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Logo of ajrccmIssue Featuring ArticlePublisher's Version of ArticleSubmissionsAmerican Thoracic SocietyAmerican Thoracic SocietyAmerican Journal of Respiratory and Critical Care Medicine
 
Am J Respir Crit Care Med. 2007 October 15; 176(8): 768–777.
Published online 2007 June 28. doi:  10.1164/rccm.200701-161OC
PMCID: PMC2020830

Mitochondrial Biogenesis Restores Oxidative Metabolism during Staphylococcus aureus Sepsis

Abstract

Rationale: The extent, timing, and significance of mitochondrial injury and recovery in bacterial sepsis are poorly characterized, although oxidative and nitrosative mitochondrial damage have been implicated in the development of organ failure.

Objectives: To define the relationships between mitochondrial biogenesis, oxidative metabolism, and recovery from Staphylococcus aureus sepsis.

Methods: We developed a murine model of fibrin clot peritonitis, using S. aureus. The model yielded dose-dependent decreases in survival and resting energy expenditure, allowing us to study recovery from sublethal sepsis.

Measurements and Main Results: Peritonitis caused by 106 colony-forming units of S. aureus induced a low tumor necrosis factor-α state and minimal hepatic cell death, but activated prosurvival protein kinase A, B, and C sequentially over 3 days. Basal metabolism by indirect calorimetry was depressed because of selective mitochondrial oxidative stress and subsequent loss of mitochondrial DNA copy number. During recovery, mitochondrial biogenesis was strongly activated by regulated expression of the requisite nuclear respiratory factors 1 and 2 and the coactivator peroxisome proliferator-activated receptor γ coactivator-1α, as well as by repression of the biogenesis suppressor nuclear receptor interacting protein-140. Biogenesis reconstituted mitochondrial DNA copy number and transcription, and restored basal metabolism without significant hepatocellular proliferation. These events dramatically increased hepatic mitochondrial density in transgenic mice expressing mitochondrially targeted green fluorescent protein.

Conclusions: This is the first demonstration that mitochondrial biogenesis restores oxidative metabolism in bacterial sepsis and is therefore an early and important prosurvival factor.

Keywords: gram-positive bacteria, biogenesis, oxidative stress

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

Mitochondrial biogenesis has been described as an adaptive cellular response in conditions such as exercise and hormone exposure. Emerging data now suggest that mitochondrial biogenesis might play a role in inflammatory conditions.

What This Study Adds to the Field

Bacterial sepsis induces mitochondrial injury resulting in depressed metabolism and biogenesis restores mitochondrial content and function.

Sepsis is the tenth leading cause of death in the United States and its incidence has tripled since 1979. At the same time, the microbiology of sepsis has shifted toward gram-positive bacteria, primarily Staphylococcus aureus, which caused 52% of episodes of sepsis in 2000 (1, 2). The virulence of S. aureus is conveyed by release of proinflammatory products and toxins that cause cell damage and contribute to progressive dysfunction of one or more vital organs (3). Fatalities are often preceded by the multiple organ dysfunction syndrome (MODS) and emerging data implicate mitochondrial damage and dysfunction as critical factors in the pathogenesis of sepsis-induced MODS (4).

MODS is frequently characterized by compromise of the liver's central role in the immune response to systemic infection. The liver clears microbial products and elaborates inflammatory mediators such as tumor necrosis factor (TNF)-α and IL-1, enhancing the immune response (5). However, hepatocytes also undergo oxidative and nitrosative stress during systemic infection through a variety of mechanisms including activation of innate immunity (6). These conditions produce reactive oxygen and nitrogen species primarily within mitochondria through disruption of electron transport chain (ETC) function, resulting in sepsis-induced damage to mitochondrial DNA (mtDNA) and proteins, including ETC components (712). However, the fate of damaged and dysfunctional mitochondria and their effect on the respiratory capacity of affected tissues are unknown.

