Published online before print November 30, 2006, doi: 10.1161/01.RES.0000253888.09574.7a
© 2007 American Heart Association, Inc.
Integrative Physiology |
Cardiomyocyte-Specific Overexpression of Nitric Oxide Synthase 3 Prevents Myocardial Dysfunction in Murine Models of Septic Shock
From the Department of Anesthesia and Critical Care (F.I., R.J.S., M.K., K.D.B.), Massachusetts General Hospital, Boston; Cardiovascular Research Center (F.I., E.S.B., E.M.F., A.R.G., M.S.-C., K.D.B.) and Cardiac Ultrasound Laboratory (T.G.N., J.G.M., D.S.J., M.H.P., M.S.-C.), Cardiology Division, Department of Medicine, Massachusetts General Hospital, Boston Cardiac Muscle Research Laboratory (R.L.), Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; Boston University School of Medicine (C.C.L.), Mass; Department of Cardiology and Center for Transgene Technology and Gene Therapy (S.J.), University of Leuven, Belgium.
Correspondence to Fumito Ichinose, MD, Department of Anesthesia and Critical Care, Massachusetts General Hospital, 55 Fruit St, Boston, MA 02114. E-mail fichinose{at}partners.org
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Abstract |
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Myocardial dysfunction contributes to the high mortality of patients with endotoxemia. Although nitric oxide (NO) has been implicated in the pathogenesis of septic cardiovascular dysfunction, the role of myocardial NO synthase 3 (NOS3) remains incompletely defined. Here we show that mice with cardiomyocyte-specific NOS3 overexpression (NOS3TG) are protected from myocardial dysfunction and death associated with endotoxemia. Endotoxin induced more marked impairment of Ca2+ transients and cellular contraction in wild-type than in NOS3TG cardiomyocytes, in part, because of greater total sarcoplasmic reticulum Ca2+ load and myofilament sensitivity to Ca2+ in the latter during endotoxemia. Endotoxin increased reactive oxygen species production in wild-type but not NOS3TG hearts, in part, because of increased xanthine oxidase activity. Inhibition of NOS by NG-nitro-L-arginine-methyl ester restored the ability of endotoxin to increase reactive oxygen species production and xanthine oxidase activity in NOS3TG hearts to the levels measured in endotoxin-challenged wild-type hearts. Allopurinol, a xanthine oxidase inhibitor, attenuated endotoxin-induced reactive oxygen species accumulation and myocardial dysfunction in wild-type mice. The protective effects of cardiomyocyte NOS3 on myocardial function and survival were further confirmed in a murine model of polymicrobial sepsis. These results suggest that increased myocardial NO levels attenuate endotoxin-induced reactive oxygen species production and increase total sarcoplasmic reticulum Ca2+ load and myofilament sensitivity to Ca2+, thereby reducing myocardial dysfunction and mortality in murine models of septic shock.
Key Words: nitric oxide • endotoxin • reactive oxygen species • calcium handling • myofilament
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Introduction |
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Septic shock is a complex syndrome that claims more than 200 000 lives per year in the United States.1 Endotoxemia occurs frequently in septic shock, and severe manifestations of this syndrome include cardiac depression.2 Although cytokines and nitric oxide (NO) have been implicated in the pathogenesis of septic shock, the underlying mechanisms of myocardial depression of sepsis remains incompletely understood.
High levels of NO produced by NO synthase 2 (NOS2) contribute to the systemic hypotension and myocardial dysfunction associated with sepsis.3 However, despite the prominent role of NOS2 in cardiovascular dysfunction of sepsis, clinical trials using NOS inhibitors that are not isoform specific have been associated with increased mortality in septic patients, presumably caused by further impairment of cardiac function.4 Although these observations indirectly suggest that NOS1 and/or NOS3 may have beneficial effects on myocardial function in sepsis, other studies using NOS isoform–deficient mice reported variable results. For instance, NOS3-deficient mice (NOS3–/–) were reported to have higher mortality at 1 day after endotoxin challenge compared with wild-type (WT) mice.5 In contrast, another study showed that endotoxin-induced hypotension was more pronounced in WT than in NOS3–/–,6 possibly because of increased NOS3 uncoupling leading to the production of superoxide in the former.7 It is noteworthy that NOS3 overexpression in vascular endothelial cells attenuated endotoxin-induced lung injury and mortality in mice.8 Nonetheless, the role of NOS3 in the myocardial dysfunction associated with sepsis remains incompletely defined.
