Published online before print September 25, 2008, doi: 10.1161/CIRCRESAHA.108.186015
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© 2008 American Heart Association, Inc.
Cellular Biology |
Inducible Nitric Oxide Synthase Expression and Cardiomyocyte Dysfunction During Sustained Moderate Ischemia in Pigs
From the Institute of Pathophysiology (F.R.H., P.G., K.B., A.D., I.K., J.S., S.M., A.S., G.H., R.S.), University of Essen Medical School, Germany; Department of Cardiology, Pneumology, and Vascular Medicine (T.R., M.K.), University Hospital Aachen, Germany; and Abteilung für Kardiologie (F.R.H.), Medizinische Universität Graz, Austria.
Correspondence to Dr Rainer Schulz, University of Essen Medical School, Institute of Pathophysiology, Hufelandstrasse 55, Essen 45122, Germany. E-mail rainer.schulz{at}uk-essen.de
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Abstract |
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In acute myocardial ischemia, regional blood flow and function are proportionally reduced. With prolongation of ischemia, function further declines at unchanged blood flow. We studied the involvement of an inflammatory signal cascade in such progressive dysfunction and whether dysfunction is intrinsic to cardiomyocytes. In 10 pigs, ischemia was induced by adjusting inflow into the cannulated left anterior coronary artery to reduce coronary arterial pressure to 45 mm Hg (ISCH); 4 pigs received the inducible nitric oxide synthase (iNOS) inhibitors aminoguanidine or L-N6-(1-iminoethyl)-lysine during ISCH (ISCH+iNOS-Inhib); 6 pigs served as controls (SHAM). Anterior (AW) and posterior (PW) systolic wall thickening (sonomicrometry) were measured. After 6 hours, nitric oxide (NO) synthase (NOS) protein expression, NOS activity, and NO metabolites (nitrite/nitrate/nitroso species) were quantified in biopsies isolated from AW and PW. Cardiomyocyte shortening and intracellular calcium (Indo-1 acetoxymethyl ester) were measured without and with the NOS substrate L-arginine (100 µmol/L). In ISCH, AW wall thickening decreased from 42±4% (baseline) to 16±3% (6 hours). Wall thickening remained unchanged in ISCH-PW and SHAM-AW/PW. NOS2 (iNOS) protein expression and activity, but not NOS3 (endothelial NO synthase), were increased in ISCH-AW and ISCH-PW. iNOS expression correlated with increased nitrite contents. Cardiomyocyte shortening was reduced in ISCH-AW versus SHAM-AW (4.4±0.3% versus 5.6±0.3%). L-Arginine reduced cardiomyocyte shortening further in ISCH-AW (to 2.8±0.2%) and ISCH-PW (3.4±0.4% versus 5.4±0.4%) but not in SHAM or in ISCH+iNOS-Inhib; intracellular [Ca2+] remained unchanged. With L-arginine, in vitro AW cardiomyocyte shortening correlated with in vivo AW wall thickening (r=0.72). In conclusion, sustained regional ischemia induces myocardial iNOS expression in pigs, which contributes to contractile dysfunction at the cardiomyocyte level.
Key Words: myocardial ischemia • nitric oxide • iNOS • sphingosine • TNF-
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Introduction |
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Acute reduction of regional coronary blood flow decreases regional myocardial contractile function.1 With prolonged moderate ischemia (
6 hours), contractile function further deteriorates despite a stable reduction in coronary blood flow.2,3 A reduction in cardiac contractile function can result from changes in external mechanical loading. Impaired myocardial function may also reflect alterations within the myocardium, such as changes in extracellular space composition, nonmyocytic cellular compartments, or the cardiomyocyte itself. Indeed, during sustained ischemia, deterioration of contractile function is associated with a decreased responsiveness to external calcium and reduced postextrasystolic potentiation in vivo,2,4 suggesting dysfunction at the level of the cardiomyocyte. The aim of our present study was to identify first whether or not contractile dysfunction after sustained ischemia is intrinsic to cardiomyocytes.
