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Am J Physiol Heart Circ Physiol 293: H3673-H3684, 2007. First published October 5, 2007; doi:10.1152/ajpheart.00284.2007
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Mechanisms of acrolein-induced myocardial dysfunction: implications for environmental and endogenous aldehyde exposure

Jianzhu Luo,1 Bradford G. Hill,1,2 Yan Gu,1 Jian Cai,2 Sanjay Srivastava,1 Aruni Bhatnagar,1,2 and Sumanth D. Prabhu1,3

1Institute of Molecular Cardiology, Department of Medicine, and 2Department of Pharmacology, University of Louisville, and 3Medical Service, Louisville Veterans Affairs Medical Center, Louisville, Kentucky

Submitted 7 March 2007 ; accepted in final form 29 September 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aldehydes are ubiquitous pollutants generated during the combustion of organic materials and are present in air, water, and food. Several aldehydes are also endogenous products of lipid peroxidation and by-products of drug metabolism. Despite well-documented high reactivity of unsaturated aldehydes, little is known regarding their cardiovascular effects and their role in cardiac pathology. Accordingly, we examined the myocardial effects of the model unsaturated aldehyde acrolein. In closed-chest mice, intravenous acrolein (0.5 mg/kg) induced rapid but reversible left ventricular dilatation and dysfunction. In mouse myocytes, micromolar acrolein acutely depressed myofilament Ca2+ responsiveness without altering catecholamine sensitivity, similar to the phenotype of stunned myocardium. Immunoblotting revealed increased acrolein-protein adducts and protein-carbonyls in both acrolein-exposed myocardium (1.8-fold increase, P < 0.002) and myocytes (6.4-fold increase, P < 0.02). Both the contractile dysfunction and adduct formation were markedly attenuated by pretreatment with the thiol donor N-acetylcysteine (5 mM). Two-dimensional gel electrophoresis and mass-assisted laser desorption/ionization time-of-flight mass spectrometry analysis revealed two groups of adducted proteins, sarcomeric/cytoskeletal proteins (cardiac {alpha}-actin, desmin, myosin light polypeptide 3) and energy metabolism proteins (mitochondrial creatine kinase-2, ATP synthase), indicating site-specific protein modification that was confirmed by immunohistochemical colocalization. We conclude that direct exposure to acrolein induces selective myofilament impairment, which may be, in part, related to the modification of proteins involved in myocardial contraction and energy metabolism. Myocardial dysfunction induced by acrolein and related aldehydes may be symptomatic of toxicological states associated with ambient or occupational exposures or drug toxicity. Moreover, aldehydes such as acrolein may mediate cardiac dysfunction in pathologies characterized by high-oxidative stress.

oxidative stress; myocardial contraction; protein adducts; aldehydes

ALDEHYDES ARE UBIQUITOUS AIR and water pollutants generated by burning fossil fuels such as coal and gasoline (11). Aldehydes also occur naturally in plant and animal material and during the heating of cooking oils; consequently, they are significant constituents of human food. Large amounts of aldehydes are present in cigarette smoke, and aldehyde-DNA adduct formation has been linked to the development of lung cancer in smokers (10). At least 36 different aldehydes have been found in US drinking water at levels often exceeding maximal recommended concentrations (6). Accidental occupational exposure to high levels of aldehydes is frequent (nearly 45,000 cases/yr) (6). Aldehydes are highly reactive and toxic. They react with cell thiols to form Michael adducts or hemithioacetals and also react with amine groups in sugars, phospholipids, proteins, and DNA bases (9). Epidemiological data suggest that workers exposed to aldehydes have an elevated risk of cardiovascular disease and death (14, 23). Nevertheless, the direct cardiovascular effects of aldehydes remain unknown, and the mechanisms by which they could induce or exacerbate cardiovascular disease and dysfunction remain unclear.

Toxicological profiles of complex aldehyde mixtures in food, water, and smoke are difficult to establish. Hence, to assess prototypic aldehyde toxicity, we studied the cardiac effects of acrolein, a highly reactive C3 {alpha},β-unsaturated aldehyde (CH2 = CH – CHO). Acrolein is classified by the Environmental Protection Agency as a high-priority air and water toxic (7). It is generated in concentrations ranging from 10 to 140 µg per cigarette, and its levels in several foods range from 10 to 600 µg/kg (11, 13). Acrolein is also generated endogenously from the oxidation of polyunsaturated fatty acids and the metabolism of cyclophosphamide (9, 36, 37). It is one of the most reactive biological aldehydes and readily forms covalent adducts with proteins, nucleic acids, and phospholipids. DNA adducts of acrolein and related aldehydes are the most common background DNA lesions in humans (23) and protein adducts of acrolein have been detected in several chronic diseases, including Alzheimer's disease (18), atherosclerosis (27), and renal failure (25). Nonetheless, in most cases, the proteins susceptible to acrolein modification have not been identified, and the pathological significance of protein-acrolein adducts remains unclear.