To evaluate the impact of varying levels of NOS3 expression on cardiac function during endotoxemia, we studied WT, NOS3–/–, and mice with cardiomyocyte-specific NOS3 overexpression (NOS3TG).9 In addition, we generated NOS3-deficient mice with myocyte-specific NOS3 overexpression (NOS3–/–TG) by mating NOS3–/– and NOS3TG. We examined myocardial function in vivo and in isolated cardiomyocytes at baseline and after endotoxin challenge. The impact of NOS3 on cardiac function was further examined in a model of polymicrobial sepsis. We report that cardiomyocyte-specific NOS3 overexpression prevented endotoxin- or sepsis-induced myocardial dysfunction in mice.
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Materials and Methods |
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Genetically Modified Mice
After approval by the Massachusetts General Hospital Subcommittee on Research Animal Care, we studied 2- to 4-month-old male WT, NOS3–/–, NOS3TG,9 and NOS3–/–TG backcrossed 10 generations onto a C57BL/6J background.
Sepsis Models
Two complementary models of sepsis were used in this investigation.
Endotoxin Challenge Model
Endotoxin challenge was performed by administering Escherichia coli 0111:B4 endotoxin (lipopolysaccharide [LPS] 50 mg/kg; Sigma) intraperitoneally.
Colon Ascendens Stent Peritonitis Model
We performed Colon ascendens stent peritonitis (CASP) as described previously.10
For additional details regarding generation of NOS3–/–TG, administration of allopurinol and molsidomine, CASP model, echocardiography, in vivo hemodynamics, Western blotting, RT-PCR, measurement of contractility and calcium handling in isolated cardiomyocytes, skinned cardiomyocytes experiments, measurement of tissue NO, reactive oxygen species (ROS) measurement, detection of tyrosine-nitrated proteins, and survival analysis, see the expanded Materials and Methods section and the supplemental Figures, available in the online data supplement at http://circres.ahajournals.org.
Statistical Analysis
All data are expressed as mean±SEM. Data were analyzed using ANOVA for repeated measures or 2-way ANOVA with Statistica statistical software package (Statsoft Inc). Probability values were adjusted using Scheffe’s method.
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Results |
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Cardiomyocyte-Specific NOS3 Overexpression Attenuates Endotoxin-Induced Myocardial Dysfunction
At baseline, cardiac function parameters were similar among the 4 genotypes, except that left ventricular (LV) end-systolic pressure was higher in NOS3–/– and NOS3–/–TG than in WT and NOS3TG (Figure 1A and Table). Endotoxin progressively impaired LV fractional shortening (FS) in WT and NOS3–/– but not in mice with the transgene (Figure 1B and supplemental Figure I). Endotoxin markedly depressed cardiac output and load-independent parameters of LV contractility (peak rate of pressure rise [dP/dtmax] divided by instantaneous pressure [dP/dtmax/IP] and preload-recruitable stroke work) in WT and NOS3–/– but not in NOS3TG and NOS3–/–TG. Although measures of contractility (dP/dtmax and end-systolic elastance) that are relatively afterload-sensitive were preserved in NOS3–/– after endotoxin challenge, myocardial mechanical efficiency (estimated by the ratio of arterial elastance to end-systolic elastance) was markedly impaired in both NOS3–/– and WT but not in NOS3TG and NOS3–/–TG (Table). The relaxation time constant
, a measure of diastolic function, was markedly prolonged in WT and NOS3–/– after endotoxin challenge but not in mice with the transgene.