Nitric oxide (NO) is generated in cardiomyocytes from L-arginine by NO synthases (NOS) and has been identified as an endogenous regulator of myocardial function.5 Increased NO production, as a result of increased expression of the inducible NOS isoform (iNOS), decreases myocardial function following myocardial infarction,6 in heart failure,7–9 and in human hibernating myocardium.10 iNOS expression can be triggered by proinflammatory cytokines such as tumor necrosis factor (TNF)-,11,12 which is functionally important in myocardial ischemia.13 Although increased levels of TNF- may be a trigger for increased iNOS activity, TNF- also exerts iNOS-independent negative inotropic effects via sphingosine, an intermediate of the sphingomyelin pathway,14 as observed following coronary microembolization.15,16
Thus, as a second aim, we examined whether a reduction in cardiomyocyte function depends on increased iNOS expression and quantified TNF- tissue content as a potential trigger of iNOS or sphingosine expression during sustained ischemia.
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Materials and Methods |
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In Vivo Model
The experimental protocols used in this study were approved by the local bioethics committee and adhered to the guiding principles of the American Physiological Society.
Sixteen Göttinger miniswine (20 to 40 kg) of either sex were anesthetized and instrumented as described previously2 and as detailed in the online data supplement, available at http://circres.ahajournals.org. Left ventricular pressures were measured by a micromanometer, and anterior wall (AW) and posterior wall (PW) thickening (WTh) were measured by sonomicrometry. Heart rate was controlled by atrial pacing. The proximal left anterior descending coronary artery was cannulated, and coronary blood flow was adjusted so that the minimum coronary artery pressure was not less than 70 mm Hg under control conditions to avoid initial hypoperfusion. Regional myocardial work was estimated by a regional myocardial work index, as described in the online data supplement.
After measurements of systemic hemodynamics and regional function at baseline, left anterior descending inflow was decreased to achieve a reduction in coronary artery pressure to 45 mm Hg (ISCH, N=10), whereas coronary artery pressure was maintained at baseline levels in SHAM (N=6). Measurements were repeated at 5 minutes and 6 hours. In a subgroup of ISCH animals (ISCH+iNOS-Inhib), continuous application of an iNOS inhibitor [aminoguanidine (AG), 1 mg/kg per minute, N=3; L-N6-(1-Iminoethyl)lysine (L-NIL), 1 mg/kg per hour following a bolus of 0.5 mg/kg; N=1] through a peripheral vein was initiated after the baseline measurement, 30 minutes before the start of hypoperfusion, and continued until the end of the experiment. At 6 hours, myocardial biopsies were taken from the anterior and posterior (control) wall and an apical section of the heart containing parts of the hypoperfused AW and the PW was embedded in paraffin for immunohistochemistry.
In parallel experiments, sham-operated animals (N=4) received a continuous application of the iNOS inhibitor AG (1 mg/kg per minute) for 6 hours through a peripheral vein following the baseline measurements, and the coronary endothelial response to bradykinin, a potent agonist of endothelial (e)NOS-mediated vasodilatation,17 was assessed as detailed in the online data supplement.
Isolation of Cardiomyocytes
Ventricular cardiomyocytes were obtained by enzymatic digestion simultaneously from the AW and PW (see the online data supplement for details) and used for measurements within 12 hours after isolation.18,19
Measurement of Contractile Function in Isolated Cardiomyocytes
As detailed in the online data supplement, cardiomyocytes were loaded with the Ca2+ indicator Indo-1 acetoxymethyl ester (2.5 µmol/L), superfused with Tyrode’s solution at 37°C and electrically stimulated (1 Hz). Cell shortening was quantified as percentage of total cell length. L-Arginine (100 µmol/L, Sigma-Aldrich, Seelze, Germany) was washed in via a rapid perfusion system, and recordings were performed during cell shortening steady state after 5 minutes. In cardiomyocytes isolated from ISCH+iNOS-Inhib, aminoguanidine was added to the bath solution following L-arginine and another measurement was recorded.