Given the importance of oxidative stress in cardiac diseases, such as heart failure (30), ischemia-reperfusion (11, 38), and myocardial stunning (3), acrolein and related aldehydes may be important, albeit unrecognized, contributors to their pathophysiology. Accordingly, we examined the functional and cellular effects of environmentally and endogenously relevant concentrations of acrolein in murine myocardium. We demonstrate, for the first time, that acrolein induces profound and, to a large extent, reversible myocardial dysfunction, and that these effects are related, at least in part, to acrolein-triggered modification of select proteins regulating myocardial contraction and energy metabolism. Preliminary findings of this study have been reported previously (19).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Acrolein preparation. Acrolein was prepared daily and used within 4 h. Free acrolein was released by the acid hydrolysis (pH 3.0) of diethyl acetal acrolein (Sigma) in 0.1 N HCl for 1 h at room temperature (RT). Acrolein was resuspended in distilled water at 10 mM, filtered through a 0.22-µm sterile syringe filter, and stored at 4°C until used.

Acrolein and in vivo left ventricular function. All studies were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHHS publication No. [NIH] 85-23, revised 1996). All of the animal protocols in the paper were approved by the Institutional Animal Care and Use Committee at the University of Louisville. Adult C57/BL6 mice, 8–10 wk old, weighing 25–30 g, were used for closed-chest left ventricular (LV) pressure-volume studies (n = 8). Mice were anesthetized with 80 µg/g ip pentobarbital, intubated, and ventilated with 95% O2 using a small-rodent ventilator (Harvard Apparatus) at 155–160 breaths/min and tidal volume of 15 µl/g. Body temperature was maintained at 37°C using a heating pad and lamps. The left jugular vein was cannulated for fluid and drug administration. A Millar 1.4-Fr conductance catheter (SPR-839) was inserted into the LV via the carotid artery, and pressure and conductance signals were visualized online using the ARIA-1 system (Millar). After baseline steady-state measurements, acrolein (0.5–10 mg/kg) was administered via the jugular vein, and hemodynamic recordings were recorded for 20 min. Intravenous (IV) hypertonic saline (0.5–1 µl/g) was then given to determine parallel conductance, and LV volume (µl) was derived from the parallel conductance and ex vivo cuvette calibration with heparinized, warm blood.

LV systolic function was indexed by maximum rate of change in LV pressure (dP/dtmax; mmHg/s) in relation to end-diastolic volume (EDV; µl), and by preload adjusted maximal power (PAMP; mW/µl2). Maximal power is the peak value of the product of LV pressure and flow (dV/dt, where V is LV volume) (28). PAMP (maximal power divided by the square of EDV) is a steady-state index of LV function that is both preload and afterload independent and correlates with end-systolic elastance (28). Diastolic function was assessed by the LV end-diastolic pressure (EDP) (mmHg) and {tau}, the time constant of LV relaxation (ms) (30). Myocardial protein-acrolein adduct formation following acute exposure was evaluated in eight additional mice. Mice were anesthetized as above and administered either 0.5 mg/kg acrolein or PBS via the jugular vein (n = 4/group). After 20 min, the chest was opened, and the hearts were rapidly excised. The LV was separated, snap frozen, and saved at –80°C until used for determination of protein-acrolein adducts.

Isolation of murine cardiomyocytes. Mouse LV myocytes were isolated by modified Langendorff perfusion and collagenase digestion, as described by the Alliance for Cellular Signaling (26). Mice were anesthetized with ketamine (43.5 mg/kg im), acepromazine (1.5 mg/kg), and xylazine (1.7 mg/kg) and given heparin (10 U/g ip). After median sternotomy, the heart was rapidly excised and rinsed with physiological saline. The aortic lumen was tied to a 18-g cannula and perfused with oxygenated (95% O2–5% CO2) Ca2+-free modified Tyrode bicarbonate buffer (buffer A in mM: 126 NaCl, 4.4 KCl, 1.0 MgCl2, 18 NaHCO3, 11 glucose, 4 HEPES, 10 2,3-butanedione monoxime, 30 taurine, pH 7.35) at 37°C for 5 min. The perfusate was then changed to 50 ml of recirculating digestion buffer [buffer A with 0.25 mg/ml Liberase Blendzyme type 1 (Roche), 0.14 mg/ml trypsin (GIBCO), and 12.5 µM CaCl2] for 12–15 min. The heart was removed, and the LV was separated with blunt forceps in 2-ml digestion buffer. The minced tissue was gently agitated by repeated pipette aspiration, filtered through a 140-µm nylon mesh, and transferred into a conical tube containing 10 ml of buffer B (buffer A with 10% FCS and 12.5 µM CaCl2) and allowed to sediment for 15 min at RT. The supernatant was transferred to another tube and gently centrifuged at 90 g for 2 min. The pellets from both tubes were combined and resuspended in buffer C (buffer A with 5% FCS and 12.5 µM CaCl2) and transferred to a 100-mm culture dish. CaCl2 was then added in a graded fashion at 4-min intervals (five total steps) to sequentially increase the Ca2+ concentration to 500 µM. The suspension was then placed in a 15-ml conical tube and allowed to sediment for 10 min at RT. The supernatant was transferred to another tube and gently centrifuged at 90 g for 2 min, and myocytes contained in both pellets were combined and resuspended in modified serum-free DMEM medium supplemented with 0.2% albumin, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 0.1 µM insulin, 0.1 nM triiodothyronine, 10 mM 2,3-butanedione monoxime, and 0.1% penicillin/streptomycin and plated on laminin-coated culture dishes overnight at 37°C in a 5% CO2 incubator. The media was replaced before experimentation to wash away unattached cells and ensure that only rod-shaped myocytes were used for subsequent studies.

