AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 281: H2261-H2269, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (23)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dai, L.
Right arrow Articles by Anderson, P. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dai, L.
Right arrow Articles by Anderson, P. G.
Vol. 281, Issue 6, H2261-H2269, December 2001

SPECIAL TOPIC
Bioenergetics in cardiac hypertrophy: mitochondrial respiration as a pathological target of NO·

Lijun Dai*, Paul S. Brookes*, Victor M. Darley-Usmar, and Peter G. Anderson

Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A rat aortic banding model of cardiac hypertrophy was used to test the hypothesis that reversible inhibition of mitochondrial respiration by nitric oxide (NO·) elicits a bioenergetic defect in the hypertrophied heart. In support of this hypothesis, the respiration of myocytes isolated from hypertrophied hearts was more sensitive to exogenous NO· (IC50 200 ± 10 nM vs. 290 ± 30 nM in controls, P = 0.0064). Hypertrophied myocytes also exhibited significantly elevated inducible NO· synthase (iNOS). Consistent with this endogenous source for NO·, the respiration of hypertrophied myocytes was significantly inhibited at physiological O2 tensions versus controls. Both the nonspecific NOS inhibitor nitro-L-arginine and the iNOS-specific inhibitor N-[3-(aminomethyl)- benzyl]acetamidine · 2HCl reversed this inhibition, with no effect on respiration of control myocytes. Consistent with an NO·-mediated mitochondrial dysfunction, the ability of intact perfused hearts to respond to a pacing workload was impaired in hypertrophy, and this effect was reversed by NOS inhibition. We conclude that endogenously generated NO· can modulate mitochondrial function in the hypertrophied heart and suggest that this bioenergetic defect may underlie certain pathological features of hypertrophy.

heart failure; cardiomyocytes; iNOS; oxidative phosphorylation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC HYPERTROPHY is a complex pathological process, contributing to the development of congestive heart failure (19, 23). It is reported that cardiac hypertrophy results in a modified cardiac metabolism including elevated glycolysis and lactate production (1-3), over-expression of fetal isozymes (48), and impaired ATP regeneration and functional recovery after ischemia-reperfusion (4, 22, 34, 38). These metabolic changes are consistent with a defect at the mitochondrial level (22, 24, 47), and several studies have reported dysfunctional mitochondria in hypertrophy (5, 37, 41). Paradoxically, it has also been reported that mitochondria isolated from the hypertrophied heart are normal regarding maximal activities necessary for oxidative phosphorylation (8, 14, 27). In support of the hypothesis that a mitochondrial dysfunction may contribute to the pathology of cardiac hypertrophy, several heritable mitochondrial DNA mutations result in a hypertrophic cardiomyopathy phenotype (42-44).

In a recent study (8), we hypothesized that the reversible inhibition of mitochondrial cytochrome-c oxidase (complex IV) by nitric oxide (NO·) might account for this apparent paradox between in vitro and in vivo findings. It was found that mitochondria isolated from hypertrophied hearts exhibited an increased sensitivity to inhibition of cytochrome-c oxidase by NO·. Investigating this in a cellular or organ setting is complex because NO· has multiple effects both in normal cardiac function and in disease. For example, NO· produced by the constitutive endothelial form of NO· synthase (eNOS) can modulate cardiomyocyte energetics (28, 50), contractility, blood flow, and platelet aggregation (17). In contrast, high levels of NO· from inducible NO· synthase (iNOS) are associated with several diseases including cardiac allograft rejection, dilated cardiomyopathy, and congestive heart failure (16, 21, 31, 33, 46). Little is known of the factors that establish responses to NO· as protective or pathological. These may include the sensitivity of mitochondrial respiration to NO· and the bioavailability of NO· controlled by its reactions with other species such as superoxide.

To further understand the role of NO· and mitochondrial dysfunction in cardiac hypertrophy, this study directly addresses the question of whether the increased sensitivity to NO· revealed in isolated mitochondria from hypertrophied hearts (8) is present in both the cellular and whole organ milieu. To avoid confounding effects of NO· on factors such as platelet aggregation and neutrophil recruitment, these experiments were performed in isolated cardiomyocytes and in buffer-perfused hearts. Herein it is reported that hypertrophied cardiomyocytes exhibit a greater sensitivity of respiration to NO· inhibition. It is also shown that iNOS protein and activity are upregulated in hypertrophy and that NO· from this iNOS can inhibit respiration at physiological O2 tensions. Finally, it is reported that impaired contractile function in hypertrophy, specifically the ability to respond to a pacing workload, is restored by NOS inhibition. The implications for these findings are discussed in the context of the control of respiration in response to stress in the hypertrophied heart.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and supplies. Male Sprague-Dawley rats were from Harlan (Indianapolis, IN); NO· synthase (NOS) inhibitors nitro-L-arginine and N-[3-(aminomethyl)benzyl]acetamidine · 2HCl (1400W) were from Aldrich (Milwaukee, WI); and 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) was from Alexis (San Diego, CA). Type II collagenase was from Worthington (Freehold, NJ). Antibodies to iNOS and eNOS were from BD Bioscience (San Diego, CA). L-[2,3-3H]arginine was from New England Nuclear (Boston, MA). Enhanced chemiluminescence (ECL) reagents were from Amersham-Pharmacia (Piscataway, NJ). ATP assay reagents were from Promega (Madison, WI). All other biochemicals and reagents, analytical grade or higher, were from Sigma (St. Louis, MO). Stock solutions of DETA NONOate were prepared in 10 mM NaOH and stored at -20°C. Their decomposition, assayed spectrophotometrically at 251 nm, was not significant during 1 wk.

Animal model of cardiac hypertrophy. Animals were housed with food and water available ad libitum, according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1996). Cardiac hypertrophy was elicited by aortic banding as previously described (1, 3). Briefly, after anesthesia with methohexital sodium (0.5 mg/100 g ip), a 0.6-mm band was placed around the ascending aorta of 23-day-old rats (~50 g body mass). Control animals underwent identical surgery without placement of the band. Aortic-banded (Band) and control (Sham) animals were studied at 8-12 wk postsurgery. In agreement with previous studies (1, 3, 8), the heart weight-to-body weight ratio was increased 46.6 ± 6.3%, and there was no evidence of fibrosis. This degree of hypertrophy has previously been shown not to elicit impaired contractile function (1, 3). In this particular model of hypertrophy, congestive heart failure typically develops around 20-24 wk postsurgery.