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Because endotoxin depressed load-independent measures of myocardial function in WT and NOS3–/– but not in NOS3TG and NOS3–/–TG, we focused on the comparison between WT and NOS3TG in the rest of the study.
Effects of Endotoxin on Expression of NOS and NO Production
Whereas only very low levels of NOS2 mRNA were detected in cardiac tissue extracts of WT and NOS3TG at baseline, NOS2 gene expression was induced to a similar degree in cardiac tissue of both genotypes 7 hours after endotoxin challenge (
10-fold; supplemental Figure II). Cardiac levels of mouse NOS3 mRNA were similar in the 2 genotypes and were not altered by endotoxin. Cardiac levels of human NOS3 mRNA were markedly higher in NOS3TG than in WT at baseline and 7 hours after endotoxin challenge. This pattern of gene expression was paralleled by NOS2 and NOS3 protein levels (Figure 1C). Whereas the cardiac tissue NO production levels, as measured by diaminofluorescein (DAF) staining, were markedly higher (7 fold) in NOS3TG than in WT at baseline, endotoxin increased cardiac NO levels to the similar extent in both genotypes (supplemental Figures III and IV).
NOS3 Overexpression Ameliorates Contractile Dysfunction of Isolated Cardiomyocytes From Endotoxin-Challenged Mice
In cardiomyocytes isolated from saline-challenged WT and NOS3TG, increasing the pacing rate from 1 to 6 Hz resulted in a progressive increase in percentage cell shortening (%CS) (Figure 2B) and peak calcium transient amplitude (
[Ca2+]i; Figure 2D) as well as a progressive decrease in the time constant of cell relengthening (Figure 2C) and Ca2+ transients ([Ca2+]i) decay (Figure 2E). Although endotoxin decreased [Ca2+]i in both genotypes (Figure 2A and 2D), magnitude of reduction was greater in WT than in NOS3TG. Endotoxin markedly depressed %CS in WT but not in NOS3TG (Figure 2A and 2B). Endotoxin shifted the relationship between peak systolic [Ca2+]i and %CS to the left in NOS3TG, suggesting increased myofilament sensitivity to Ca2+ (Figure 2F). Time constants of cell relengthening (Figure 2C) and of [Ca2+]i decay (Figure 2E) were similar in saline-challenged WT and NOS3TG cardiomyocytes but prolonged only in the former after endotoxin challenge, suggesting that endotoxin impaired relaxation in WT but not in NOS3TG cardiomyocytes.
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Effects of Endotoxemia and NOS3 Overexpression on Ca2+ Handling in Cardiomyocytes
To examine the impact of endotoxemia and NOS3 overexpression on Ca2+ handling in cardiomyocytes, we measured total sarcoplasmic reticulum (SR) calcium load ([Ca2+]SRT), fractional SR Ca2+ release, sodium/calcium exchanger activity, and SR Ca2+ leak in cardiomyocytes isolated from mice 7 hours after saline or endotoxin challenge. Although [Ca2+]SRT was lower in NOS3TG than in WT cardiomyocytes after saline (Figure 3A), fractional SR Ca2+ release during a normal twitch was significantly greater in the former (Figure 3B). Endotoxin markedly decreased [Ca2+]SRT in WT but increased in NOS3TG cardiomyocytes, rendering the [Ca2+]SRT levels greater in NOS3TG than in WT after endotoxin. Whereas endotoxin decreased fractional SR Ca2+ release in NOS3TG to the level similar to that of WT, the magnitude of twitch Ca2+ release was larger in NOS3TG because of the greater [Ca2+]SRT. Sodium/calcium exchanger activity levels paralleled the [Ca2+]SRT levels (Figure 3C). Interestingly, SR Ca2+ leak was significantly less in NOS3TG than in WT cardiomyocytes after saline, and it markedly increased only in the latter after endotoxin (Figure 3D). Taken together, these observations suggest that the greater impairment of contractile function in WT than in NOS3TG after endotoxin is, at least in part, attributable to smaller [Ca2+]SRT and increased SR Ca2+ leak in WT cardiomyocytes.
