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. Author manuscript; available in PMC: 2018 Jul 13.
Published in final edited form as: Surg Infect (Larchmt). 2007 Feb;8(1):41–54. doi: 10.1089/sur.2006.033

Cardiac Mitochondrial Damage and Inflammation Responses in Sepsis

Qun Zang 1, David L Maass 1, Sue Jean Tsai 1, Jureta W Horton 1
PMCID: PMC6044285  NIHMSID: NIHMS465969  PMID: 17381396

Abstract

Background and Purpose

Studies in sepsis suggest that mitochondria mediate multiple organ dysfunction, including cardiac failure; however, the underlying molecular mechanisms remain elusive. This study examined changes in mitochondrial membrane integrity, antioxidant activities, and oxidative stress in the heart after infectious challenge (intratracheal Streptococcus pneumoniae, 4 × 106 colony-forming units). Inflammation responses also were examined.

Methods

Cardiac tissues were harvested from Sprague-Dawley rats 4, 8, 12, and 24 h after bacterial challenge (or intratracheal vehicle for sham-treated animals) and homogenized, followed by preparation of subcellular fractions (mitochondrial, cytosol, and nuclei) or whole-tissue lysate. We examined mitochondrial outer membrane damage and cytochrome C translocation to evaluate mitochondrial integrity, mitochondrial lipid and protein oxidation to assess oxidative stress, and mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities to estimate antioxidant defense. In addition, we measured nuclear factor-kappa B (NF-κB) activation in myocardium and cytokine production to investigate inflammatory responses to septic challenge.

Results

Oxidation of mitochondrial protein and lipid was evident 4 h through 24 h after bacterial challenge. Mitochondrial outer membrane damage and cytochrome C release were accompanied by down-regulation of mitochondrial SOD and GPx activity. After bacterial challenge, systemic and myocardial cytokine production increased progressively, and NF-κB was activated gradually.

Conclusion

Sepsis impaired cardiac mitochondria by damaging membrane integrity, increasing oxidative stress, and altering defenses against reactive oxygen species. These alterations occur earlier than or simultaneously with inflammatory responses in myocardium after infectious challenge, suggesting that mitochondria play a role in modulating inflammation in sepsis.

Sepsis-induced multiple organ failure is a major cause of death, yet the mechanisms of the biological responses remain unclear. Mitochondrial dysfunction has been hypothesized as a potential cause of sepsis-mediated organ injury through accelerated oxidative damage and apoptosis [1]. Studies of human skeletal muscle biopsies suggested an association between mitochondrial dysfunction and the severity of sepsis [2]. In the heart, impaired energy generation and elevated oxidative products in mitochondria were detected in several animal models of sepsis [3].

A major source of mitochondrial injury is reactive oxygen species (ROS). In mitochondria, ROS are by-products of energy production; under various pathological conditions, excess ROS accumulate secondary to an imbalance between ROS generation and scavenging. Reactive oxygen species impair the function of proteins, lipids, and DNA by structural modifications [4,5], and numerous studies have confirmed that ROS alter various aspects of mitochondrial function. For example, in animal models, ROS-related modifications have been described in both mitochondrial aconitase, an enzyme participating in energy metabolism, and adenine nucleotide translocase, a factor regulating the permeability transition pore [6,7]. These structural modifications were associated with loss of enzymatic activities. Mitochondrial nicotinamide adenine dinucleotide (NADP)+ -isocitrate dehydrogenase, an important enzyme in the regulation of energy and redox status, was found to be inactivated by 4- hydroxynonenal modification in hypertensive rat hearts [8]. Increased mitochondrial lipid peroxidation has been described in rat hearts subjected to ischemia and reperfusion [9], and alterations in the lipid content of mitochondrial membranes has been proposed to contribute to decreased mitochondrial respiratory function [10]. Oxidative damage to mitochondrial DNA (mtDNA) has been reported in heart tissue from rats challenged with lipopolysaccharide (LPS) [11], and mtDNA damage contributes to decreased mtDNA copy number and altered mtDNA transcription [12].