Cardiomyocyte isolation. Calcium-tolerant ventricular myocytes were isolated from the hearts of 8- to 12-wk postsurgery rats (250-350 g) by collagenase perfusion according to Powell (35) with minor modifications. After pentobarbital anesthesia (50 mg/kg ip), hearts were rapidly extirpated and perfused with 37°C oxygenated (95% O2-5% CO2) Krebs-Henseleit (KH) buffer, composed of (in mM) 118 NaCl, 2.6 KCl, 14.5 NaHCO3, 1.2 MgSO4, 11 glucose, and 1.2 KH2PO4. Perfusion was at 12 ml · min-1 · g wet wt-1 for 5 min. All other buffers were based on modified KH with 50 µM Ca2+ (KHCa2+). The nominally Ca2+-free perfusion was followed by 6 min/g perfusion at 6 ml · min-1 · g-1 with KHCa2+ plus fat-free BSA (0.1% wt/vol), taurine (10 mM), and type II collagenase (140 U/ml). Ventricular tissue was then removed, chopped, and incubated for 4 min/g at 37°C in an aerated conical flask in 10 ml/g of fresh collagenase buffer plus fat-free BSA (2% wt/vol). Tissue was disaggregated by pipetting and then filtered through 300-µm nylon mesh, and 10-20 ml of KHCa2+ plus BSA (2% wt/vol) was added. Cells were centrifuged at 30 g for 1 min. The cell pellet was resuspended in 10 ml of KHCa2+, layered onto 30 ml of KHCa2+ plus BSA (4% wt/vol), and centrifuged again. The layering/centrifugation step was repeated, and the cell pellet was finally resuspended in KH with 1 mM CaCl2 plus BSA (2% wt/vol). Cell count was adjusted to 106 cells/ml, and the suspension was kept for up to 1.5 h at room temperature with 95% O2-5% CO2 blowing over the surface and gentle agitation. This protocol yielded 4-6 × 106 cells/heart with 70-80% of cells being rod shaped and excluding Trypan blue. Consistent with a hypertrophic response at the individual cell level, the protein content of Band cells was significantly higher than that of Sham cells (22.3 ± 2.0 vs. 17.0 ± 0.9 mg protein/106 cells, respectively; P = 0.028). Protein concentration of cell suspensions was determined using Folin-phenol reagent against a standard curve constructed using BSA (29).

Detection of NOS protein by Western blot. Within 1 h of isolation, myocytes (106 cells) were pelleted and resuspended in 0.5 ml of ice-cold lysis buffer composed of 10 mM Tris, 1% SDS, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na2VO3, 1 mM EDTA, and 1 mM benzamidine, pH 7.4. Lysates were sonicated, incubated on ice for 20 min, and then centrifuged at 14,000 g for 20 min. Supernatants were boiled in denaturing sample buffer and electrophoresed on 7.5% polyacrylamide SDS gels (100 µg protein/well) followed by transfer to nitrocellulose. Membranes were blocked with Tris-buffered saline plus 0.05% Tween 20 (TBST) containing 10% nonfat dry milk and then probed using rabbit polyclonal antibodies against iNOS or eNOS, dissolved in TBST-5% milk. A peroxidase-linked goat anti-rabbit IgG secondary antibody was used, and detection was by ECL. Densitometry was performed on developed blots using public domain Scion software (National Institutes of Health). Saturation of the ECL signal was determined by analyzing cross-sectional band density plots, and only blots with no saturated bands were used for quantitative analyses. These methods gave a linear response over a approx 20-fold range of protein concentrations for appropriate positive controls provided by the antibody manufacturer.

Measurement of enzyme activities. NOS activity was measured by the conversion of [3H]arginine to [3H]citrulline using a NOS assay kit (Calbiochem, San Diego, CA) according to the manufacturer's instructions. Myocytes (106 cells) were washed with PBS, washed with PBS containing 1 mM EDTA, and then pelleted by centrifugation for 5 min at 1,000 g. The pellet was resuspended in 200 µl homogenization buffer composed of 25 mM Tris, 1 mM EDTA, and 1 mM EGTA, pH 7.4, and the cells were disrupted by pipetting and freeze-thawing. Protein content of homogenates was then determined (29). Homogenate (10 µl) was added to 40 µl reaction mix containing (final concentrations) 25 mM Tris, 3 µM tetrahydrobiopterin, 1 µM flavin adenine dinucleotide, 1 µM flavine mononucleotide, 1 mM NADPH, and 0.375 µM [3H]arginine (specific activity 53.4 Ci/mmol), pH 7.4. After 1 h at 37°C, reactions were stopped by adding 400 µl stop buffer composed of 50 mM HEPES and 5 mM EDTA, pH 5.5. Parallel reactions were performed in the presence of either 0.6 mM CaCl2 or 1 mM NG-nitro-L-arginine methyl ester (L-NAME). [3H]citrulline was separated from the reaction mixture by cation-exchange chromatography using equilibrated Dowex 50W-X8 resin and quantified by liquid scintillation counting. Total NOS activity was determined by subtracting L-NAME-blocked counts from counts in the presence of CaCl2. Calcium-independent activity (iNOS activity) was determined by subtracting L-NAME-blocked counts from counts in the absence of CaCl2 (16, 45).

Measurement of oxygen consumption and NO·. Oxygen and NO· were measured simultaneously using appropriate Clark-type electrodes in a 1-ml magnetically stirred 37°C chamber as previously described (8). The NO· electrode was calibrated using authentic NO· gas at both high (~240 µM) and low (~25 µM) concentrations of O2. Because the reaction between NO· and O2 in the buffer is faster at high O2 concentrations, the NO· electrode gives a smaller current response at high O2 for the same amount of added NO·. The ratio of the electrode current response at high versus low O2 was used as a correction factor to determine actual concentrations of NO· from recorded values as recently described (40). Myocytes were incubated at 105 cells/ml in KH plus BSA (2% wt/vol) and Ca2+ (1 mM). Because quiescent myocytes respire slowly, the protonophore FCCP (2 µM) was added to initiate uncoupled respiration. Stirring speeds were optimized to avoid mechanical disruption of cells without affecting electrode response. Data were collected with the use of a digital recording device (Dataq, Akron, OH) connected to a personal computer.