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P<0.05 vs endotoxin-challenged WT. Fraction of total PLN phosphorylated at Ser16 (PLN Ser16) (E) or Thr17 (PLN Thr17) (F) in LV protein extracts from WT and NOS3TG at 7 hours after saline (LPS–) or endotoxin (LPS+) challenge, detected by immunoblot and normalized to WT baseline. Representative blots from 2 mice in each group are shown. N=8 to 11 mice in each group. *P<0.05 vs saline-challenged NOS3TG, #P<0.05 vs saline-challenged WT.
Endotoxin Increases Phosphorylation of Phospholamban in NOS3TG but Not in WT
Because we found differences in [Ca2+]SRT between WT and NOS3TG and because myocardial diastolic function was preserved in NOS3TG but not in WT after endotoxin, phosphorylation of phospholamban (PLN) at Ser16 and Thr17 was examined in cardiac tissue extracts. At baseline, there was no difference in the fraction of PLN phosphorylation on Ser16 between genotypes, but the fraction of PLN phosphorylation on Thr17 was greater in WT than in NOS3TG, possibly consistent with greater [Ca2+]SRT in WT than in NOS3TG (Figure 3E and 3F). Endotoxin increased phosphorylation of PLN at both Ser16 and Thr17 in NOS3TG mice but not in WT mice. It is possible that the change in PLN phosphorylation at Ser16 or Thr17 via SR Ca2+-ATPase (SERCA2a) activation may have contributed to the augmented [Ca2+]SRT and preserved diastolic function found in endotoxin-challenged NOS3TG mice.
NOS3 Overexpression Increases Sensitivity of Skinned Cardiomyocytes to Ca2+ After Endotoxin Challenge
To further examine the sensitivity of the contractile machinery to Ca2+, contraction of chemically skinned cardiomyocytes was studied. Myofilament sensitivity to Ca2+ was similar in cardiomyocytes from saline-challenged WT and NOS3TG (Figure 4A and 4B). Endotoxin increased the sensitivity of myofilaments to Ca2+ in both genotypes with or without the thiol-reducing agent dithiothreitol (DTT). In the absence of DTT, endotoxin-challenged NOS3TG cardiomyocytes showed a greater sensitivity to Ca2+ than did endotoxin-challenged WT cardiomyocytes (Figure 4A and 4C). In the presence of DTT, however, myofilament sensitivity to Ca2+ did not differ between endotoxin-challenged WT and NOS3TG cardiomyocytes (Figure 4B). Taken together, these observations suggest that, following endotoxin challenge, NOS3 overexpression further increased myofilament sensitivity to Ca2+, perhaps by preventing endotoxin-induced thiol modification.
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Endotoxin-Induced ROS Accumulation Is Blunted by NOS3 Overexpression
To explore the mechanisms responsible for the differing function of WT and NOS3TG cardiomyocytes during endotoxemia, we investigated the ability of endotoxin to induce oxidative stress in the myocardium. We found that cardiac tissue ROS production in WT, as assessed by lucigenin-enhanced chemiluminescence, peaked at 1 hour after endotoxin challenge (3-fold; supplemental Figure V) and then declined. Accordingly, we examined ROS production at 1 hour after saline or endotoxin challenge in heart and lung tissues of WT and NOS3TG. Endotoxin increased myocardial superoxide production in WT hearts; this was not observed in endotoxin-challenged NOS3TG hearts (Figure 5A). Of note, endotoxin increased lung tissue superoxide production similarly in both genotypes. Incubation of tissues with superoxide dismutase (SOD) and cell-permeable SOD mimetic Tiron largely abolished the chemiluminescence signals. Importantly, administration of NG-nitro-L-arginine-methyl ester (L-NAME) 1 hour before endotoxin increased superoxide production in NOS3TG hearts to the levels measured in endotoxin-challenged WT hearts (with or without L-NAME), demonstrating that the attenuation of superoxide production in NOS3TG hearts is NO dependent.