Immunohistochemistry
Confocal images were obtained with a laser scanning microscope (Zeiss LSM Pascal) at x400 magnification from iNOS, eNOS, phospho-eNOS, and TNF- antibody-stained biopsy sections, and leukocytes were quantified in additional, hematoxylin/eosin-stained tissue sections from the myocardial biopsies (see the online data supplement for a detailed description).
iNOS, eNOS, and TNF- Protein Expression
Western blots of iNOS, eNOS, and phospho-eNOS were performed as described previously,8 and results were normalized to GAPDH protein expression (iNOS) or to Ponceau S staining (eNOS). TNF- content was determined by enzyme linked immunosorbent assay (ELISA) using a commercially available porcine ELISA kit (R&D Systems, Minneapolis, Minn) (see also the online data supplement for details).
NOS Activity Measurements
The activities of NOSs were determined in myocardial tissue samples using a commercially available NOS activity assay kit (No 781001, Cayman Chemical Company, Ann Arbor, Mich). Total NOS activity is expressed as percent of [3H]-arginine (substrate) converted to [3H]-citrulline (product). 1400W (a selective iNOS inhibitor20) was used in parallel experiments to determine iNOS-dependent activity (see the online data supplement for details).
Determination of Tissue Nitroso Species and Nitrite/Nitrate Content
Nitrite (NO2–) is a major oxidative metabolite of NO21 and nitrite levels represent a sensitive measure of tissue NO formation.22 Myocardial nitrite and nitroso species contents were determined using a triiodide/ozone-based reductive gas phase chemiluminescence assay, essentially as described previously23 and in the online data supplement. Nitrate was quantified after enzymatic reduction to nitrite by nitrate reductase.22
Sphingosine
Myocardial sphingosine content was measured by high-performance liquid chromatography using the method of Merrill et al.24 See the online data supplement.
Data Analysis and Statistics
Data are reported as mean values±SEM. Statistical analysis comprised ANOVA for repeated measures for the time course of hemodynamic parameters (ISCH); 2-way ANOVA was used to compare hemodynamic data from all groups (SHAM, ISCH, ISCH+iNOS-Inhib) at baseline and 6 hours, to compare iNOS protein expression, NOS activities, and immunohistochemistry data, as well as cell shortening data (within each group, and also separate analyses for AW and PW across groups). When a significant overall effect was detected, Fisher least-significant tests were performed to compare single mean values. Correlation analyses were performed using Pearson’s correlation coefficients. P<0.05 was considered significant.
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Results |
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Hemodynamics and Regional Myocardial Function
During hypoperfusion, AW WTh rapidly decreased by 23.2±3.6% of baseline at 5 minutes and had further deteriorated at 6 hours of hypoperfusion (supplemental Table I). Anterior wall work index (AW work index) decreased to 76.8±3.6% and 28.8±5.6% of baseline at 5 minutes and 6 hours of hypoperfusion, respectively (both P<0.05), associated with a reduction in left ventricular peak pressure (LVPP) and LV dP/dt and a pronounced increase in left ventricular end-diastolic pressure (LVEDP) (supplemental Table I). Posterior (remote) wall function remained stable early during hypoperfusion. At 6 hours of hypoperfusion, however, moderate dysfunction of the PW was reflected by a decrease in work index to 73.9±13.4% of baseline (P<0.05). A slight increase in LVEDP was also found in sham-operated animals. Except for a small decrease in work index (to 82.7±3.9% of baseline) of the cannulated AW (P<0.05 versus 6 hours of ISCH), no changes in regional myocardial function were observed in SHAM.
In the presence of the iNOS inhibitors (ISCH+iNOS-Inhib), AW and PW function tended to be lower than in ISCH (at 6 hours: AW WTh, 8.9±2; PW WTh, 16.0±1.8%; compare with supplemental Table I). Two of the animals receiving AG developed severe systemic lactic acidosis (arterial lactate rose to 7.7 and 11.8 mmol/L, respectively, at 6 hours), despite adequate oxygenation.