Myocytes were incubated as described above in the presence or absence of the NOS inhibitor nitro-L-arginine (1 mM). When the O2 concentration reached 90% saturation (216 µM; assuming 100% = 240 µM), DETA NONOate (0.9 mM) was added, resulting in a steady release of NO· and progressive inhibition of myocyte respiration. Oxygen consumption rate and NO· concentration data were taken at several corresponding time points and used to construct graphs showing respiration rate as a function of NO· concentration. Data were expressed as a percentage of the maximal rate in each experiment, i.e., normalized to the initial rate before the addition of NO·.

To measure the effects of endogenously produced NO· on respiration, myocytes were also incubated as described, and their respiration was monitored until all O2 in the chamber had been consumed. Experiments were performed in random order with buffer alone or in the presence of the nonspecific NOS inhibitor nitro-L-arginine (1 mM) or the specific iNOS inhibitor 1400W (400 µM) (18). The low solubility of these NOS inhibitors in buffer precluded experiments in which NOS inhibitors could be added halfway through an incubation period to monitor acute responses. In addition, these compounds exhibit slow onset of inhibition (18) such that their addition during an incubation would not elicit a response within the time course of a typical experiment. For these reasons, NOS inhibitors were predissolved in the incubation buffer at the desired concentrations.

Measurement of ATP. Myocytes were incubated at 105 cells/ml as detailed above. After 1 min, 100-µl aliquots of suspension were taken, and protein was removed by perchloric acid precipitation. The ATP content of supernatants was assayed using a luciferase assay kit (Enliten reagent, Promega).

Heart perfusion/pacing studies. To examine the effects of endogenously generated NO· on cardiac contractile function, hearts were rapidly extirpated and perfused at 12 ml · min-1 · g wet wt-1 with KH buffer (see Cardiomyocyte isolation) containing 2.5 mM CaCl2 bubbled with (95% O2-5% CO2). Contractile function was monitored with a latex balloon in the left ventricle connected to a pressure transducer, and coronary vascular resistance was measured by using a pressure transducer in-line with the perfusion cannula. Both transducers were connected to a digital recording device (Dataq) and a personal computer. Hearts were electrically stimulated at 300 beats/min. After a 15-min equilibration period, the pacing frequency was increased to 360, 420, and 480 beats/min for 3 min at each pacing point. Hearts were then returned to 300 beats/min, and after a 5-min recovery period, the buffer was changed for one containing 1 mM of nitro-L-arginine. After a 15-min wash-in period to allow NOS inhibition to occur, the pacing regimen was repeated (300 right-arrow 360 right-arrow 420 right-arrow 480 right-arrow 300 beats/min). Data were averaged from the final 30 s at each pacing point. Left ventricular (LV) developed pressure was calculated as systolic minus diastolic pressure (in mmHg) and expressed as a percentage of the initial 300 beats/min value under each condition.

Statistics. All data are results from at least four independent experiments. Statistical significance between experimental groups was determined using Student's t-test, with P < 0.05 taken as the significance limit.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of exogenous NO· on cardiomyocyte respiration. In the first series of experiments, the effect of exogenous NO· on the respiration of myocytes isolated from Sham and Band hearts was investigated. To achieve this, myocytes were incubated in a combined NO· and O2 electrode chamber, and NO· was added from DETA NONOate. Under these conditions of minimal work load, cardiomyocyte respiration is relatively slow due to a low ATP demand. Therefore, the uncoupler FCCP was added to stimulate mitochondrial respiration close to its maximum level. This treatment allows examination of one aspect of mitochondrial oxidative phosphorylation where the electron transport chain is placed under maximal electron flux. This method of stimulating cardiomyocyte respiration is preferential to either field stimulation or infusion of ADP and substrates by permeabilizing the plasma membrane. For example, field stimulation may interfere with electrodes used to measure NO· and O2, and detergents may lead to loss of cytosolic factors controlling respiration such as substrates for endogenous NO· synthesis. A potential drawback of using FCCP is that uncoupling mitochondria may shift the control pattern of oxidative phosphorylation, making respiration more sensitive to respiratory chain inhibitors (15). However, experiments with isolated heart mitochondria showed that the control by complex IV over respiration was identical in either state 3 respiration or with FCCP in both Band and Sham myocytes (not shown). These data support the use of FCCP because they suggest that the effects of inhibitors such as NO· on uncoupled respiration measured here can be recapitulated into effects on fully coupled, ATP-synthesizing mitochondria.

Results from a typical myocyte incubation are shown in Fig. 1A. Addition of the NO· donor DETA NONOate (indicated by the arrow) resulted in a steady release of NO· and progressive inhibition of respiration rate (the slope of the O2 trace). NO· inhibitor titration curves (Fig. 1, B and C) were constructed by measuring the respiration rate and NO· concentration at paired time points and plotting the corresponding values against each other.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1.   Respiration of hypertrophied cardiomyocytes is more sensitive to nitric oxide (NO·) inhibition. Ca2+-tolerant myocytes were isolated from control (Sham) or aortic-banded (Band) rats at 8-12 wk postsurgery and incubated (105 cells/ml) in Krebs-Henseleit (KH) buffer with BSA (2% wt/vol), Ca2+ (1 mM), and FCCP (2 µM). A: representative O2 consumption and NO· trace. When O2 reached 90% saturation, 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate, 0.9 mM) was added, as indicated by the arrow, resulting in a steady release of NO· and progressive inhibition of myocyte respiration. B: response of respiration to NO·, calculated from many experiments of the type shown in A. Data are shown for Band () and Sham (open circle ) myocytes. C: same as in B but in the presence of nitro-L-arginine (1 mM). D: calculated IC50 for NO· from data in B and C. All data are means ± SE of at least 4 independent experiments. *NO · IC50 was significantly lower in Band vs. Sham myocytes.