To isolate myocytes for studies of cell contraction and immunofluorescence, the above protocol was modified as follows. First, the digestion buffer used contained buffer A with 1 mg/ml collagenase II (Worthington), 1 mg/ml albumin, and 25 µM CaCl2 and was recirculated for 20 ~ 30 min until hearts became flaccid. Second, the heart was separated in mincing buffer (10 ml digestion buffer with 9 mg/ml albumin). Finally, in place of buffer C in the above protocol, we used buffer A with 1% albumin and 25 µM CaCl2. After the final step, myocytes were resuspended in modified serum-free DMEM medium (37°C, 5% CO2) and maintained under resting conditions for at least 1 h before experimentation.

Studies of acrolein and cardiomyocyte contraction. Myocytes were loaded with the fluorescent Ca2+ indicator fura-2 AM (1 µM, Molecular Probes) at RT for 15 min and washed three times (10 min/wash) with modified Tyrode solution (in mM: 137 NaCl, 1.2 MgSO4, 4.9 KCl, 1.2 NaH2PO4, 20 HEPES, 15 D-glucose, 1.8 CaCl2, pH 7.35). Myocytes were then placed in a closed-cell chamber (Warner) on an inverted stage microscope with epifluorescence objectives (Nikon Eclipse TE 200) and superfused at 1 ml/min with modified Tyrode solution. Field stimulation (MyoPacer Field Stimulator, IonOptix) was performed at 0.5 Hz (pulse duration 4 ms) via platinum electrodes in the cell chamber. Rod-shaped myocytes with clear striations and stable contraction for 20 min were selected for study using an IonOptix StepperSwitch system. Sarcomere shortening was determined using video-based (x40) digitized sarcomere spacing (SarcLen Acquisition, IonOptix). Fura-2 Ca2+ transients were measured by dual excitation at 365 and 380 nm, with excited fluorescence measured at 510 nm using a photomultiplier tube, and expressed as the 365-to-380-nm fluorescence ratio after background subtraction. Parameters were recorded during superfusion, either without or with acrolein (0.1–2 µM), and before and after the addition of 1 µM isoproterenol (ISO, Sigma). In some experiments, myocytes were pretreated with 5 mM N-acetylcysteine (N-AC) in modified serum-free DMEM medium 1 h before fura-2 loading.

Measurement of intracellular GSH and GSSG. After the appropriate exposure, mouse myocytes were homogenized in 5% sulfasalicylic acid, and reduced (GSH) and oxidized glutathione (GSSG) concentrations were determined as previously described (31).

Immunofluorescent detection of acrolein-protein adducts. Mouse cardiomyocytes were washed with PBS and allowed to attach to Lab-Tek II glass slides. Cells were fixed with methanol at –20°C for 5 min, blocked with PBS with 10% goat serum and 0.5% Triton X-100 at RT for 30 min, and incubated with 1:2,000 polyclonal rabbit anti-mouse keyhole limpet hemocyanin (KLH)-acrolein antibody (raised in house) at 4°C overnight. The cells were washed several times with PBS containing 10% goat serum and 0.5% Triton X-100, incubated with goat anti-rabbit FITC-conjugated secondary antibody (1:1,000, Santa Cruz) for 1 h at RT in the dark, and washed with PBS. Immunohistochemical colocalization studies required subsequent additional steps. For troponin I, slides were blocked with 10% bovine serum and 0.5% Triton X-100 for 30 min and incubated with goat-anti-mouse troponin I (1:500, Santa Cruz) for 2 h at RT followed by Texas Red conjugated chicken-anti-goat IgG secondary antibody (1:1,000) for 1 h. For actin, slides were incubated with PBS with 1% BSA for 30 min and then stained with Alexa 568 phalloidin (Molecular Probes) for 30 min at RT. Mitochondria were labeled by incubating the slides for 30 min in PBS with 100 nM MitoFluor Red 589 (Molecular Probes) for 30 min at RT. After dual labeling, the slides were washed several times with PBS and incubated at 37°C for 12 min with 4',6-diamidino-2-phenylindole (1:5,000, Molecular Probes) to label nuclei, washed further with PBS, covered with FluorSave reagent (Calbiochem), and imaged after 2 h with either epifluorescence objectives (Zeiss) or confocal laser scanning microscopy (Zeiss LSM510).

Western blotting and two-dimensional gel analysis for acrolein-modified proteins. Mouse cardiomyocytes were superfused (1 ml/min, 37°C) for 20 min with modified Tyrode solution, with or without 10 µM acrolein. Cardiomyocytes were then washed with modified Tyrode solution three times and centrifuged for 5 min at 500 g. The cell pellet was isolated and solubilized with Laemmli buffer, pH 6.8, containing 62.5 mM Tris·HCl and 2% SDS. Protein was loaded onto 12% SDS-polyacrylamide gels, electrophoresed, and electroblotted to polyvinylidene difluoride membranes. Western blotting was performed with IgG-purified polyclonal anti-KLH-acrolein antibodies using standard SDS-PAGE immunoblotting techniques and enhanced chemiluminescence plus detection (Amersham), as previously described (30). A parallel method (Oxyblot) was also used to detect protein-bound carbonyls formed after Michael addition of acrolein to amino or thiol groups or after protein fragmentation (4). Before electrophoresis, 5 µg protein from control and treated groups were derivatized for 15 min with 2,4-dinitrophenylhydrazine; the reaction was stopped by the addition of neutralization solution. The dinitrophenol (DNP) tag was then probed by Western blotting using anti-DNP antibodies (Chemicon International).