In parallel experiments on sham-operated animals, continuous infusion of AG for 6 hours had no effect on AW WTh, LVPP, or coronary artery pressure at constant cerebral blood flow (Figure IB in the online data supplement). The dose–response relationship during incremental intracoronary bradykinin application at baseline and at 6 hours of AG infusion did not differ in EC50 and maximal bradykinin-induced coronary blood flow (supplemental Figure IC through IE). During AG infusion, we observed a time-dependent increase in the arterial lactate concentration (supplemental Figure III).
Contractile Function in Isolated Cardiomyocytes
The isolation procedure yielded 30% to 70% rod-shaped, well-striated quiescent cardiomyocytes, with no obvious differences between groups. In the absence of L-arginine, in vitro cell shortening in cardiomyocytes from the AW, but not from the PW, was slightly reduced in ISCH as compared to SHAM (Figure 1; ncells
20 for each group and region). Addition of 100 µmol/L L-arginine to the bath led to a significant further reduction in cell shortening in both ISCH-AW and ISCH-PW cells but not in SHAM. In cardiomyocytes from ISCH+iNOS inhibitor (ncells11 for each region from N=4 animals), cell shortening was not reduced versus SHAM, and L-arginine had no effect (Figure 1). Also, in the presence of L-arginine, cell shortening was not changed by wash-in of aminoguanidine (5.3±0.6% with 1 mmol/L AG+L-arginine versus 5.2±0.5% with L-arginine alone).
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P<0.05 vs ISCH.
The diastolic and systolic [Ca2+]i in cardiomyocytes isolated from the AW following 6 hours of hypoperfusion were not different from those measured in cardiomyocytes isolated from the PW (remote) myocardium (96±12% and 93±9% of PW, respectively); similarly, systolic and diastolic [Ca2+]i did not differ in cardiomyocytes isolated from the AW and PW of sham animals (AW: 87±9% and 91±9% of PW, respectively). [Ca2+]i in both groups of pigs was unaffected by L-arginine (ncells6 from Nanimals3 for each group and myocardial region, with and without L-arginine).
iNOS and eNOS Expression
Following 6 hours of hypoperfusion, histological sections showed a homogeneously distributed increase in iNOS-dependent immunofluorescence in the myocardial tissue of the hypoperfused AW, as well as in the remote PW, as compared to sham-operated animals (Figure 2A and 2B). eNOS-dependent immunofluorescence was not different between the groups (in arbitrary units): for ISCH: 1336±31 in AW and 1214±34 in PW; for SHAM 1436±38 in AW and 1310±46 in PW. No significant changes in phospho-eNOS–dependent immunofluorescence were detected: for ISCH, 942±21 in AW and 933±21 in PW; for SHAM, 911±11 in AW and 966±21 in PW.
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iNOS protein expression, as measured by Western blot densitometry and normalized to GAPDH protein expression, was 2.5-fold higher in the AW of ISCH versus sham-operated animals (P<0.05) and tended to be elevated in the remote PW of ISCH animals (Figure 2D). GAPDH signal density was not significantly different between groups. The ratio of phospho-eNOS over eNOS between AW and PW was not different between SHAM (2.46±0.72) and ISCH (2.60±0.94). Similarly, the ratio of eNOS in AW over PW was not different between ISCH and SHAM (supplemental Figure II).
Leukocyte counts within the myocardial biopsies from a subgroup of animals (N=5 in SHAM, N=4 in ISCH) were not different between SHAM and ISCH or between AW and PW (SHAM-AW, 186±26/mm2; SHAM-PW, 169±10/mm2; ISCH-AW, 138±11/mm2; ISCH PW, 156±6/mm2).
iNOS Activity
iNOS activity amounted to approximately one-third of total NOS activity in SHAM (35.9±11.4% in SHAM-AW and 29.9±17% in SHAM-PW). iNOS activity tended to be higher in ISCH-AW and was significantly higher in ISCH-PW (Figure 2E). Total NOS activity was not significantly different between groups (SHAM-AW, 24.6±5.0%; SHAM-PW, 18.3±3.6%; ISCH-AW, 26.3±4.5%; ISCH-PW, 28.8±5.5%).