Figure 1B shows that the respiration of Band myocytes is more sensitive to NO· than that of Sham myocytes, i.e., less NO· is required to inhibit respiration in Band myocytes. Because iNOS is upregulated in the hypertrophied heart (8), these experiments were also conducted in the presence of nitro-L-arginine to eliminate any effects due to endogenous NO·. Figure 1C shows that NOS inhibition did not affect the response to exogenous NO· in Sham or Band myocytes, suggesting that this phenomenon is a property of the mitochondrial respiratory system. From the data used to construct Fig. 1, B and C, the concentration of NO· that resulted in IC50 respiration was determined for each curve and was significantly lower in Band versus Sham myocytes, as shown in Fig. 1D.

Because the NO· inhibition of mitochondrial respiration is competitive with O2, it is possible that different O2 tensions may elicit the different response to NO· between Sham and Band. However, the O2 tension at which the NO· IC50 occurred was not significantly different in the Sham and Band incubations (124 ± 12 vs. 128 ± 9 µM O2, respectively), removing this as a possible mechanism.

Another important factor that may contribute to the different responses of Sham and Band myocytes to NO· is their consumption of the NO· itself (40). However, the rates of NO· evolution from DETA NONOate, measured by the NO· electrode, were the same in Sham and Band incubations (0.21 ± 0.05 vs. 0.18 ± 0.03 µM/min, respectively, not significant). In addition, the steady-state plateau concentrations of NO· achieved were similar (1.04 ± 0.12 vs. 1.02 ± 0.11 µM in Sham and Band, respectively, not significant). These data indicate that the greater apparent sensitivity of Band myocyte respiration to NO· (Fig. 1) is not due to an increased availability of NO· in these cells.

A third factor that may underlie the increased sensitivity of respiration to NO· in Band myocytes is the initial maximum respiration rate (before NO· addition). When normalized to protein, initial respiration rate was significantly different between Sham and Band myocytes (23.1 ± 2.9 vs. 13.6 ± 1.8 µmol · min-1 · mg-1 protein, respectively, P = 0.014). However, when normalized to cell number, respiration rate was not significantly different between Sham and Band myocytes (33.7 ± 3.4 vs. 28.2 ± 1.9 µmol · min-1 · 10% cells-1, respectively, P = 0.127). The finding that initial respiration normalized to protein is lower in Band cells is unlikely to contribute to the increased sensitivity to NO·, because in isolated mitochondria from Band or Sham hearts, maximal respiration rates are identical (8). In the intact cell, additional control could derive from either substrate delivery or other contributions to total cell protein that are nonmitochondrial in origin.

NOS protein content of cardiomyocytes. Having demonstrated that exogenously added NO· can modulate cardiomyocyte respiration, we next investigated the content of endogenous NO·-generating machinery in cardiomyocytes. Myocyte lysates were prepared and analyzed for their content of iNOS and eNOS protein. Figure 2A shows results of a typical iNOS Western blot, with quantitation of several such blots in Fig. 2B. In agreement with our previous findings in whole heart homogenates (8), iNOS is upregulated in Band versus Sham myocytes, by 56 ± 14%. This result also confirms our previous immunohistochemical data (8), showing that iNOS in hypertrophy is primarily inside myocytes and not the endothelium. Some eNOS expression was detected in isolated myocyte preparations (Fig. 2C) although this was not different between Sham and Band. In addition, NOS activity results (below) suggest that iNOS accounts for the majority of the NOS activity in myocyte preparations.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 2.   Inducible NO· synthase (iNOS) protein is upregulated in hypertrophied cardiomyocytes. A: representative developed Western blot of isolated myocytes, showing iNOS at 130 kDa. S, Sham; B, Band. B: densitometric analysis of iNOS Western blots. Data are means ± SE of at least 4 independent experiments. C: representative developed Western blot of isolated myocytes showing endothelial NOS (eNOS) at 140 kDa. Data are representative of 4 similar independent experiments. *iNOS protein was significantly higher in Band vs. Sham myocytes.

NOS activity of cardiomyocytes. To determine whether the iNOS measured by Western blotting (Fig. 2) was enzymatically active, NOS activity was measured by the conversion of [3H]arginine to [3H]citrulline (16, 45). The results in Table 1 indicate that iNOS activity was significantly greater in Band versus Sham myocytes. This increase (65 ± 15%) agreed well with the increase in iNOS protein level measured by Western blot (Fig. 2). Total NOS activity (in the presence of Ca2+) was also higher in Band versus Sham myocytes (Fig. 2B), although not significantly. These data suggest the majority of NOS in the myocyte preparation is iNOS.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   iNOS activity is greater in hypertrophied cardiomyocytes

Effects of endogenous NO· on cardiomyocyte respiration. Having demonstrated an effect of exogenously added NO· on cardiomyocyte respiration and showing that both iNOS protein and activity are upregulated in Band versus Sham myocytes, it is important to determine whether NO· from this iNOS can inhibit hypertrophied myocyte respiration. The inhibition of respiration by NO· is competitive with O2 (9-11). Thus when cells producing NO· are incubated in a sealed chamber, their respiration rate gradually decelerates with time, as they utilize the finite amount of O2 in the chamber. This has been demonstrated in endothelial cells that constitutively express eNOS (12, 13) and provides a convenient method to assay the effect of endogenously produced NO· on respiration.

Sham and Band myocytes were incubated in the O2 electrode chamber (1 ml volume = 240 nmol O2), and O2 consumption was monitored continuously down to the baseline. Figure 3A shows typical respiration profiles for Sham and Band myocytes, indicating that the respiration of Band but not Sham myocytes becomes progressively slower as O2 tension falls (solid lines). Consistent with this effect being due to endogenously produced NO·, the nonspecific NOS inhibitor nitro-L-arginine (1 mM) eliminated the deceleration of O2 consumption rate, resulting in a respiration profile that was almost linear down to the baseline (dotted lines). Nitro-L-arginine had no effect on Sham myocyte respiration. The selective iNOS inhibitor 1400W (400 µM) also gave identical results to those seen with nitro-L-arginine (Fig. 3B) indicating that the principal source of NO· in these myocytes is iNOS. This is consistent with iNOS immunoblot and activity results shown in Fig. 2 and Table 1.