Whereas mitochondria are a target of ROS-related damage, mitochondria also provide an ROS defense mechanism through production of non-enzymatic and enzymatic antioxidants. Three major families of intracellular antioxidant enzymes are glutathione peroxidase (GPx) [13], catalase (CAT) for detoxification of hydrogen peroxide, and superoxide dismutase (SOD) for removal of superoxide radicals (O2−). All three activities have been identified in mitochondria [14–16]. Various studies have described ROS-related modulation of gene expression in animal models and cell lines, confirming the important roles of antioxidant enzymes in maintaining mitochondrial integrity, protecting cells from apoptosis, and improving organ function under disease conditions [16,17].

That oxidative stress occurs after infectious challenge has been recognized widely. In the livers of rats subjected to cecal ligation and puncture (CLP), alterations in mitochondrial respiratory function contribute to overproduction of ROS [18]. In a similar model, elevated oxidative stress and abnormal mitochondrial morphology were observed [19]. Significant mtDNA damage was detected in the hearts of septic rats [12] and the livers of septic mice [20]. Furthermore, administration of natural antioxidant to LPS-challenged rats suppressed lipid oxidation, increased SOD activity in the heart, and improved survival [11].

Recent investigations suggested that mitochondria participate in the regulation of inflammatory responses, and mitochondrial oxidative damage-mediated inflammation has been detected both in vivo and in vitro [20–23]. The mitochondrial matrix proteins MAVS [24] and DOK-4 [25] were identified as signaling factors that regulate NF-κB activation, and changes in mitochondrial Ca2+ have been suggested to modulate cytokine production in cardiomyocytes [26]. Recently, we showed that antioxidant therapy prevented burn-related mitochondrial damage, attenuated inflammatory responses in myocardium, and improved cardiac function [27], suggesting a role for mitochondria in myocardial inflammation and cardiac dysfunction.

Previously, we developed a pneumonia-related sepsis model in rats based on the finding that pneumonia and subsequent sepsis occur frequently in burn units [28,29]. In the current study, we used this animal model to examine mitochondrial integrity, oxidative stress, and ROS defense in myocardium at various times after infectious challenge. We determined that sepsis initiated both systemic cytokine production and inflammation responses in the heart. We speculated that sepsis causes cardiac mitochondrial damage that leads to inflammation, which in turn contributes to the myocardial dysfunction that is characteristic of sepsis.

MATERIALS AND METHODS

Experimental model

Adult male Sprague-Dawley rats (320–350 g) (Harlan Laboratories, Houston, TX) were conditioned in-house for 5–6 days after arrival, with commercial rat chow and tap water available at will. All experiments performed in this study were reviewed and approved by The University of Texas Southwestern Medical Center’s Institutional Review Board for the care and handling of laboratory animals and conformed to all guidelines for animal care, as outlined by the American Physiology Society and the National Institutes of Health.

Sepsis protocol

Preparation of inoculum

Streptococcus pneumoniae type 3 (ATCC 6303) was obtained in lyophilized form from the American Type Culture Collection (Rockville, MD). Bacteria were reconstituted and then injected into the lungs of a rat to increase their virulence; lung lavage liquid was plated and purified, and aliquots were prepared and stored at −80 °C until use. Before each experiment, individual aliquots were thawed, inoculated onto trypticase soy blood agar plates, and incubated overnight at 37 °C. The plate was washed with sterile endotoxin- free phosphate-buffered saline (PBS), and a concentration of 1 × 107 colony forming units (CFU)/mL was determined by absorbance at 540 nm. The bacteria were agitated and drawn up into sterile tuberculin syringes in 0.4-mL aliquots. Bacterial CFUs were determined by plating 10 microliters of the diluted (1 ×107 CFU/mL) bacterial suspension onto blood agar plates and incubating the plates overnight at 37 °C. The number of viable bacteria inoculated into each animal was approximately 4 ×106. Studies have shown that the surgical procedure alone (injection of PBS and no bacteria) produces no ill effects or cardiac dysfunction.