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Two additional independent methods were used to evaluate cardiac oxidative stress in endotoxin-challenged mice. Oxidative fluorescent imaging using the fluorescent probe dihydroethidium (orange staining in Figure 5B) revealed that endotoxin increased superoxide production in WT but not in NOS3TG hearts. Incubation of cardiomyocytes isolated from saline- or endotoxin-challenged mice with dichlorodihydrofluorescein revealed that endotoxin induced ROS production (green staining in Figure 5C) in WT but not in NOS3TG. Dichlorodihydrofluorescein fluorescence was markedly attenuated by coincubation of the cells with Tiron and cell-permeable polyethylene-glycol (PEG)-SOD.
NO interacts with superoxide to form peroxynitrite, a potent oxidant whose presence may be reflected by an increase in nitrotyrosine formation. At baseline, myocardial nitrotyrosine levels were modestly higher in NOS3TG than in WT (Figure 5D and 5E). Seven hours after endotoxin challenge, nitrotyrosine levels increased to a greater extent in WT than in NOS3TG myocardium, suggesting that higher myocardial peroxynitrite levels were present in the former.
Inhibition of Xanthine Oxidase Prevented Myocardial Dysfunction in Mice
Endotoxin-induced superoxide production in the heart and lung of WT was prevented by the xanthine oxidase (XO) inhibitor allopurinol (Figure 5A). Cardiac XO gene expression did not differ between WT and NOS3TG myocardium at baseline and increased similarly 7-fold in both genotypes 7 hours after endotoxin challenge (supplemental Figure II). Cardiac XO activity, as reflected by xanthine-stimulated uric acid production, was similar in the 2 genotypes at baseline and increased in WT but not in NOS3TG 1 hour after endotoxin challenge (Figure 5F). Of note, pretreatment with L-NAME 1 hour before endotoxin abrogated the difference in XO activity in endotoxin-challenged WT and NOS3TG, suggesting that XO inhibition by NOS3 overexpression is NO dependent. Because these results suggested NOS3 overexpression attenuates the endotoxin-induced activation of XO in the heart, we tested whether treatment with allopurinol would prevent endotoxin-induced myocardial dysfunction. Pretreatment with allopurinol attenuated the endotoxin-induced reduction in FS in WT (Figure 5G). Furthermore, compared with cardiomyocytes from endotoxin-challenged vehicle-treated WT, cardiomyocytes from endotoxin-challenged allopurinol-treated WT had greater %CS (0.6±0.2 versus 2.7±0.4%, P<0.05) and modestly higher [Ca2+]i (0.22±0.06 versus 0.46±0.20 mmol/L, P=0.12). We also found that pretreatment with a NO donor, molsidomine, prevented endotoxin-induced cardiac dysfunction in WT (Figure 5G). These protective effects of molsidomine are unlikely to be attributable to its vasodilating effects because gavage feeding of molsidomine only transiently (within the first 60 minutes) and modestly (20%) reduced blood pressure (BP) (data not shown). These results suggest that NOS3 overexpression and increased cardiac NO levels prevented endotoxin-induced myocardial dysfunction, at least partially, via inhibition of XO by NO.
NOS3 Overexpression Attenuates Endotoxin-Induced Death
To further examine the impact of endotoxemia in mice, heart rate (HR) and BP were continuously measured in freely moving WT and NOS3TG (6 mice in each genotype). At baseline, values and apparent variability of HR and BP were similar between the genotypes (Figure 6A). All 6 WT lost variability of HR and BP immediately after endotoxin challenge, showed progressive reduction of HR, and died approximately 12 hours after endotoxin challenge (Figure 6A). In contrast, 3 of 6 NOS3TG did not lose HR and BP variability and survived indefinitely (>120 hours after endotoxin challenge), whereas the other 3 NOS3TG showed hemodynamic changes similar to those observed in endotoxin-challenged WT but appeared to live longer (died 18 to 36 hours after endotoxin challenge). This marked improvement in survival associated with NOS3 overexpression was confirmed in larger groups of uninstrumented, volume-resuscitated mice (Figure 6B).