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 3.   Endogenously generated NO· can inhibit cardiomyocyte respiration. Ca2+-tolerant myocytes were incubated at 105 cells/ml in KH buffer with 2% (wt/vol) BSA, 1 mM Ca2+, and 2 µM FCCP (see MATERIALS AND METHODS) A: representative O2 electrode traces showing respiration of Sham and Band myocytes in the presence (dotted lines) or absence (solid lines) of 1 mM nitro-L-arginine. For the Band incubations, slopes of the lines at points indicated are expressed as percentages of the initial slope (means ± SE, n = 5 experiments) demonstrating the magnitude of deceleration in rate as O2 tension falls. B: representative O2 electrode traces showing respiration of Band myocytes in the presence (dotted lines) or absence (solid lines) of 1400W (400 µM). Traces are representative of at least 5 similar experiments. N-L-Arg, nitro-L-arginine.

Figure 4 shows a quantitative representation of several experiments of the type in Fig. 3 in which respiration rates were calculated at various time points and expressed as a percentage of the maximum rate as a function of O2 tension. This linear-fit analysis of the data indicates significant differences in the respiration profiles of Sham (Fig. 4A) and Band (Fig. 4B) cardiomyocytes (filled symbols) that are normalized by nitro-L-arginine (open symbols). It is interesting to note that the respiration rate of Band myocytes is sensitive to the NOS inhibitor across a wide range of O2 tensions, up to ~200 µM. This suggests that the effects of NO· on respiration may not only be important at low but also at high O2 tensions.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Quantitation of the effects of endogenous NO· on cardiomyocyte respiration as a function of O2 tension. Data from several experiments of the type shown in Fig. 3A were quantified by measuring the respiration rate (slope of the O2 consumption curve) at various concentrations of O2 and graphed for Sham (A) and Band (B) cells in the absence (open circle ) or presence () of nitro-L-arginine. Saturating O2 (240 µM) is indicated by the arrow. All data are means ± SE of at least 5 independent experiments. *P < 0.05, Band vs. Sham myocytes.

In further support of a bioenergetic defect at the mitochondrial level in cardiac hypertrophy, the steady-state ATP level of Band myocytes was significantly lower than that of Sham myocytes (0.22 ± 0.03 vs. 0.43 ± 0.03 µmol/105 cells, respectively, P = 0.0015).

Effects of endogenous NO· on contractile function in hypertrophy. Because the experiments in isolated myocytes were performed in the presence of a mitochondrial uncoupler, the possibility remains that the effects of NO· on mitochondrial respiration may be unique to the uncoupled system. Therefore, in the next series of experiments, a NOS inhibitor was used to assess the effects of endogenously generated NO· on mitochondrial function in the intact working heart. We examined the contractile function of Sham and Band hearts, specifically their ability to respond to a pacing workload directly related to mitochondrial ATP-generating capacity. Hearts were paced from 300 to 480 beats/min, allowed to recover, and then perfused with 1 mM nitro-L-arginine and paced again. As the data in Fig. 5 show, pacing resulted in a progressive decline in LV developed pressure, which was more pronounced in Band than Sham hearts but not significantly different. Most strikingly, the NOS inhibitor resulted in a significant improvement in cardiac function in Band hearts. No such improvement was seen in Sham hearts with the NOS inhibitor eliciting a slightly poorer response to pacing and ruling out a contribution from eNOS in this perfusion system. These results are consistent with the hypothesis that endogenously generated NO· is able to regulate mitochondrial ATP generation at the intact organ level, and that in the hypertrophied heart, this has a direct impact on cardiac function.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of endogenous NO· on the ability of the intact perfused heart to handle a pacing workload in cardiac hypertrophy. Hearts were perfused and subjected to a pacing workload, as detailed in MATERIALS AND METHODS, in the absence (open circle ) or presence () of nitro-L-arginine. Data are presented for Sham (A) and Band (B) hearts and represent means ± SE of at least 5 independent experiments. *P < 0.05 between nitro-L-arginine and control experimental groups. LV, left ventricular; bpm, beats per minute.

The addition of nitro-L-arginine elicited similar increases in coronary vascular resistance in both groups (not shown). This result indicates that in this system, the NOS inhibitor can enter endothelial cells and inhibit eNOS, and thus by inference can enter myocytes and inhibit iNOS. It is interesting to note that at the highest pacing point (480 beats/min), the NOS inhibitor was unable to significantly improve the function of Band hearts. This is possibly due to other factors that limit LV function at high heart rate but are not NO· sensitive. In contrast to the NOS inhibitor, the NOS substrate arginine (1 mM) had no effect on cardiac function in either Sham or Band hearts (results not shown), suggesting no substrate limitation for NO· production.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The principal findings of this study are as follows: 1) myocytes from hypertrophied hearts exhibit elevated iNOS protein and activity; 2) respiration of hypertrophied myocytes is more sensitive to NO·; and 3) endogenously produced NO· can inhibit mitochondrial respiration both in intact cells and at the whole organ level.

In a previous study (8), we determined that mitochondria isolated from hypertrophied hearts are more sensitive to inhibition of cytochrome c oxidase. Because NO· is a physiological inhibitor of this enzyme (9-11), and iNOS is upregulated in hypertrophied hearts (8), it was hypothesized that the combination of more NO· and a greater sensitivity to it may result in a bioenergetic defect at the tissue level. However, the possibility remained that this phenomenon was unique to isolated mitochondria and may have been due to the mitochondrial isolation procedure itself. In addition, these previous experiments were performed at saturating O2 tensions (~240 µM) i.e., far higher than those experienced at the cell level. In the current investigation, myocytes were isolated from hypertrophied and control hearts and various NO· and mitochondrial parameters were studied to determine whether NO· can regulate mitochondrial respiration at the intact cell level in hypertrophy. In addition, intact perfused hearts were studied to test this hypothesis at the whole organ level.

The most important finding in our study was that endogenously formed NO· was increased and could inhibit hypertrophied myocyte respiration. When NO· synthesis was blocked by both nonspecific (nitro-L-arginine) and specific (1400W) iNOS inhibitors (Figs. 3 and 4), O2 consumption in Band myocytes was restored to the level seen in Sham myocytes, suggesting that endogenous NO· from iNOS has an important physiological role in the control of myocyte O2 consumption in hypertrophy.