Tissue Nitroso Species and Nitrite/Nitrate Content
Tissue concentrations of NO-derived nitroso species were significantly increased in the AW versus PW in animals with hypoperfusion (292±96 versus 190±48 nmol/L, P<0.05) but not in SHAM (158±21 versus 123±19 nmol/L). Chemiluminescence measurements of the NO metabolite nitrite correlated with iNOS protein expression measured in histological sections (Figure 3A) and homogenates (Figure 3B) of the myocardial regions, reflecting increased NO production with increased iNOS expression. Regional myocardial WTh and work index were inversely correlated with myocardial nitrite levels (Figure 3C and 3D). The cellular nitrate pool remained unchanged.
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In cardiomyocytes isolated from the anterior wall, addition of the iNOS substrate L-arginine established a closer relationship between AW WTh in vivo and cardiomyocyte shortening in vitro (Figure 4).
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TNF- and Sphingosine
In the myocardium of sham-operated animals, TNF- was mainly distributed around vessels, as shown by immunohistochemistry (Figure 5). In hearts subjected to 6 hours of ischemia, the histological sections revealed a patchy distribution of TNF- within the myocardial tissue of the AW and PW (Figure 5, center images).
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Ischemia, however, did not significantly affect the total TNF- protein concentration (in pg/g: SHAM, 218.4±37.3 in AW and 231.4±66.8 in PW; ISCH, 187.7±68.3 in AW and 136.2±48.9 in PW) or the sphingosine concentration (in pmol/Lxg–1 wet weight: SHAM, 298±27 in AW and 294±31 in PW; ISCH, 331±19 in AW and 314±27 in PW) in the myocardial tissue.
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Discussion |
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In this study, we demonstrate an increase in myocardial iNOS expression following 6 hours of regional ischemia and a L-arginine–dependent contractile dysfunction intrinsic to cardiomyocytes from the ischemic region.
During acute myocardial ischemia, contractile function in the ischemic region rapidly decreases. With prolongation of ischemia, contractile function further decreases despite a constantly reduced coronary blood flow and preserved tissue viability.2 By measuring contractile function of isolated cardiomyocytes, this study focused on cellular changes that occur during more sustained ischemia independent from the interstitial milieu. With the supplementation of 100 µmol/L of the NOS substrate L-arginine to the cell bath, a concentration measured in human blood plasma,25 a close relationship between contractile function in situ and in isolated cardiomyocytes was observed (Figure 4). The cardiomyocytes showing the largest decrease in cell shortening in response to L-arginine (left most data point in Figure 4), thereby having the largest isolated impact on the in vivo/in vitro contractile function relationship were from the animal with the most pronounced ischemia-induced regional dysfunction (AW WTh from 35.8% at baseline to 3.5% at 6 hours of ischemia). Our findings suggest a key role of NOS activity for inducing contractile dysfunction on the level of the cardiomyocyte during sustained ischemia. Decreased contractile function of cardiomyocytes from the hypoperfused AW during stimulation at 1 Hz was not associated with alterations in diastolic or peak systolic [Ca2+]i. In line with an NO-dependent reduction in myofilament Ca2+ sensitivity,26 previous studies using the same animal model showed reduced Ca2+ responsiveness in vivo without alterations in Ca2+ handling proteins or troponin I levels following 90 minutes or 12 hours of moderate hypoperfusion.2,4
NO has evolved as an important modulator of cardiac contractile function.5 eNOS and iNOS, 2 NOS isoforms, which can modulate myocardial function, are expressed within cardiomyocytes. iNOS (in contrast to eNOS) is substrate-limited, and once activated, it can produce high amounts of NO, which may account for the reduced contractile function observed in vitro in the presence of L-arginine. Increased concentrations of iNOS mRNA and iNOS protein have been found in many, albeit not all,27 studies on nonischemic,8,28 as well as ischemic,6,29 heart failure, including human cardiomyopathy9,30,31 and human hibernating myocardium.32 During regional myocardial ischemia, an initial increase in NOS activity was observed within minutes following coronary artery occlusion.33,34 In a rat model of septic cardiomyopathy, induction of iNOS mRNA was detected as early as 30 minutes following injection of endotoxin, resulting in a peak iNOS enzyme activity after 6 hours.28