For two-dimensional (2D) analysis of protein-bound carbonyls, protein was precipitated by the addition of trichloroacetic acid (10% vol/vol) and centrifuged at 14,000 g for 15 min. The pellet was washed three times with acetone, dried with N2, and resuspended in 2D electrophoresis sample buffer, pH 6.8, containing 20 mM Tris, 8 M urea, 2% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate, and 1 mM EDTA. Protein (8 µg) was loaded on 7-cm, pH 3–10, immobilized pH gradient strips for isoelectric focusing (Bio-Rad) and focused for 26,000 V/h, with a maximum current of 50 µA/strip. After isoelectric focusing, strips were immersed in 5% trifluoroacetic acid containing 10 mM 2,4-dinitrophenylhydrazine for 20 min. Each strip was then washed for 20 s in 0.63 M Tris·HCl, pH 8.8, containing 2% SDS, and incubated with equilibration buffer, pH 8.8, containing 6 M urea, 50 mM Tris, 20% glycerol, and 2% SDS for 10 min. The washing and equilibration steps were repeated three times, with the last equilibration performed in the presence of 2.5% iodoacetamide for 15 min. Strips were briefly rinsed in 1x SDS-PAGE running buffer and applied to a 12% SDS-polyacrylamide gel for electrophoresis in the second dimension. Separated proteins were either silver stained or transferred to polyvinylidene difluoride membranes and probed with anti-DNP antibodies. All secondary antibodies were horseradish peroxidase linked, and membranes were developed using enhanced chemiluminescence plus reagents. Developed images were visualized by use of a Typhoon 9400 Variable Mode Imager (Amersham). Parallel silver-stained gels were used for protein excision and identification by mass-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS).

MALDI-TOF/MS identification of modified proteins. For MALDI-TOF/MS, protein spots that were immunoreactive with anti-DNP antibodies were excised from parallel silver-stained gels and digested with trypsin, using a modification of the method of Jensen et al. (15). Peptide masses obtained by MALDI-TOF/MS analysis were used to search the National Center for Biotechnology Information database to identify the intact proteins.

Statistical analysis. Data are presented as means ± SD. Two-group comparisons were performed using an unpaired or paired t-test, as appropriate. Multiple-group comparisons were performed using two-way ANOVA and Student-Newman-Keuls post-test. The null hypothesis was rejected with a P value <0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acrolein induces LV dysfunction in vivo. IV acrolein (0.5–10 mg/kg) induced progressive dose-dependent LV dilatation with little change in dP/dtmax (Fig. 1A, a rightward shift of the dP/dtmax-EDV relation) and profoundly reduced PAMP (Fig. 1B), both indicating depressed contractility. Based on these pilot studies, the 0.5 mg/kg dose was used in subsequent time course experiments. As shown in Fig. 1C, a single dose of 0.5 mg/kg acrolein induced a rapid and marked rightward shift of the pressure-volume loop and elevation of LV EDP, indicating acute LV dilatation and systolic and diastolic dysfunction. All of these effects returned toward baseline over 20 min. Figure 1D depicts group data for the dP/dtmax-EDV relation. Despite augmented EDV, there were immediate acrolein-mediated reductions in dP/dtmax, which returned toward baseline over time. Table 1 shows group data for IV acrolein and LV performance. Acrolein consistently induced marked, rapid, and, to a large extent, reversible contractile depression, as evidenced by significant LV dilatation (increased EDV), systolic dysfunction (reduced dP/dtmax and PAMP), and diastolic dysfunction (increased EDP and {tau}) at 1 and 5 min postadministration, but with loss of statistical significance by 20 min as these parameters returned toward their baseline values. There was also a nonsignificant decrease in heart rate and a delayed mild increase in peak LV pressure, perhaps reflecting an autonomic reflex response.


Figure 1
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  		Fig. 1. Acrolein (ACR) depresses left ventricular (LV) function in vivo. A: maximum rate of change in LV pressure (dP/dtmax) vs. LV end-diastolic volume (EDV) following single-dose intravenous (IV) ACR revealed dose-dependent LV dilatation with only minor changes in dP/dtmax, suggesting depressed contractility. B: preload-adjusted maximal power (PAMP) under the same conditions, confirming marked contractile depression. C: pressure-volume loops after 0.5 mg/kg IV ACR demonstrated a rapid rightward shift and elevation of LV end-diastolic pressure, followed by return toward baseline by 20 min. D: group data for dP/dtmax vs. EDV after 0.5 mg/kg IV ACR revealed immediate reductions in dP/dtmax, despite increased EDV. This effect was reversible over time.