The effects of NOS inhibitors on cell respiration (Figs. 3 and 4) are similar to those seen in endothelial cells (12) that constitutively express eNOS. It has been proposed that eNOS can modulate respiration in myocytes and whole tissues (6, 25, 26, 28, 39, 49, 50). However, the observation that 1400W (18) has the same effect as nitro-L-arginine suggests iNOS is the sole source of NO· in hypertrophied myocytes. Nevertheless, by using Western blotting, we determined which isoforms of NOS were expressed in myocytes. This method revealed an upregulation in iNOS in Band myocytes (Fig. 2A), in agreement with our previous findings in whole heart homogenates (8). NOS activity results also showed enhanced iNOS activity in Band myocytes, demonstrating that the upregulated iNOS protein was enzymatically active.

The conventional view of iNOS is as a pathological NOS induced by cytokines such as interleukin-1beta , interferon-gamma , and tumor necrosis factor-alpha (16, 30, 45). However, more recently, low-level constitutive expression of iNOS has been demonstrated in several tissues including the kidney, heart, and blood vessels under normal conditions (30, 32). Our previous report (8) and our current findings show that low-level iNOS is expressed in control heart tissue and myocytes and is elevated in the hypertrophied heart. Interestingly, the observation that fetal isozymes are over expressed in hypertrophy (48) is corroborated by recent findings that iNOS is abundantly expressed in the fetus (7), suggesting common gene regulatory pathways.

It is striking that the difference in the effect of NOS inhibitors on the respiration of Band and Sham myocytes is far greater than would be predicted from the difference in iNOS level and activity. However, it must be noted that the effect of NO· on respiration is the result of not only iNOS activity, but also the sensitivity of respiration to NO·. Previously, we observed that isolated mitochondria from Band hearts exhibit greater sensitivity to NO· (8). The results in Fig. 1 demonstrate that this phenomenon is maintained at the myocyte level, i.e., mitochondria within hypertrophied myocytes are more sensitive to NO· inhibition. The result of this increased sensitivity is that a small change in the level of endogenously produced NO·, such as would be experienced with iNOS up-regulation in hypertrophy, can have large effects on respiration rate.

Importantly, it appears that the effects of endogenously generated NO· observed in intact myocytes can be recapitulated at the intact organ level, as exemplified by the experiments shown in Fig. 5. These results are consistent with an effect of NO· on mitochondrial respiration, as measured previously in isolated mitochondria and here in myocytes. There is still considerable debate as to whether NO· is a physiological inhibitor of mitochondrial respiration. Addition of exogenous NO· to cells certainly inhibits respiration, although the concentrations required are relatively high (IC50 ~250 nM in the current investigation), possibly due to the high O2 tensions in these experiments. Even when the effects of endogenous NO· are examined, stimulation of the NO· producing machinery (e.g., iNOS with cytokines, eNOS with bradykinin) is usually required (13, 45). This study is among the first to show an effect of baseline levels of NO· on cellular respiration, i.e., without NOS stimulation. To reveal this effect, low O2 tensions were required, as shown in Figs. 3 and 4. Although these results were obtained in a pathological condition, cardiac hypertrophy, they have implications for the interaction between NO· and mitochondria under physiological conditions, where low fluxes of NO· may be continuously generated.

The precise role of NO· in cardiac hypertrophy is also controversial. It has been reported that endothelial NO· generation is elevated during the initial phases of pacing induced cardiac hypertrophy (31) but that NO· production is lowered during the decompensation phase of pacing induced heart failure (36). The initial elevation in NO· may be a compensatory mechanism to elevate coronary blood flow, due to the increased metabolic demand on the heart caused by greater workload. Indeed, Heymes et al. (20) suggest that elevated iNOS and eNOS in dilated cardiomyopathy may have beneficial effects on LV diastolic function by enhancing preload reserve. This may be accompanied by an NO·-induced lowering of the inotropic response to beta 1-adrenergic stimulation (16, 45).

These results are consistent with our previous study, suggesting that NO·-mediated mitochondrial dysfunction is only apparent under conditions of stress (8). As hypertrophy progresses, the initial benefits of elevated NO· may be lost, and NO· may then contribute to the pathology of hypertrophy when its preservation of mitochondrial function begins to interfere with ATP synthesis. Indeed, as end-stage heart failure develops, a pathological role for NO· appears to prevail (16, 21, 31, 46). The results presented are consistent with the hypothesis that during the stable, nonfailing phase of cardiac hypertrophy, NO· has a pathological role targeted at the level of mitochondrial respiration and ATP synthesis. This is, then, expected to directly impact on contractile function under stress, as suggested by the data from working hearts (Fig. 5). Overall, it appears that mitochondria in hypertrophy are subject to a double insult, in that they are both more sensitive to NO· and exposed to more of it. Such an insult may contribute to certain pathologies of cardiac hypertrophy.


    ACKNOWLEDGEMENTS

We thank Junxuan Zhang for technical assistance.


    FOOTNOTES

* L. Dai and P. S. Brookes contributed equally to this work.

P. S. Brookes received American Heart Association Postdoctoral Fellowship 9920144. This work was funded in part by National Institutes of Health Grants RO1-HL-058895 (to P. G. Anderson) and RO3-AA-12613 (to V. M. Darley-Usmar).

Address for reprint requests and other correspondence: P. G. Anderson, Dept. of Pathology, Univ. of Alabama at Birmingham, Volker Hall Rm. G046, 1670 Univ. Blvd., Birmingham, AL 35294-0019 (E-mail: pga{at}uab.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.

Received 31 May 2001; accepted in final form 13 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allard, MF, Emanuel PG, Russell JA, Bishop SP, Digerness SB, and Anderson PG. Preischemic glycogen reduction or glycolytic inhibition improves postischemic recovery of hypertrophied rat hearts. Am J Physiol Heart Circ Physiol 267: H66-H74, 1994[Abstract/Free Full Text].

2.   Allard, MF, Schonekess BO, Henning SL, English DR, and Lopaschuk GD. Contribution of oxidative metabolism and glycolysis to ATP production in hypertrophied hearts. Am J Physiol Heart Circ Physiol 267: H742-H750, 1994[Abstract/Free Full Text].

3.   Anderson, PG, Allard MF, Thomas GD, Bishop SP, and Digerness SB. Increased ischemic injury but decreased hypoxic injury in hypertrophied rat hearts. Circ Res 67: 948-959, 1990[Abstract/Free Full Text].