Induction of aspiration pneumonia

To produce sepsis, animals were anesthetized with isoflurane and placed supine, and the area over the trachea was prepared with 10% povidone– iodine solution. A midline cervical incision was made, and the trachea was identified and isolated via blunt dissection. Bacterial suspension containing 0.4 mL of a suspension 1 × 107 CFU/mL for a final of 4 ×106 bacterial injection per rat or sterile endotoxin-free PBS was injected directly into the trachea using a 30-ga needle. The incision was then closed with surgical staples, and the animals were positioned 30° with the head up to ensure that the injected fluid entered the lungs. All rats were given 10 mL of lactated Ringer’s solution intraperitoneally while anesthetized to ensure hydration. All animals given intratracheal S. pneumoniae challenge had blood samples and hearts collected for assays at different time points after challenge.

Preparation of blood serum and subcellular fractionation of cardiac tissue

Cardiac tissues and blood samples were collected from sham-treated and septic rats 4, 8, 12, and 24 h after infectious challenge (n = 3–6 at each time point). To prepare serum, blood was centrifuged at 3000 × g for 15 min.; supernatant liquids were stored at −80 °C until used. To isolate the cytosolic and mitochondrial fractions, cardiac tissue was homogenized in 20 mM HEPES-KOH (30)/250 micromolar sucrose/10 mM KOH/1.5 mM MgCl2/1 mM ethylenediaminetetraacetic acid (EDTA)/1 mM ethylene glycol tetraacetic acid (EGTA)/1 micromolar dithiothreitol (DTT)/0.1 mM phenylmethylsulfonyl fluoride and subjected to differential centrifugation (600 × g for 5 min., 10,000 × g for 30 min., and 100,000 × g for 30 min.)[31]. The final supernatant liquids were the cytosol fractions. Mitochondrial pellets, obtained after the second centrifugation, were resuspended in 10 mM HEPES-KOH (30)/200 mM mannitol/70 mM sucrose/1 mM EGTA. To isolate the nuclear fractions, cardiac tissue was homogenized in 10 mM HEPES (pH 7.9)/1.5 mM MgCl2/10 mM KCl/1 micromolar DTT/protease inhibitors and centrifuged at 10,000 × g for 30 min. The pellets were resuspended in 20 mM HEPES (pH 7.9)/1.5 mM MgCl2/0.42 M NaCl/0.2 mM EDTA/25% (v/v) glycerol/l micromolar DTT/protease inhibitors and centrifuged at 20,000 × g for 10 min. The supernatant liquids were the nuclear fractions. To prepare tissue lysates, cardiac tissue was homogenized in CelLyticMT reagent (Sigma, Saint Louis, MO) and centrifuged at 20,000 × g for 10 min. The lysate supernatant liquids were collected. All procedures were carried out at 4°C. The protein concentrations were determined using an assay kit (BioRad, Hercules CA).

Assessment of outer mitochondrial membrane damage

Mitochondrial outer-membrane integrity was assessed by measuring cytochrome C oxidase activity in the presence or absence of the detergent n-dodecyl β-D-maltoside (Sigma). Experimental procedures were performed according to the manufacturer’s protocol; 20 mcg of freshly isolated mitochondrial fraction was used for each of the reactions, which were conducted in duplicate. Briefly, the mitochondrial fraction was diluted in the enzyme dilution buffer (10 mM Tris HCl, pH 7.0, containing 250 mM sucrose) with 1 mM n-dodecyl β-D-maltoside and incubated on ice for 30 min. The reaction was initiated by adding freshly prepared ferrocytochrome C substrate solution (0.22 mM) to the sample. The decrease in absorbance at 550 nm related to oxidation of ferrocytochrome C by cytochrome C oxidase was recorded using a kinetic program (5 sec delay; 10 sec interval; six readings). Cytochrome C oxidase activities were calculated and normalized for the amount of protein per reaction, and the results were expressed as units/mg of mitochondrial protein. The mitochondrial outer-membrane damage was calculated from the ratio of cytochrome C oxidase activity without and with the detergent.

Measurement of SOD activity in mitochondria

The SOD activity was measured according to an original method [32] using an SOD assay kit (Calbiochem, San Diego, CA). Approximately 100 mcg of mitochondrial protein was used for each reaction; all assays were performed at least in duplicate. According to the vendor’s protocol, sample was first incubated with 1-methyl-2-vinylpyridinium in assay buffer at 37 °C for 1 min. to eliminate interference. Immediately after addition of substrate (5,6,6a,1,1b-tetrahydro-3,9,10-trihydroxybenzo[ c]fluorine, the oxidation of which is regulated by SOD), absorbance at 525 nm was recorded using a kinetic program: 10 sec interval; six readings. The SOD activity was determined from the ratio of the auto-oxidation rates measured in the presence and absence of SOD. Results were normalized by protein amount per reaction and expressed as units/mg of mitochondrial protein.