 

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  		Table 1. Acrolein and LV function in vivo

 
Acrolein depresses cardiomyocyte contractility. We next evaluated the effects of acrolein on cardiomyocyte contraction, using concentrations of 0.01–2 µM based on studies documenting similar serum levels in human disease (25). Figure 2 shows sarcomere shortening and Ca2+ transients upon exposure to 0.01 and 2 µM acrolein. Low nanomolar acrolein induced a biphasic contractile response with an immediate and brief augmentation of contraction followed by a monotonic decline to baseline over 20 min. The Ca2+ transients showed no appreciable change. In contrast, micromolar acrolein induced a monophasic and progressive depression of shortening. Again, no changes were seen in the Ca2+ transients, indicating a loss of myofilament Ca2+ responsiveness. At both concentrations, the contractile/Ca2+ transient responses to 1 µM ISO remained robust. Figure 3, A and B, displays group data for normalized shortening and Ca2+ release ({Delta}fura-2 ratio) early (5 min) and late (20 min) after acrolein exposure. These indicate concentration- and time-dependent effects of acrolein on contraction: low nanomolar levels induce a brief, early increase in contractility, whereas longer durations and/or higher (micromolar) concentrations induce significant contractile depression without changing Ca2+ availability. Table 2 shows group data early and late after acrolein exposure, confirming that these concentration- and time-dependent contractile effects were unaccompanied by changes in peak or diastolic fura-2 ratio, maximal rate of Ca2+ release, or rates of Ca2+ reuptake. Group data in Fig. 3, C and D, demonstrate preservation of β-adrenergic responsiveness after 20-min exposure to 2 µM acrolein, indicating that the contractile deficit was overcome upon increasing myocyte Ca2+ availability. Figure 3E displays typical phase loops of normalized sarcomere length vs. fura-2 ratio before and after 20 min of 2 µM acrolein. The length-ratio relation during relaxation (when myofilaments are in dynamic equilibrium with Ca2+) was markedly shifted to the right by micromolar acrolein, indicating reduced myofilament Ca2+ responsiveness. Figure 3F depicts the fura-2 ratio corresponding to sarcomere relaxation to 1% above baseline length. For the group, this parameter was significantly increased in acrolein-treated myocytes (and unchanged in control myocytes), confirming a shift to the right of the length-ratio relation and reduced myofilament responsiveness.


Figure 2
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  		Fig. 2. Sarcomere shortening and Ca2+ transients upon myocyte exposure to 0.01 µM (A) and 2 µM (B) ACR. 0.01 µM ACR induced a biphasic contractile response with brief augmentation of contraction followed by a decline to baseline over 20 min. In contrast, 2 µM ACR induced progressive depression of shortening. The Ca2+ transients did not change significantly, suggesting underlying changes in myofilament Ca2+ responsiveness. At either concentration, the contractile and Ca2+ responses to 1 µM isoproterenol (ISO) remained robust.

 

Figure 3
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  		Fig. 3. Group data for early (5 min) and late (20 min) ACR effects on sarcomere shortening (normalized as percentage of initial baseline) (A) and Ca2+ release (normalized net change in fura-2 ratio) (B) at all concentrations of ACR tested and ISO-induced contractile (C) and calcium (D) response after 20 min of exposure to 2 µM ACR. E: phase loops of normalized sarcomere length vs. fura-2 ratio before and after 20 min of 2 µM ACR. F: group data for fura-2 ratio at sarcomere relaxation to 1% above baseline length establishing a shift to the right of the length-ratio relation and reduced myofilament responsiveness. See text for details. In A: *P < 0.05 and **P < 0.01 vs. baseline. In C and D: *P < 0.05 and **P < 0.01 vs. pre-ISO.

 

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  		Table 2. Acrolein and myocyte sarcomere shortening and Ca2+ transients

 
Thiol repletion attenuates acrolein-mediated contractile depression and acrolein-protein adducts. Acrolein can be rapidly incorporated into proteins via Michael addition, prototypically an acrolein-sulfhydryl interaction that yields exposed carbonyl groups (9, 36, 37). Accordingly, we examined the formation of acrolein-protein adducts in our myocyte model and the effects of the thiol donor N-AC. Figure 4A shows shortening traces and Ca2+ transients after exposure to 2 µM acrolein, with and without 1-h pretreatment with 5 mM N-AC. N-AC abrogated acrolein-induced contractile depression while maintaining catecholamine responsiveness. Group data (Fig. 4B) confirmed that N-AC normalized the contractile depression induced by acrolein, with shortening unchanged from that of control superfusion, indicating that thiol interactions are required for acrolein-mediated dysfunction. Figure 5A shows acrolein-protein adduct formation in myocytes exposed to acrolein (2 µM, 20 min), with and without N-AC pretreatment, as determined by immunostaining (anti-acrolein-KLH antibody). Immunofluorescence intensity in acrolein-exposed myocytes was markedly reduced on N-AC pretreatment, indicating that thiol loading prevented adduct formation, together with the observed improvement in contractility. Figure 5B shows myocyte GSH and GSSG levels and the GSH-to-GSSG ratio (GSH/GSSG). Acrolein significantly decreased myocyte GSH concentration (P < 0.001), whereas GSSG levels and the GSH/GSSG were not affected. This pattern is consistent with our laboratory's previous studies showing that {alpha},β-unsaturated aldehydes spontaneously conjugate with nucleophiles such as GSH and that the aldehyde-GSH conjugate is then extruded from the cell (32). In myocytes pretreated with N-AC, although the magnitude of effect was smaller, acrolein still significantly diminished cellular GSH (P < 0.05, N-AC vs. N-AC/acrolein). Thus, under these experimental conditions, acrolein depleted cellular GSH but did not affect the overall redox state. Moreover, N-AC prevented acrolein-induced dysfunction but only modestly improved GSH, suggesting that redox-independent mechanisms were also contributing to acrolein-induced myofilament impairment.