4.   Anderson, PG, Bishop SP, and Digerness SB. Transmural progression of morphologic changes during ischemic contracture and reperfusion in the normal and hypertrophied rat heart. Am J Pathol 129: 152-167, 1987[Abstract].

5.   Arcos, JC, Sohal RS, Sun SC, Argus MF, and Burch GE. Changes in ultrastructure and respiratory control in mitochondria of rat heart hypertrophied by exercise. Exp Mol Pathol 8: 49-65, 1968[ISI][Medline].

6.   Balligand, JL, Kobzik L, Han X, Kaye DM, Belhassen L, O'Hara DS, Kelly RA, Smith TW, and Michel T. Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes. J Biol Chem 270: 14582-14586, 1995[Abstract/Free Full Text].

7.   Bloch, W, Fleischmann BK, Lorke DE, Andressen C, Hops B, Hescheler J, and Addicks K. Nitric oxide synthase expression and role during cardiomyogenesis. Cardiovasc Res 43: 675-684, 1999[Abstract/Free Full Text].

8.   Brookes, PS, Zhang J, Dai L, Zhou F, Parks DA, Darley-Usmar VM, and Anderson PG. Increased sensitivity of mitochondrial respiration to inhibition by nitric oxide in cardiac hypertrophy. J Mol Cell Cardiol 33: 69-82, 2001[ISI][Medline].

9.   Brown, GC. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett 369: 136-139, 1995[ISI][Medline].

10.   Brown, GC, and Cooper CE. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett 356: 295-298, 1994[ISI][Medline].

11.   Cleeter, MW, Cooper JM, Darley-Usmar VM, Moncada S, and Schapira AH. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett 345: 50-54, 1994[ISI][Medline].

12.   Clementi, E, Brown GC, Feelisch M, and Moncada S. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci USA 95: 7631-7636, 1998[Abstract/Free Full Text].

13.   Clementi, E, Brown GC, Foxwell N, and Moncada S. On the mechanism by which vascular endothelial cells regulate their oxygen consumption. Proc Natl Acad Sci USA 96: 1559-1562, 1999[Abstract/Free Full Text].

14.   Cooper, G, Puga FJ, Zujko KJ, Harrison CE, and Coleman HN. Normal myocardial function and energetics in volume-overload hypertrophy in the cat. Circ Res 32: 140-148, 1973[Abstract/Free Full Text].

15.   Dos, SP, Aliev MK, Diolez P, Duclos F, Besse P, Bonoron-Adele S, Sikk P, Canioni P, and Saks VA. Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction:diffusion mathematical model. J Mol Cell Cardiol 32: 1703-1734, 2000[ISI][Medline].

16.   Fukuchi, M, Hussain SN, and Giaid A. Heterogeneous expression and activity of endothelial and inducible nitric oxide synthases in end-stage human heart failure: their relation to lesion site and beta -adrenergic receptor therapy. Circulation 98: 132-139, 1998[Abstract/Free Full Text].

17.   Furlong, B, Henderson AH, Lewis MJ, and Smith JA. Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol 90: 687-692, 1987[ISI][Medline].

18.   Garvey, EP, Oplinger JA, Furfine ES, Kiff RJ, Laszlo F, Whittle BJ, and Knowles RG. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem 272: 4959-4963, 1997[Abstract/Free Full Text].

19.   Ghali, JK, Liao Y, Simmons B, Castaner A, Cao G, and Cooper RS. The prognostic role of left ventricular hypertrophy in patients with or without coronary artery disease. Ann Intern Med 117: 831-836, 1992.

20.   Heymes, C, Vanderheyden M, Bronzwaer JG, Shah AM, and Paulus WJ. Endomyocardial nitric oxide synthase and left ventricular preload reserve in dilated cardiomyopathy. Circulation 99: 3009-3016, 1999[Abstract/Free Full Text].

21.   Ikeda, U, and Shimada K. Nitric oxide and cardiac failure. Clin Cardiol 20: 837-841, 1997[ISI][Medline].

22.   Ingwall, JS, Atkinson DE, Clarke K, and Fetters JK. Energetic correlates of cardiac failure: changes in the creatine kinase system in the failing myocardium. Eur Heart J 11 Suppl B: 108-115, 1990[Abstract/Free Full Text].

23.   Katz, AM. Cardiomyopathy of overload. A major determinant of prognosis in congestive heart failure. N Engl J Med 322: 100-110, 1990[ISI][Medline].

24.   Katz, AM. Is the failing heart energy depleted? Cardiol Clin 16: 633-644, 1998[Medline].

25.   Kelly, RA, Balligand JL, and Smith TW. Nitric oxide and cardiac function. Circ Res 79: 363-380, 1996[Free Full Text].

26.   Laycock, SK, Vogel T, Forfia PR, Tuzman J, Xu X, Ochoa M, Thompson CI, Nasjletti A, and Hintze TH. Role of nitric oxide in the control of renal oxygen consumption and the regulation of chemical work in the kidney. Circ Res 82: 1263-1271, 1998[Abstract/Free Full Text].

27.   Leichtweis, SB, Leeuwenburgh C, Chandwaney R, Parmelee DJ, and Ji LL. Ischaemia-reperfusion induced alterations of mitochondrial function in hypertrophied rat heart. Acta Physiol Scand 156: 51-60, 1996[ISI][Medline].

28.   Loke, KE, McConnell PI, Tuzman JM, Shesely EG, Smith CJ, Stackpole CJ, Thompson CI, Kaley G, Wolin MS, and Hintze TH. Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ Res 84: 840-845, 1999[Abstract/Free Full Text].

29.   Lowry, OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

30.   Mohaupt, MG, Elzie JL, Ahn KY, Clapp WL, Wilcox CS, and Kone BC. Differential expression and induction of mRNAs encoding two inducible nitric oxide synthases in rat kidney. Kidney Int 46: 653-665, 1994[ISI][Medline].

31.   O'Murchu, B, Miller VM, Perrella MA, and Burnett JC, Jr. Increased production of nitric oxide in coronary arteries during congestive heart failure. J Clin Invest 93: 165-171, 1994.