Measurement of glutathione peroxidase activity in mitochondria

The GPx activity was measured using a specific assay kit (Calbiochem, San Diego, CA). Mitochondrial extracts were diluted with assay buffer, and approximately 50 mcg of protein was used per reaction; all assays were performed in duplicate. The sample was added to a solution containing 1 mM glutathione, glutathione reductase ≥0.4 unit/mL, and 0.2 mM reduced NADP (NADPH). The reaction was initiated by adding substrate, tert-butyl hydroperoxide (final concentration 0.22 mM), and reduction was recorded at A340 nm using a kinetic program: 30 sec interval; 6 readings. The GPx activity was determined by the rate of decrease in A340 (GPx 1 mU/mL = (A340/min)/ 0.0062). Results were normalized by protein amount per reaction and expressed as microunits/ mg of mitochondrial protein.

Measurement of lipid peroxidation in mitochondria

Lipid peroxidation was assessed by the concentration of malondialdehyde (MDA), a marker of lipid oxidation, using a peroxidation assay kit (Calbiochem) [33]. To prevent sample oxidation, all mitochondrial extracts were normalized to 1–1.5 mg/mL in resuspension buffer with butylated hydroxyl toluene to achieve a final concentration of 5 mM. Standards were prepared and experimental procedures performed according to the manufacturer’s protocol. For each reaction, a 200 microliter sample or standard was added to 650 microliters of chromogenic reagent (provided by the manufacturer) and 150 microliters of 12 N HCl. After reaction at 45 °C for 60 min., the samples were cooled to 4 °C and centrifuged at 10,000 × g for 5 min. The supernatant liquids were collected, and absorbance at 586 nm was recorded. The MDA concentration was calculated using a standard curve. All measurements were performed in duplicate.

Measurements of protein oxidation in mitochondria

Oxidative modifications of proteins introduce carbonyl groups into side chains. Protein oxidation can be monitored by depriving those carbonyl groups of 2,4-dinitrophenylhydrazone (DNP-hydrozone), which can be detected by DNP-specific antibody [34]. The extents of protein oxidation in the mitochondrial fractions from the cardiac tissue of sham-treated and septic rats were examined by the OxyBlot assay (Chemicon, Temecula, CA). Briefly, 20 mcg of protein from each sample was applied to the derivatization reaction and separated by 4–15% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (BioRad) followed by Western blotting using anti-DNP antibody. All results were quantified by densitometry.

Measurements of cytokine production by enzyme-linked immunosorbent assay

The concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in serum and myocardial tissue of sham-treated and septic rats were examined using immunoassay kits (Biosource, Camarillo, CA). For tissue lysates, samples were diluted to 1 mcg/ microliter before assay. Results in the tissue were normalized by the amount of protein, and the results in serum were normalized by volume.

Western blots

Protein samples from mitochondrial or cytosolic fractions were mixed with Laemmli’s loading buffer, boiled for 5 min., subjected to 15% SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk–PBS at room temperature for 1 h and probed with one of the following antibodies: Anti-cytochrome C or antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon), or anti-NF-κB p65 and anti-cJun (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then rinsed and incubated with the corresponding horseradish peroxidase-conjugated anti-mouse IgG (Roche, Indianapolis, IN) or anti-rabbit IgG (Stressgen Bioreagents, Victoria, B.C., Canada). Antibody dilutions and incubation times were according to the manufacturer’s instructions. The membranes were then rinsed, and bound antibodies were detected using enhanced chemiluminescence [27] (Amersham, Piscataway, NJ).

Statistical analysis

All values are expressed as mean ± standard error of the mean. Analysis of variance (ANOVA) was used to assess an overall difference among the groups for each of the variables. Levene’s test for equality of variance was used to suggest the multiple-comparison procedure to be used. If equality of variance among the four groups was suggested, multiple- comparison (Bonferroni) procedures were performed; if inequality of variance was suggested, Tamhane multiple comparisons were performed. Probability values <0.05 were considered statistically significant. Analysis was performed using SPSS for Windows, Version 7.5.1; SPSS Inc., (Chicago, IL).

