content calculated using the formula: −1 −1 absorption at 360 nm × 45.45 nmol mL protein content (mg).

Immunodetection of oxidized protein Oxidized proteins in cell extracts were revealed immunochemically by their carbonyl content after derivatization with dinitrophenylhydrazine (Yan et al., 1998). Immunodetection was performed according to the instructions for the OxyBlot kit (Chemicon International, Inc., Temecula, CA, USA).

Tissue methylglyoxal (MG) determination MG level in cardiac tissue was measured using the most widely accepted o-phenylenediamine (o-PD)-based assay (Chaplen et al., 1996). Briefly, cardiac tissue was homogenized on ice, followed by sonication (3 × 5 s) and centrifugation (12 000 g, 10 min). The supernatant was derivatized with 125 nmol of o-PD (derivatizing agent) at 20 °C for 4 h. The quinoxaline derivative of MG (2-MQ) and the quinoxaline internal standard (5-MQ) was measured using a Beckman GOLD system HPLC. © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005


AGE-ELISA

Acknowledgments

The degree of AGE-modified proteins in total protein lysate was assayed by AGE-ELISA (Li et al., 2005). Briefly, after coating with AGE sample and blocking, each well was washed and incubated with 6D12 monoclonal antibody (1 : 2000 dilution, Trans Genic Inc., Kumamoto, Japan) overnight at 4 °C. Following three rinses, HRP-conjugated secondary antibody (1 : 2000) was added to each well and incubated at 37 °C for 1 h. After rinsing six times, 100 µL per well of NeA-Blue TMB substrate (Clinical Science Products Inc., MA, USA) was added to each well and the optical density at 450 nm was determined by micro plate reader after 10 –30 min of incubation at 37 °C following addition of 1 N H2SO4 to stop the color development reaction.

We thank Dr Mark Quinn from Montana State University (Bozemann) for kindly providing antibodies for NAD(P)H oxidase phox phox subunits gp91 and p47 . This work was supported in part by grants from the American Diabetes Association (7-00-RA-21) and NIH/NIA 1 R03 AG21324-01 to J.R.

Western blot analysis of NAD(P)H oxidase subunits Western blot analysis of NAD(P)H oxidase subunits was performed on total protein lysate prepared from cardiac tissue. Briefly, freshly dissected hearts were homogenized on ice with a tissue tearer in 1 mL RIPA lysis buffer containing 150 mM NaCl, 0.25 deoxycholic acid, 1% NP-40, 1 mM EDTA, 50 mM Tris/HCl, pH 7.4, and 1% protease inhibitor cocktail, followed by sonication (3 × 5 s, 10 W) over ice, and then by centrifugation (12 000 g, 10 min) to remove the precipitated material. Protein concentration was determined in the supernatant containing the soluble proteins using a Bradford assay. Samples containing equal amounts of proteins were separated on 10% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in TBS-T, and then incubated with monoclonal antibodies to NAD(P)H oxidase subunits gp91phox and p47phox (kindly provided by Dr Mark Quinn, Montana State University, Bozemann). After immunoblotting, the film was scanned and the intensity of immunoblot bands was detected with a Bio-Rad Calibrated Densitometer (Model: GS-800) (Li et al., 2004).

Two-dimensional gel electrophoresis and Western blot analysis of cardiac AGEs Protein lysates prepared from cardiac tissue were precipitated with the ReadyPrep™ 2-D Cleanup kit (Bio-Rad), and re-suspended in ReadyPre Rehydration Buffer (Bio-Rad). Isoelectric focusing (20 –50 µg of protein) was performed in ReadyStrip™ immobilized pH gradient (IPG) strips (7 cm, pH 3 –10, Bio-Rad) according to the manufacturer’s instructions. Second dimension SDS-PAGE was performed on 10% acrylamide gels followed by anti-AGEs Western blot (Schutt et al., 2003).

Statistics For each experimental series, data are reported as mean ± SEM. Differences between groups was assessed using Student’s t-test. A P-value of less than 0.05 was considered significant. © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005

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