32.   Park, CS, Park R, and Krishna G. Constitutive expression and structural diversity of inducible isoform of nitric oxide synthase in human tissues. Life Sci 59: 219-225, 1996[ISI][Medline].

33.   Paulus, WJ, Kastner S, Pujadas P, Shah AM, Drexler H, and Vanderheyden M. Left ventricular contractile effects of inducible nitric oxide synthase in the human allograft. Circulation 96: 3436-3442, 1997[Abstract/Free Full Text].

34.   Pike, MM, Luo CS, Yanagida S, Hageman GR, and Anderson PG. 23Na and 31P nuclear magnetic resonance studies of ischemia-induced ventricular fibrillation. Alterations of intracellular Na+ and cellular energy. Circ Res 77: 394-406, 1995[Abstract/Free Full Text].

35.   Powell, T. The isolation and characterization of calcium-tolerant myocytes. Basic Res Cardiol 80, Suppl 2: 15-18, 1985.

36.   Recchia, FA, McConnell PI, Bernstein RD, Vogel TR, Xu X, and Hintze TH. Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog. Circ Res 83: 969-979, 1998[Abstract/Free Full Text].

37.   Schwartz, A. Electron transport and mitochondria. Cardiology 56: 35-42, 1971[ISI][Medline].

38.   Shen, W, Asai K, Uechi M, Mathier MA, Shannon RP, Vatner SF, and Ingwall JS. Progressive loss of myocardial ATP due to a loss of total purines during the development of heart failure in dogs: a compensatory role for the parallel loss of creatine. Circulation 100: 2113-2118, 1999[Abstract/Free Full Text].

39.   Shen, W, Hintze TH, and Wolin MS. Nitric oxide. An important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption. Circulation 92: 3505-3512, 1995[Abstract/Free Full Text].

40.   Shiva, S, Brookes PS, Patel RP, Anderson PG, and Darley-Usmar VM. Nitric oxide partitioning into mitrochondrial membranes and the control of respiration at cytochrome c oxidase. Proc Natl Acad Sci USA 98: 7212-7217, 2001[Abstract/Free Full Text].

41.   Sordahl, LA, McCollum WB, Wood WG, and Schwartz A. Mitochondria and sarcoplasmic reticulum function in cardiac hypertrophy and failure. Am J Physiol 224: 497-502, 1973.

42.   Tokoro, T, Ito H, Maenishi O, and Suzuki T. Mitochondrial abnormalities in hypertrophied myocardium of stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol Suppl 1: S268-S269, 1995.

43.   Tokoro, T, Ito H, and Suzuki T. Alterations in mitochondrial DNA and enzyme activities in hypertrophied myocardium of stroke-prone SHRS. Clin Exp Hypertens 18: 595-606, 1996.

44.   Ueno, H, and Shiotani H. Cardiac abnormalities in diabetic patients with mutation in the mitochondrial tRNA[Leu(UUR)] gene. Jpn Circ J 63: 877-880, 1999[Medline].

45.   Ungureanu-Longrois, D, Balligand JL, Simmons WW, Okada I, Kobzik L, Lowenstein CJ, Kunkel SL, Michel T, Kelly RA, and Smith TW. Induction of nitric oxide synthase activity by cytokines in ventricular myocytes is necessary but not sufficient to decrease contractile responsiveness to beta -adrenergic agonists. Circ Res 77: 494-502, 1995[Abstract/Free Full Text].

46.   Vejlstrup, NG, Bouloumie A, Boesgaard S, Andersen CB, Nielsen-Kudsk JE, Mortensen SA, Kent JD, Harrison DG, Busse R, and Aldershvile J. Inducible nitric oxide synthase (iNOS) in the human heart: expression and localization in congestive heart failure. J Mol Cell Cardiol 30: 1215-1223, 1998[ISI][Medline].

47.   Vogt, AM, and Kubler W. Heart failure: is there an energy deficit contributing to contractile dysfunction? Basic Res Cardiol 93: 1-10, 1998[ISI][Medline].

48.   Waldenstrom, A, Schwartz K, and Swynghedauw B. Cardiac hypertrophy: from fetal to fatal? Clin Physiol 9: 315-320, 1989[ISI][Medline].

49.   Xie, QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, and Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256: 225-228, 1992[Abstract/Free Full Text].

50.   Xie, YW, Shen W, Zhao G, Xu X, Wolin MS, and Hintze TH. Role of endothelium-derived nitric oxide in the modulation of canine myocardial mitochondrial respiration in vitro. Implications for the development of heart failure. Circ Res 79: 381-387, 1996[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 281(6):H2261-H2269
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
M. van Bilsen, F. A. van Nieuwenhoven, and G. J. van der Vusse
Metabolic remodelling of the failing heart: beneficial or detrimental?
Cardiovasc Res, November 6, 2008; (2008) cvn282v2.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
J. Gutierrez, S. W. Ballinger, V. M. Darley-Usmar, and A. Landar
Free Radicals, Mitochondria, and Oxidized Lipids: The Emerging Role in Signal Transduction in Vascular Cells
Circ. Res., October 27, 2006; 99(9): 924 - 932.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
S. M. Davidson and M. R. Duchen
Effects of NO on mitochondrial function in cardiomyocytes: Pathophysiological relevance
Cardiovasc Res, July 1, 2006; 71(1): 10 - 21.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Benderdour, G. Charron, B. Comte, R. Ayoub, D. Beaudry, S. Foisy, D. deBlois, and C. Des Rosiers
Decreased cardiac mitochondrial NADP+-isocitrate dehydrogenase activity and expression: a marker of oxidative stress in hypertrophy development
Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2122 - H2131.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
W. J. Paulus and J. G. F. Bronzwaer
Nitric oxide's role in the heart: control of beating or breathing?
Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H8 - H13.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Benderdour, G. Charron, D. deBlois, B. Comte, and C. Des Rosiers
Cardiac Mitochondrial NADP+-isocitrate Dehydrogenase Is Inactivated through 4-Hydroxynonenal Adduct Formation: AN EVENT THAT PRECEDES HYPERTROPHY DEVELOPMENT
J. Biol. Chem., November 14, 2003; 278(46): 45154 - 45159.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. H. Hintze
Prologue: Nitric oxide-hormones, metabolism, and function
Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2253 - H2255.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services