RESULTS

Infectious challenge impaired mitochondrial integrity in the heart

Leak of mitochondrial protein cytochrome C into the cytosol provides a measure of mitochondrial injury. As shown in Figure 1, Western blot analysis demonstrated that the release of cytochrome C was significant 4 h after infectious challenge and increased further over the first 24 h. Expression of the control cytosolic marker protein, GAPDH, was unchanged.

FIG. 1.

FIG. 1

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Infectious challenge-induced cytochrome C release from mitochondria to cytosol. Protein samples were extracted from hearts of sham-treated and infected rats at multiple times after challenge, as indicated. Cytosolic marker GAPDH was used as control. Western blots were analyzed by densitometry. All values are means ± SEM. *Significant difference from sham-treated rats at p < 0.05 (n ≥3).

Using an alternative approach to evaluate mitochondrial integrity, we examined mitochondrial outer-membrane damage, as determined by cytochrome C oxidase activity in mitochondrial fractions with and without the detergent n-dodecyl β-D-maltoside. Cytochrome C oxidase is located on the inner membrane of mitochondria. The detergent n-dodecyl β-D-maltoside can solubilize mitochondrial matrix but does not interfere with measurement of cytochrome C oxidase activity [35]. Therefore, the ratio of cytochrome C oxidase activity measured in the presence/ absence of detergent represents the percentage of mitochondrial outer-membrane damage. As shown in Figure 2, we detected a dramatic increase in cardiac mitochondrial outer-membrane damage 4–24 h after infectious challenge.

FIG. 2.

FIG. 2

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Infectious challenge increased mitochondrial outer-membrane damage in heart. Membrane damage was measured in cardiac mitochondrial preparations from sham-treated and infected rats sacrificed at different time points post-challenge. All values are means ± SEM. *Significant difference from sham-treated rats at p < 0.05 (n ≥3).

Infectious challenge increased oxidative stress in myocardial mitochondria

To assess oxidative stress in cardiac mitochondria after sepsis, we first examined the extent of lipid peroxidation in mitochondria from cardiac tissue of sham-treated and septic challenged rats. As shown in Figure 3A, MDA was increased significantly in the cardiac mitochondria prepared from all septic rats.

FIG. 3.

FIG. 3

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Infectious challenge increased lipid oxidation (MDA) and protein oxidation in cardiac mitochondria. (A) Concentrations of MDA in cardiac mitochondrial preparations from sham-treated and infected animals. All measurements were normalized for amount of protein per reaction and are expressed as nanomoles MDA/mg of mitochondrial protein. (B) Oxidatively modified proteins in cardiac mitochondria from sham-treated and infected rats detected by Western blot with anti-DNP. Results were quantified by densitometry. All values are means ± SEM. *Significant difference from sham-treated rats at p < 0.05 (n ≥3).

We evaluated mitochondrial oxidative stress in the hearts of sham-treated and septic challenged rats further by examining protein oxidation using Western blotting with DNP-specific antibody [34]. As shown in Figure 3B, protein oxidation in cardiac mitochondria was dramatically induced immediately after infectious challenge and reached a maximum 24 h later.

Infectious challenge altered mitochondrial defense against ROS in the heart

In order to determine whether bacterial challenge altered ROS defenses in cardiac mitochondria, we compared the enzymatic activities of SOD and GPx [13] in the mitochondrial fractions from sham-treated and septic rats. Both SOD and GPx activities were decreased significantly after sepsis (Fig. 4), with SOD activity being reduced maximally 4–8 h after septic challenge but returning to sham-treatment values later. The GPx activity fell to ~70% of sham-treatment levels 12–24 h after bacterial challenge.

FIG. 4.

FIG. 4

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Activities of SOD (panel A) and GPx (panel B) in cardiac mitochondria from sham-treated and infected rats. All measured activities were normalized by amount of mitochondrial protein per reaction. All values are means ± SEM. *Significant difference from sham-treated rats at p < 0.05 (n ≥3).