Figure 4
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  		Fig. 4. A: myocyte sarcomere shortening and Ca2+ transients upon exposure to 2 µM ACR, without and with pretreatment with 5 mmol/l N-acetylcysteine (N-AC) for 1 h. N-AC pretreatment prevented ACR-induced contractile depression without altering catecholamine responsiveness. B: group shortening data over 20 min of perfusion, normalized to initial baseline, from control, 2 µM ACR, N-AC alone, and N-AC pretreatment followed by 2 µM ACR. Compared with control, there was reduced contractility in the ACR group after 15 min that was significantly attenuated by N-AC pretreatment. *P < 0.05, **P < 0.01 vs. control; #P < 0.05 vs. ACR.

 

Figure 5
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  		Fig. 5. A: immunofluorescent staining of ACR adducts in mouse myocytes. Compared with control, 20-min exposure to 2 µM ACR increased ACR-protein adduct formation (green fluorescence) that was reduced by 1-h pretreatment with 5 mmol/l N-AC. B: myocyte GSH and GSSG levels and GSH-to-GSSG ratio (GSH/GSSG) from similarly treated cells. ACR significantly decreased myocyte GSH (P < 0.001), whereas GSSG and the GSH/GSSG were not affected, indicating cellular GSH depletion but no change in the overall redox state. In myocytes pretreated with N-AC, ACR still significantly diminished cellular GSH but to a smaller extent (P < 0.05) and did not significantly affect GSSG or GSH/GSSG. *P < 0.001 vs. control, N-AC, and N-AC/ACR. #P < 0.05 vs. N-AC.

 
Abundance and localization of acrolein-modified proteins. Figure 6A displays Western blots for acrolein-protein adducts and Oxyblots for protein-carbonyls from myocytes superfused in the presence or absence of 10 µM acrolein for 20 min. Acrolein-exposed myocytes exhibited a marked increase in immunoreactivity with both techniques, indicating adducts with proteins over a broad molecular mass range (5–125 kDa). Figure 6B shows Oxyblots of protein extracts from hearts 20 min after exposure to one dose of 0.5 mg/kg acrolein, as used in our in vivo experiments. While the response was less robust than in isolated myocytes, there was an obvious significant increase in protein-carbonyls in vivo (1.8-fold over control). Figure 7 depicts representative 2D Oxyblots and parallel silver stains of total myocyte protein, with and without 20 min of acrolein (10 µM) exposure. Acrolein consistently modified protein spots 1–5 and 7 (indicated on the Oxyblot) with disappearance of spot 6 (indicated on the corresponding silver stain). These spots were excised, trypsin digested, and subjected to MALDI-TOF/MS analysis for protein identification. The corresponding proteins were either components of the sarcomere/cytoskeleton (cardiac {alpha}-actin, desmin, myosin light polypeptide 3), or involved in mitochondrial energy metabolism [mitochondrial creatine kinase (CK)-2, ATP synthase] (Table 3), indicating site-specific, rather than diffuse, protein modification via Michael addition. Immunohistochemical colocalization studies were performed to confirm the sites of acrolein-protein adducts. Acrolein adducts were labeled with anti-KLH-acrolein antibodies. Actin staining was used to label the cytoskeleton/myofibrils, troponin I to label the myofilaments, and MitoFluor Red 589 to label the mitochondria. As seen in the confocal overlay images in Fig. 8, acrolein-protein modifications exhibited colocalization with all three sites, further supporting the MALDI-TOF/MS results.


Figure 6
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  		Fig. 6. A: abundance of ACR-protein adducts (Western blot) and protein-carbonyls in control (CTRL) myocytes and ACR-treated (10 µmol/l, 20 min) myocytes. ACR-exposed myocytes exhibited increased immunoreactivity with both techniques over a broad molecular mass range. B: protein-carbonyl abundance (Oxyblot) in hearts 20 min after 0.5 µg/kg IV ACR. There was increased protein-carbonyl abundance (1.8-fold over control) consistent with ACR-protein adduct formation in vivo. AB, antibody.

 

Figure 7
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  		Fig. 7. Two-dimensional Oxyblots and parallel silver stains of total protein from control and ACR-treated (10 µmol/l, 20 min) myocytes demonstrating the modification of several proteins (arrows 1–5 and 7) and one degraded protein (circle, arrow 6). Massassisted laser desorption/ionization time-of-flight mass spectrometry identification of these spots is shown in Table 3.

 

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  		Table 3. MALDI-TOF/MS protein identification

 

Figure 8
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  		Fig. 8. Immunohistochemical colocalization of ACR-protein adducts by confocal microscopy. ACR adducts (anti-keyhole limpet hemocyanin-ACR) are indicated by green fluorescence (left). Actin was labeled with Alexa 568 phalloidin in red (top middle), mitochondria with MitoFluor Red 589 in red (middle), and troponin I with anti-troponin I with secondary antibodies conjugated with Texas-Red (bottom middle). Nuclei were stained with 4',6-diamidino-2-phenylindole. Overlay images are shown on the right.