iNOS protein expression was increased during sustained ischemia in the present study, and the concomitantly increased levels of nitrite and nitroso species are in line with a higher iNOS activity and NO production. A specific activation of the inducible NOS isoform is supported by the iNOS activity measurements (Figure 2E) using the iNOS-specific inhibitor 1400W.35 Furthermore, following in vivo application of AG, contractile function at the cardiomyocyte level was preserved in ISCH, whereas the eNOS-mediated endothelial response to bradykinin was not affected. AG had no effect on cerebral blood flow, AW WTh, and LVPP in sham-operated animals. This contrasts well with the effects of L-NNA, a NOS inhibitor also directed against eNOS,35 in the same animal model in an earlier study,36 which showed pronounced effects on endothelial and regional myocardial function (supplemental Figure IA). In line with an iNOS-mediated dysfunction, we demonstrated increased iNOS activity during sustained ischemia and found that tissue nitrite concentrations as a surrogate of NO production correlated inversely with in vivo regional function (Figure 3C and 3D). Total NOS activity was not different between groups, implying that the activity of the constitutive NOS isoforms eNOS and neuronal NOS in the tissue homogenates may have been reduced during sustained ischemia. Indeed, accumulation of asymmetric dimethylarginine, which occurs after 30 minutes of ischemia, could have suppressed eNOS activity, thereby shifting L-arginine toward iNOS and increasing its activity.37
Apart from NOS-derived NO, we have recently demonstrated NOS-independent NO accumulation within the myocardial interstitium during myocardial ischemia in vivo.38 Although NOS-independent NO production may thus have contributed to nitrite formation during ischemia, cellular contractile dysfunction triggered by L-arginine in ISCH was reversible by iNOS inhibition, suggesting that iNOS-dependent NO formation confined to the cardiomyocyte was most important for the measured functional alterations.
iNOS protein content was also increased in confocal cardiomyocyte cross-sections from the posterior (remote) wall of ischemic hearts. Myocardial nitrite levels in ISCH-PW were ranging between values from sham-operated animals and the hypoperfused AW. Thus, not only regional metabolic processes during hypoperfusion but also increased mechanical stress (as reflected by the pronounced increase in LVEDP during sustained ischemia, supplemental Table I) may have triggered increased iNOS expression, analogous to observations in human dilative cardiomyopathy.9
Whereas regional function in the remote region initially remained stable, increased iNOS levels at 6 hours of hypoperfusion were associated with a decrease in contractile function in the PW. Systolic WTh was higher in the AW versus PW, a physiological phenomenon that has been reported earlier and is discussed in more detail in the online data supplement.
We and others have previously shown that systemic NOS inhibition (with L-NNA) decreases myocardial contractile function during normoperfusion and myocardial ischemia in vivo.39–41 In a previous study, L-NNA decreased regional myocardial function to a similar extent during normoperfusion and ischemia (as indicated by a parallel downward shift of the relationship between external work and transmural blood flow), suggesting that NO production from constitutively active NOS has a beneficial effect on myocardial function in vivo (as recently confirmed also in humans42) independent of moderate ischemia.
On the other hand, in the present study, iNOS inhibition with AG in sham-operated animals had no effect on myocardial function. Our results indicate that iNOS activity does not modulate myocardial function during normoperfusion and that the negative inotropic effects of L-NNA during normoperfusion described earlier are related to eNOS inhibition.