In these experiments, the SOD activity was contributed by the mitochondria-specific manganese- dependent enzyme (Mn-SOD). The GPx activity was from isoforms GPx1 and GPx4, both of which are expressed in mitochondria and utilize H2O2 as substrate [13].

Myocardial NF-κB activation

We next determined the time course of inflammation responses in the myocardium after infectious challenge. As NF-κB is a crucial mediator of inflammation, we first examined the activation status of NF-κB in cardiac tissue from sham-treated and challenged rats. Activation of NF-κB is associated with its translocation from the cytosol to the nucleus. We therefore isolated the cytosolic and nuclear fractions and examined NF-κB concentrations by Western blot; cytosolic marker GAPDH and nuclear marker c-Jun, respectively, were used as controls. As shown in Figure 5, the concentration of NF-κB p65 decreased gradually in the cytosol and increased in the nucleus during sepsis, indicating that bacterial challenge provokes progressive activation of NF-κB in the heart.

FIG. 5.

FIG. 5

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Infectious challenge-activated NF-κB in myocardium. (A) NF-κB p65 in cytosolic fractions of hearts of shamtreated and infected rats sacrificed at various times after challenge. Cytosolic marker GAPDH was used as control. (B) NF-κB p65 in nuclear fractions of heart in sham-treated and infected rats. Nuclear marker c-Jun was used as control. Western blots were analyzed by densitometry. All values are means ± SEM. *Significant difference from shamtreated rats at p < 0.05 (n ≥3).

Cytokine production

By enzyme-linked immunosorbent assay (ELISA), we examined the systemic (serum) as well as tissue (myocardial) cytokine concentrations in sham-treated rats and rats given a bacterial challenge (4, 8, 12, and 24 h after instillation of S. pneumoniae). As shown in Figure 6, cytokines IL-1β, IL-6, IL-10 and TNF-α increased gradually, both in blood and in the cardiac tissue, after infectious challenge.

FIG. 6.

FIG. 6

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Infectious challenge-promoted cytokine production in blood and heart. (A) Measurement of systemic production of IL-1β, IL-6, IL-10 and TNF-α in sham-treated and infected rats. Values were normalized by volume of serum per reaction. (B) Measurement of myocardial production of IL-1 β, IL-6, IL-10, and TNF-α in sham-treated and septic rats. Values were normalized by the amount of protein per reaction. All values are means ± SEM. *Significant difference from sham-treated rats at p < 0.05 (n ≥3).

DISCUSSION

Although substantial evidence supports the impact of mitochondrial abnormalities in sepsis, the role of mitochondria in sepsis-mediated cardiac dysfunction remains unclear. Using our rat model of pneumonia-related sepsis, we did a time-course study to show that sepsis produces progressive mitochondrial damage in the heart, as confirmed by mitochondrial outer-membrane damage and release of cytochrome C. Furthermore, sepsis down-regulated the activities of the antioxidant enzymes SOD and GPx in cardiac mitochondria, and significantly increased oxidative damage to mitochondria, as shown by greater lipid and protein oxidation. In addition, inflammatory responses increased progressively after bacterial challenge. Our data suggest that changes in cardiac mitochondria are not a consequence of inflammatory responses in sepsis. Because sepsis-related cardiac dysfunction is believed to be mediated by inflammation, we speculate that sepsis produces a cascade of events that includes early myocardial mitochondrial damage, followed by progressive myocardial inflammation and late cardiac dysfunction (shown previously to occur 24 h after Streptococcus pneumoniae challenge [36]).