 

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There are several key findings of this study. First, we found that single-dose acrolein in vivo induced profound, rapid, and to a large extent reversible LV dysfunction. Second, studies of isolated cardiomyocytes (at clinically observed micromolar acrolein concentrations) revealed impaired myofilament Ca2+ responsiveness, rather than changes in Ca2+ cycling, but with preservation of catecholamine-induced augmentation of contraction and the Ca2+ transient. Third, acrolein-mediated contractile depression was accompanied by the formation of acrolein-protein adducts, and both the contractile dysfunction and adduct formation were markedly attenuated by thiol excess, underscoring the importance of acrolein-sulfhydryl interactions in mediating these effects. Fourth, acrolein-protein adducts revealed a predilection for contractile/cytoskeletal proteins and mitochondrial energy metabolism proteins. Taken together, the data establish that acrolein selectively impairs myofilament function and that these effects are related, at least in part, to acrolein-sulfhydryl interactions and select acrolein-protein modifications. These findings suggest that acrolein may impart analogous detrimental effects in cardiac pathologies associated with oxidative stress and enhanced lipid peroxidation, or after environmental acrolein exposure.

Acrolein and human disease. Acrolein is of importance from both environmental and pathophysiological perspectives. Acrolein poses the highest potential on a nationwide basis for significant chronic noncancer effects compared with an array of chemical pollutants (17). Endogenously, acrolein is generated as a by-product of several metabolic pathways, including threonine peroxidation by myeloperoxidase (1, 38), polyamine oxidation by amine oxidase (25), and the peroxidation of membrane lipids (9, 36, 37). Acrolein is the most strongly electrophilic of all lipid peroxidation-derived aldehydes and rapidly reacts with cysteine, histidine, and lysine residues, ultimately generating protein-carbonyl derivatives (9, 36, 37). Moreover, acrolein can deplete cellular GSH, alter redox state, and activate stress-signaling pathways (12, 35). Thus acrolein is continuously generated in biological systems subjected to oxidative stress and can potentially alter protein structure and function due to its high reactivity. Indeed, acrolein-protein modifications and increased carbonyl stress have been noted in several diseases, including Alzheimer's disease (18), atherosclerosis (27), and renal failure (25). Surprisingly, however, the cardiovascular effects of acrolein exposure and role of acrolein in cardiac pathology are largely unknown. This determination is especially important, as human studies have demonstrated that serum-free acrolein levels are not insignificant, but are in the low micromolar range, from 0.5 µM in normal persons to ~4 µM in patients with renal failure (25).

Acrolein acutely impairs myofilament Ca2+ responsiveness without altering Ca2+ handling. To evaluate the acute cardiovascular effects of acrolein in vivo, oral and inhalational routes of exposure were initially considered, since acrolein is a known contaminant of both air and water. However, with an inhalational approach, the observed responses would be confounded by pulmonary effects on acrolein clearance and systemic delivery and by acrolein-induced changes in lung function. Furthermore, variability in absorption, first-pass effects, and blood delivery of acrolein rendered the oral approach problematic for these initial proof-of-concept studies. Hence, responses to IV acrolein (via the jugular vein to avoid first-pass effects in the liver) were examined. Assuming a 25-g mouse and 2-ml blood volume, the theoretical blood level attainable with 0.5 mg/kg IV is nearly 112 µM. Measurements of bound acrolein liberated from diseased tissues suggest that >100 µM acrolein can accumulate in vivo. For example, in brains from patients with Alzheimer's disease, the concentration of acrolein was estimated at 1.45 nmol/mg protein (or ~120 µM) (40). In patients with renal failure, the accumulated protein-bound acrolein in plasma was equivalent to 180 µM, sixfold higher than in normal subjects (25). Thus we estimate that the concentration of acrolein reaching the heart in our studies is likely to be within the dose range of endogenous exposures.

Acrolein exposure in vivo produced rapid and (at least in part) reversible LV dilatation and dysfunction (Fig. 1 and Table 1). Studies of single cells indicated a direct effect on myocytes (Figs. 2–4, Table 2); acrolein, at low micromolar concentrations, induced contractile depression, despite preservation of the Ca2+ transient, indicating a selective loss of myofilament Ca2+ responsiveness. The rapidity of these effects, both in vitro and in vivo, suggested acrolein adduction of contractile proteins due to Michael addition, which is stable chemically, and sparing of Ca2+ handling systems. Despite stability of this type of chemical bond, there is significant lability of aldehyde-adducted proteins, which are readily ubiquitinated and degraded by the proteasome and the lysosome on a time scale of minutes to hours (22, 24). Since a single bolus dose of acrolein was given IV, analogous clearance of acrolein-adducted proteins may have accounted for the relatively rapid recovery of function in vivo. The myofilament impairment seen in acrolein-exposed myocytes was similar to that seen with acute oxyradical exposure (21) and in myocardial stunning (3), both of which are characterized by intense oxidative stress and lipid peroxidation. In these conditions, the contractile defect is overcome by augmenting Ca2+ release, for example by catecholamine administration or increasing stimulation rate. In acrolein-treated myocytes, the ISO response was similarly preserved, consistent with intact Ca2+ availability upstream to the myofilaments and submaximal myofilament activation. Thus acrolein (and other cytotoxic aldehydes) can amplify oxyradical-initiated contractile dysfunction and thereby may contribute importantly to ensuing myofilament impairment.