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NOS3 Overexpression Attenuates LV Dysfunction and Mortality in Experimental Sepsis
To determine whether or not NOS3 overexpression attenuates LV dysfunction and mortality associated with polymicrobial sepsis, we performed CASP surgery, a model of diffuse fecal peritonitis10 in WT and NOS3TG. Compared with sham-operated mice, 24 hours after CASP surgery, LV end-systolic pressure and dP/dtmax were markedly decreased in WT but not in NOS3TG (Figure 7), whereas cardiac output was preserved in both genotypes. Load-independent measures of LV contractile function including maximal power divided by end-diastolic volume (PMXEDV) and dP/dtmax/IP were depressed in WT but not in NOS3TG. Similarly, the relaxation time constant was markedly prolonged in WT but not in NOS3TG after CASP (Figure 7). Survival rate was higher in NOS3TG (7 of 7) than in WT (8 of 19, P<0.05 versus NOS3TG) at 24 hours after CASP surgery. In contrast, after sham CASP surgery, all mice of both genotypes survived without clinical signs of sepsis (data not shown).
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Discussion |
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The current study revealed that cardiomyocyte-specific NOS3 overexpression attenuates endotoxin-induced myocardial dysfunction in mice. The amelioration of endotoxin-induced myocardial dysfunction by the transgene was at least partially attributed to increased [Ca2+]SRT and enhanced sensitivity of myofilaments to Ca2+. Endotoxin challenge rapidly induced ROS synthesis, which was inhibited by the XO inhibitor allopurinol. Cardiomyocyte-specific NOS3 overexpression blocked endotoxin-induced ROS production in part by inhibiting XO activity. Administration of the XO inhibitor allopurinol or the NO-donor molsidomine before endotoxin challenge attenuated endotoxin-induced myocardial dysfunction in WT.
The contribution of NO to the pathogenesis of endotoxin shock has been extensively studied initially using NOS inhibitors and, more recently, genetically modified mouse models. However, because NOS3 is widely expressed in the cardiovascular system, the roles of myocardial versus extracardiac NOS3 in endotoxin shock remain incompletely defined. Accordingly, we designed this study to characterize the effects of modulating myocardial NOS3 expression on endotoxin-induced cardiac dysfunction by taking advantage of mice with a spectrum of NOS3 expression. Despite hypertension, NOS3–/– showed similar myocardial dysfunction to WT after endotoxin challenge. In contrast, the preserved cardiac function observed in endotoxin-challenged NOS3–/–TG clearly demonstrates that myocardial NOS3 prevented myocardial dysfunction. It is of note that endotoxin markedly impaired ventriculoarterial coupling (reflected by the ratio of arterial elastance to end-systolic elastance) in WT and NOS3–/– but not in mice with the transgene, suggesting a beneficial effect of NOS3 on myocardial mechanoenergetic efficiency during endotoxemia.
To examine the mechanisms responsible for the beneficial impact of NOS3 on myocardial function, we assessed Ca2+ handling and myofilament sensitivity to Ca2+ in isolated cardiomyocytes. At baseline, peak [Ca2+]i and %CS were similar between the genotypes. The higher fractional SR Ca2+ release seen in cardiomyocytes from transgenic mice may be attributable to increased NOS3 localized to the Z-disk and T-tubular structures11 and enhancement of SR Ca2+ spark frequency and Ca2+ release.12 It is possible that [Ca2+]SRT was greater in WT than in NOS3TG cardiomyocytes at baseline because increased phosphorylation of PLN at Thr17 in the former activated SERCA2a-mediated SR Ca2+ uptake. Endotoxin decreased [Ca2+]SRT in WT cardiomyocytes, leading to markedly reduced [Ca2+]i and %CS. The marked decrement in [Ca2+]SRT in WT cardiomyocytes after LPS was associated with increased SR Ca2+ leak, consistent with a previous report in septic rats.13 In contrast, the greater [Ca2+]i in NOS3TG than in WT after endotoxin was associated with an increase in [Ca2+]SRT. The increased [Ca2+]SRT in mice carrying the transgene may be attributable to less Ca2+ leak or to an increase in Ca2+ uptake associated with an increase in PLN phosphorylation at Ser16 and Thr17. Taken together, these results suggest that preserved myocardial function in NOS3TG after endotoxin are, at least in part, attributable to increased [Ca2+]SRT associated with decreased Ca2+ leak and enhanced PLN phosphorylation.