The mechanisms underlying sepsis-related mitochondrial injury in the heart remain unknown. However, an imbalance between ROS production and scavenging leads to inappropriate production of ROS and ROS-related tissue injury. In cardiac mitochondria of septic animals, we observed rapid down-regulation of SOD and GPx activities accompanied by a significant increase in lipid and protein oxidation, suggesting that sepsis altered the antioxidant capacity in the heart, likely contributing to mitochondrial oxidative damage. Considerable evidence shows that down-regulation of antioxidant activities in mitochondria increases their susceptibility to oxidative damage. In mice with either reduced expression or complete absence of mitochondrial SOD, oxidative damage to mitochondrial proteins, lipids and DNA has been documented [16,37]. Glutathione peroxidase is one of the primary enzymatic defense systems against H2O2 and lipid hydroperoxide; however, it may not be the sole regulator of oxidation in mitochondria. Recently, peroxiredoxin family proteins (Prxs) have been identified as antioxidant enzymes that protect against H2O2, peroxynitrite and a wide range of organic hydroperoxides [38,39]. We detected the expression of two Prx isoforms, PrxIII and PrxV, in cardiac mitochondria in rats (data not shown). We speculate that, along with GPx, Prxs may be involved in the regulation of the oxidation status of cardiac mitochondria. However, assays for Prxs activities have not been commercialized. Current methods involve large-scale recombinant protein production in yeast, which is experimentally difficult in our laboratory setting. Nevertheless, on the basis of our current data, we hypothesize that sepsis increases oxidative stress by down-regulating antioxidant capacity in myocardial mitochondria, which contributes to mitochondrial damage in the heart.

Although we do not have direct evidence that sepsis-induced lipid peroxidation is a causative factor of cytochrome C translocation, release of cytochrome C from mitochondria is initiated by ROS-mediated peroxidation of cardiolipin, a phospholipid component of the mitochondrial inner membrane [40]. In a recent study, we showed that antioxidant vitamin therapy improved lipid oxidation and maintained mitochondrial integrity in the heart after burn injury (Zang et al., unpublished data). In addition, a mitochondria-targeted antioxidant drug inhibited mitochondrial oxidative damage and cytochrome C release [41]. Therefore, oxidative damage is likely responsible in part for the sepsis-related loss of mitochondrial integrity in the heart.

The role of mitochondria in inflammatory responses is not clear. Mitochondria respond to cytokines such as TNF-α by releasing cytochrome C, which leads to apoptosis [42,43]. Studies suggesting that mitochondria regulate inflammation include reports that mitochondrial oxidative stress increased NF-κB activation in both animal models and cell lines [20–23]. Furthermore, mitochondrial matrix proteins such as MAVS [24] and DOK-4 [25] have been identified as signaling factors in NF- κB activation pathways. Mitochondrial calcium concentrations modulate cytokine secretion [26], as well as IL-1-induced downstream ERK activation [44].

Whether mitochondria are involved in myocardial inflammation during sepsis has not been well studied. On the basis of our timecourse study of mitochondrial damage and inflammation responses in the heart, we propose that cardiac mitochondrial damage is more a cause than a consequence of inflammation in sepsis. Our current and previous studies indicate that alterations in cardiac mitochondria occur early in the post-injury period, whereas NF-κB activation and inflammatory cytokine synthesis increase progressively after bacterial challenge, achieving a maximum inflammatory response by 24 h. Furthermore, in a burn injury model, antioxidant therapy prevented mitochondrial damage, attenuated inflammatory responses in myocardium, and improved cardiac function [27], suggesting a role for mitochondria in inflammation and cardiac dysfunction. In the present study, alterations in mitochondria occurred soon after infectious challenge and persisted throughout the study period. In contrast, NF-κB activation and cytokine production increased progressively after challenge. We speculate that sepsis-related mitochondrial abnormalities in the myocardium trigger other downstream signaling events that initiate and amplify the inflammatory responses in the heart.

In summary, we have observed that mitochondrial release of cytochrome C, damage to the mitochondrial outer membrane, increase in lipid and protein oxidation, and decrease in mitochondrial ROS defenses occurred in the heart early after infectious challenge. These alterations in mitochondrial integrity and antioxidant defense mechanisms were paralleled by gradual increases in inflammatory responses in the myocardium. Our data suggest that changes in myocardial mitochondria are a cause rather than a consequence of sepsis-related inflammation. Because numerous studies have confirmed that sepsis-mediated inflammation contributes to cardiac dysfunction, we propose that mitochondria play a pivotal role in sepsis-mediated cardiac dysfunction by modulating inflammatory cytokine responses. Further investigations, such as using mitochondrial targeting reagents, are needed to determine the precise role of mitochondria in inflammation and organ dysfunction after sepsis.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grant NIGMS R01 GM57054-08.

Footnotes

Presented at the 26th Annual Meeting of the Surgical Infection Society, San Diego, California, April 27–29, 2006.

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