Acrolein-sulfhydryl interaction and adduct formation contribute to acrolein-induced contractile dysfunction. Cytotoxic aldehyde generation and/or aldehyde-protein modifications have been demonstrated in myocardial ischemia-reperfusion (8, 38) and heart failure (30). While the formation of protein adducts with acrolein per se (as opposed to other aldehydes) in cardiac disease has not been examined previously, free acrolein in the heart has been shown to be markedly increased following both transient ischemia and myocardial infarction (38). The high reactivity of acrolein would support a strong propensity for adduct formation that can potentially alter protein function.

In our study, acrolein exposure induced robust formation of protein-carbonyls, both in isolated myocytes and in the intact heart (Figs. 6 and 7). As the thiol donor N-AC attenuated both adduct formation and myofilament impairment (Figs. 4 and 5), both responses appear mechanistically related to acrolein-sulfhydryl interactions and/or GSH depletion. As shown in Fig. 5, although acrolein significantly decreased myocyte GSH, the overall redox state (GSH/GSSG) was unchanged, likely due to extrusion of the acrolein-GSH conjugate from the cell, as our laboratory has described for other {alpha},β-unsaturated aldehydes (32). While N-AC pretreatment prevented acrolein-induced dysfunction, it only modestly improved GSH. These findings suggested that redox-independent mechanisms, such as acrolein-induced protein modification, contributed importantly to the observed myofilament impairment. Moreover, GSH depletion would be expected to cause diffuse, rather than selective, abnormalities in cellular function. As acrolein induced a highly circumscribed abnormality in myofilament Ca2+ responsiveness without impacting calcium cycling, direct modification of myofibrillar proteins is likely to play a more prominent role in acrolein-mediated contractile depression.

MALDI-TOF/MS analysis and immunohistochemical localization revealed two functional categories of modified proteins: those related to either sarcomeric structure/function or mitochondrial energy metabolism (Figs. 6–8, Table 2). We found that acrolein modified several sarcomeric proteins, including cardiac {alpha}-actin, desmin, and myosin light polypeptide 3. Cardiac {alpha}-actin is a major component of the sarcomeric thin filament and plays an essential role in contraction and maintenance of the cytoskeleton (16), and desmin is the major component of the extrasarcomeric cytoskeletal network that surrounds and interconnects sarcomeric Z-lines (5). Thus modification of either of these proteins could have profound structural and functional effects. Acrolein also modified proteins related to energy metabolism, including ATP synthase ({alpha} and β subunits) and mitochondrial CK-2. These data are consistent with prior studies of acrolein-induced myocardial toxicity (concentrations 5- to 1,500-fold higher than in the present study), demonstrating that millimolar acrolein did not produce morphological changes in the myocardium, but markedly impaired contraction and denatured CK (29) and interfered with energy metabolism and depleted ATP (34). In this context, micromolar acrolein has also been shown to directly impair mitochondrial electron transport and stimulate mitochondrial oxidative stress (20), and mitochondrial ATP synthase is a known target of modification by other lipid peroxidation-derived aldehydes (41). Thus acrolein-induced modification of structural proteins could disrupt myofibrillar and/or cytoskeletal integrity and, along with changes in mitochondrial proteins, contribute to cardiac dysfunction.

Acrolein-induced myofilament impairment also recapitulated the phenotype of stunned myocardium. Indeed, oxidative stress-induced structural modification of myofibrillar-associated proteins and loss of CK have been implicated in myocardial stunning (3). A recent proteomic analysis of the ischemic rabbit heart identified 53 proteins that were altered as a result of chemical modification or proteolytic/physical fragmentation that fell into four functional groups: the sarcomere/cytoskeleton (including {alpha}-actin), energy metabolism (including ATP synthase and CK), redox regulation, and the stress response (39). Given our findings that acrolein-induced contractile dysfunction is similarly associated with modifications of sarcomeric/cytoskeletal proteins and energy metabolism proteins, and given that acrolein is generated during ischemia-reperfusion (38), this raises the possibility that acrolein contributes to the pathophysiology of myocardial stunning. Further studies will be required to test this hypothesis.

Our findings have significant implications for understanding and managing aldehyde-induced cardiovascular toxicity. Recent evidence suggests that the cardiovascular tissues are uniquely vulnerable to environmental pollutants and that pollutant exposure triggers adverse cardiovascular events (2). Thus indexes of cardiovascular function and health should be integral to the identification and treatment of toxic syndromes associated with environmental exposures. Although follow-up human studies are required, our results showing that acrolein, a ubiquitous pollutant, causes myocardial dysfunction suggest that the evaluation of cardiac function and injury are critically important in patients accidentally exposed to acrolein and related aldehydes. Excessive exposure to aldehydes such as acrolein is frequent, most commonly due to accidental structural fires or industrial accidents. In addition, contractile changes due to acrolein, similar to those described here, may be a significant feature of acute inflammatory events that generate excessive oxidative stress or the cardiotoxic profiles of tobacco smoke or drugs such as cyclophosphamide. Thus, taken together, our data provide strong rationale for determining excessive cardiovascular risk associated with toxicological states involving exposure to acrolein and other environmental aldehydes.


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This work was supported by a Veterans Affairs Merit Award, and National Institutes of Health Grants ES11860 and HL078825.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. D. Prabhu, Medicine/Cardiology, Univ. of Louisville, ACB, 3rd Floor, 550 South Jackson St., Louisville, KY 40202 (e-mail: sprabhu{at}louisville.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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