Endotoxin enhanced myofilament sensitivity to Ca2+ in both genotypes. These observations differ from previous reports that showed endotoxin reduces myofilament Ca2+ sensitivity.14 Possible reasons for the discrepant effects of endotoxin on myofilament sensitivity include differences in severity of endotoxemia and in the time points when myocardial function was examined. A higher dose of endotoxin (50 mg/kg) used in the current study compared with previous investigations (5 to 6 mg/kg)14 may have caused a more profound modulation of mechanisms regulating myofilament Ca2+ sensitivity. It is of note that in the study by Layland et al, [Ca2+]i levels were not depressed by endotoxin.14 It is tempting to speculate that endotoxin-induced enhancement of myofilament sensitivity to Ca2+ observed in the current study is a compensatory response to the markedly decreased [Ca2+]i during endotoxemia. In WT, however, the endotoxin-induced increase of myofilament sensitivity to Ca2+ fails to fully compensate for the reduction in [Ca2+]i. Therefore, the "net" effect of endotoxemia on myocardial function in WT is severe depression.
In contrast to WT cardiomyocytes, cardiomyocytes from endotoxin-challenged NOS3TG had normal contractile function in the presence of reduced [Ca2+]i. The preserved cardiomyocyte function of endotoxin-challenged NOS3TG was associated with a more marked enhancement of myofilament sensitivity to Ca2+ compared with endotoxin-challenged WT cardiomyocytes. Interestingly, the thiol-reducing agent DTT augmented Ca2+ sensitivity in WT cardiomyocytes after endotoxin challenge to levels seen in NOS3TG cardiomyocytes but DTT did not alter Ca2+ sensitivity in the latter. These observations suggest that the transgene prevents endotoxin-induced thiol modification of contractile machinery proteins. Taken together, these results point to the intriguing possibility that redox-sensitive thiols in contractile machinery proteins may modulate Ca2+ sensitivity. Acute reversibility of the endotoxin-induced myofilament thiol modification with DTT suggests that the endotoxin-induced thiol modification of myofilament proteins is likely to be S-thiolation or S-nitrosylation, as opposed to irreversible oxidation such as formation of sulfinic (–SO2H) or sulfonic (–SO3H) acid. Given the rather harsh and nonspecific reducing effects of DTT, the precise sites and extent of thiol modification responsible for modulating cardiomyocyte Ca2+ sensitivity remain to be determined.
Because the NOS3 transgene appeared to have prevented oxidative stress–induced thiol modification in skinned cardiomyocytes, we sought to measure the impact of the transgene on endotoxin-induced ROS production. We found that endotoxin increased myocardial superoxide levels in an allopurinol-sensitive manner. Although excess NO may also exhibit direct antioxidant effects, these observations rather suggest that increased myocardial NO levels prevented endotoxin-induced XO activation, thereby reducing the oxidative stress in cardiomyocytes. XO has been detected in endothelial cells,15 cardiomyocytes,16 and inflammatory cells.17 The cellular source of XO responsible for endotoxin-induced cardiac ROS production is unknown. However, it seems likely that the high levels of membrane-permeable NO produced by transgenic cardiomyocytes are sufficient to inhibit XO activity via either autocrine or paracrine mechanisms. The key role of XO activation in endotoxin-induced myocardial dysfunction was further reinforced by our observation that administration of allopurinol prevented myocardial dysfunction in vivo and enhanced contractile function of isolated cardiomyocytes in endotoxemic WT. These observations are reminiscent of the findings by Stull et al, who reported that XO inhibition with allopurinol enhanced myofilament Ca2+ responsiveness, leading to improved myocardial mechanoenergetic coupling in murine postischemic cardiomyopathy.18 However, it remains to be formally demonstrated, likely using skinned cardiomyocytes, that allopurinol can augment calcium sensitivity in cardiomyocytes from endotoxin-challenged WT. Moreover, although the current results demonstrate that XO activation has negative impact on myocardial function during endotoxemia, XO can convert nitrite to NO, thereby conferring myocardial protective effects under ischemic condition.19 It is therefore conceivable that XO inhibition may aggravate myocardial function if sepsis is complicated with severe hypoxia or ischemia.