During moderate sustained ischemia, iNOS inhibition prevented contractile dysfunction at the level of the cardiomyocyte. However, contractile function during hypoperfusion in vivo was not improved with AG. In the presence of AG, we observed an early-onset, time-dependent metabolic deterioration, as reflected by the increase in arterial lactate (supplemental Figure III) in sham animals. Although lactate acidosis did not affect myocardial function during normoperfusion, we cannot exclude that it contributed to myocardial dysfunction during hypoperfusion. Pig mitochondria contain iNOS,43 and NOS inhibition has been shown to increase myocardial oxygen consumption,40,43 which may have contributed to the observed metabolic deterioration during moderate ischemia.
In the present study, we did not quantify transmural blood flow distribution during sustained hypoperfusion to rule out changes in tissue perfusion pattern as a source of the progressive decline in regional function, as the enzymatic isolation of cardiomyocytes following the in vivo experiments precluded the use of radioactive microspheres. However, in a previous study, we have shown that distribution of regional myocardial blood flow does not change between 5 minutes and 12 hours of moderate hypoperfusion in this model.2
In the myocardium, iNOS-dependent contractile dysfunction can be induced by a variety of cytokines and mediators released during hypoxia and the inflammatory processes triggered by ischemic myocardial damage and cell death (see the online data supplement for a more detailed discussion). TNF- is causally involved in inflammatory cardiomyopathies,44 myocardial dysfunction following ischemia/reperfusion,13,45 and also in progressive contractile dysfunction following coronary microembolization.46 TNF- exerts negative inotropic effects by 2 mechanisms: a sphingosine-mediated depression of intracellular Ca2+-release14,15 and iNOS expression and increased NO production.12 In accordance with previous reports,45 in the present study tissue sections from SHAM animals contained low levels of TNF-, which were mainly localized to the vascular endothelium (Figure 5). During sustained moderate ischemia without reperfusion (injury), myocardial infarction or overt heart failure, neither TNF- nor sphingosine was increased in the ischemic myocardium, which may reflect a less strong trigger for an inflammatory response in our model, in line with the unchanged leukocyte count in the tissue. However, other cytokines may have contributed to increased iNOS expression in our model, as discussed in more detail in the online data supplement.
In the present study, extracorporeal perfusion may have contributed to activation of cytokines and blood elements, subsequently leading to increased iNOS protein expression also in SHAM and to the small decrease in work index in the cannulated AW of SHAM (which was significantly smaller than in ISCH). Leukocytes may have contributed to the NOS activity measured in myocardial biopsies. However, similar myocardial leukocyte counts between ISCH and SHAM suggest that increased iNOS expression and activity in ISCH was not a result of increased leukocyte migration into the myocardium.
In summary, sustained moderate regional ischemia induces contractile dysfunction at the cardiomyocyte level. iNOS expression is increased in the myocardium following 6 hours of moderate ischemia, associated with an L-arginine–dependent decline in contractile function within the hypoperfused myocardium. In light of these findings, inhibition of excessive iNOS activity may prove to be beneficial in maintaining contractile function during sustained moderate ischemia. In the clinical setting, unspecific NOS inhibition has been shown to improve blood pressure and the hemodynamic response to catecholamines in sepsis47,48 and may be beneficial for cardiac function following acute myocardial infarction.49,50 However, an associated decrease in cardiac output and global oxygen delivery, and more importantly, an increase in mortality were documented in a recent study,47 emphasizing the need to investigate more specific therapeutic targets to inhibit excess NO production within cardiomyocytes.
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Acknowledgments |
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Sources of Funding
This study was supported by the Ernst und Berta Grimmke-Stiftung (Düsseldorf, Germany).
Disclosures
None.
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Footnotes |
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Original received May 19, 2006; first resubmission received October 17, 2007; second resubmission received August 25, 2008; revised second resubmission received September 15, 2008; accepted September 16, 2008.
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References |
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