The observation that cardiac-specific transgene expression could improve survival in endotoxin-challenged mice was not anticipated. Sepsis-induced mortality is usually attributed to multiorgan failure.1 On the other hand, myocardial dysfunction has been suggested to contribute to mortality in a subgroup of patients with sepsis.20 In a study of patients with septic shock, Parker et al found that some early deaths (20%) were attributable to the cardiogenic form of septic shock, with diminishing cardiac index despite volume resuscitation.21 Furthermore, presence of an abnormal LV relaxation pattern, as measured by echocardiography, was found to be an independent predictor of mortality in patients with severe sepsis.22 It is of note that, in the present study, endotoxemia markedly impaired LV diastolic function and the relaxation function of isolated cardiomyocytes in WT but not in NOS3TG (see the Table and Figure 2). Preserved LV diastolic function in endotoxin-challenged NOS3TG hearts was likely associated with enhanced SERCA2a activity caused by increased PLN phosphorylation. It is possible that the improved survival we observed in endotoxin-challenged NOS3TG was attributable to preservation of myocardial function and maintained organ perfusion.
To further characterize the impact of NOS3 on myocardial dysfunction of sepsis, we examined the effects of cardiomyocyte-specific NOS3 overexpression on cardiac function in mice subjected to CASP, a model of polymicrobial sepsis that closely mimics human peritonitis. In contrast to endotoxin bolus, CASP has been shown to gradually increase plasma endotoxin concentrations to levels similar to those found in septic patients.10 We found that CASP caused profound myocardial dysfunction in WT but not in NOS3TG 24 hours after surgery. Survival rate at 24 hours after CASP was also improved in NOS3TG compared with WT. Taken together, these findings demonstrate that cardiomyocyte-specific NOS3 overexpression in mice prevents myocardial dysfunction not only in an endotoxin bolus model but also in a more clinically relevant polymicrobial sepsis model.
In summary, our results underscore an important protective role of myocardial NOS3 against endotoxin-induced myocardial dysfunction and death. Excess NO attenuates oxidative stress, at least partially, by inhibiting XO activity in cardiomyocytes early after endotoxin challenge. The current observations also uncover an important modulatory role of myofilament protein thiol modification on cardiac contractile function. Given the prevalence of oxidative and nitrosative stress in various disease states, protective effects of NOS3 and/or NO against myofilament thiol modification may have therapeutic implications not only in septic shock but also in other cardiovascular disorders. Moreover, our results unexpectedly demonstrated that preserved myocardial function may be a key determinant of survival in septic shock. These results suggest a possibility that therapeutic approaches to enhancing myocardial function (possibly by increasing cardiac NO levels) may improve survival in patients with cardiogenic form of severe septic shock.
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Acknowledgments |
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We thank Drs Federica del Monte and Roger Hajjar for valuable advice.
Sources of Funding
This work was supported by NIH grants HL-71987 (to F.I.) and HL-70896 (to K.D.B.) and The William F. Milton Fund of Harvard University (to F.I.).
Disclosures
None.
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Footnotes |
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Original received June 15, 2006; revision received October 21, 2006; accepted November 15, 2006.
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References |
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