Normal mitochondrial number, structure, and function are supported by mitochondrial biogenesis, a cellular program that adjusts energy production by synthesis of new organelles and organelle components and mediates interorganelle interactions (13, 14). Because the mitochondrial genome encodes only a fraction of the mitochondrial proteins, mitochondrial biogenesis requires communication between mitochondria and the nucleus. For example, nuclear genes for the oxidative phosphorylation (OXPHOS) complex and other mitochondrial proteins, as well as mitochondrial transcription factor A (Tfam) and mitochondrial transcription factor B, are activated by nuclear respiratory factor (NRF)-1 and NRF-2. The mitochondrial transcription factors then direct mtDNA transcription and replication (13, 1517). Many tissues adjust mitochondrial mass and cellular mtDNA content physiologically through mitochondrial biogenesis (1822), and data suggest a role for biogenesis in the response to inflammatory conditions including sepsis (69).

In rodent studies, a single intraperitoneal LPS administration that depletes glutathione is associated with a specific deletion in an oxidant-sensitive region of the mitochondrial genome (9). However, after mitochondrial injury, mtDNA copy number is restored through up-regulation of the mitochondrial biogenesis program. In that disease model, the expression of NRF-1, NRF-2, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and Tfam accompanies restoration of mtDNA copy number and increased activation of the mitochondrial transcriptosome (79). Mice undergoing cecal ligation and puncture also exhibit decreased hepatic mitochondrial mass in association with increased Tfam and PGC-1α protein levels (23). In experiments with genetically engineered mice challenged with heat-inactivated Escherichia coli, hepatic mtDNA depletion and recovery both depended on intact innate immunity via Toll-like receptor (TLR)-4 (6). The innate immune response is therefore an important cause of oxidative mitochondrial damage as well as a critical factor in mitochondrial recovery. These investigations provide preliminary evidence linking sepsis-induced mitochondrial oxidative injury to biogenesis. However, the pathophysiology of LPS-based injury models inevitably differs from actual sepsis, in which the consequences of mitochondrial injury and repair are essentially unknown.

Given the prevalence of S. aureus in human sepsis, the central role of the liver in host defense, and the emerging implications of mitochondrial damage and repair in the pathogenesis of MODS, we developed a clinically relevant mouse model of staphylococcal sepsis characterized by depression of resting metabolism. We then specifically tested the hypothesis that staphylococcal sepsis causes mitochondrial damage and disordered energy metabolism and that recovery is linked to activation of survival signaling and mitochondrial biogenesis. Preliminary data from this study have been reported in abstract form (24).

METHODS

Mice

Young adult male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Genetically engineered mitochondrial green fluorescent protein (mt-GFP) mice (25) were obtained from Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, and bred at our facility. The studies were conducted under protocols approved by the Duke University (Durham, NC) Institutional Animal Care and Use Committee.

Bacteria

S. aureus ssp. aureus (no. 25923; American Type Culture Collection, Manassas, VA) was reconstituted according to the manufacturer's specifications and sterilely inoculated onto Trypticase soy agar (BD Diagnostic Systems, Sparks, MD) slants. The slants were incubated for 18 hours at 37°C to achieve adequate log-phase growth. Bacteria were harvested in 0.9% NaCl and centrifuged, and the pellets were resuspended in 0.9% NaCl. Bacterial suspensions were quantified with a spectrophotometer (550 nm) to generate a stock solution of 1 × 1010 viable colony-forming units per milliliter. Pour plates were used to confirm viability and accuracy of the calibration. Serial dilutions were performed to generate the desired concentrations for inoculation.

Clot Generation

To 500 μl of a 2% suspension of porcine or bovine fibrinogen (Sigma, St. Louis, MO) was added 100 μl of the desired concentration of S. aureus. Ten microliters of a 1-unit/μl solution of bovine thrombin (Sigma) was then added and the mixture was congealed for 30 minutes at room temperature.

Animal Procedures

Mice were anesthetized with an intraperitoneal injection of 0.3 mg of xylazine and 2.5 mg of ketamine. The abdomen of each animal was shaved and cleansed with povidone-iodine and ethanol. A small midline laparotomy was performed and a sterile or bacteria-impregnated fibrin clot was placed into the peritoneal cavity. The fascia and skin were closed with 3-0 silk and each animal was resuscitated with 1 ml of sterile 0.9% NaCl administered subcutaneously. The mice were monitored regularly and survival was recorded daily. Mice were killed 1, 2, or 3 days after implantation. Fresh whole blood was then collected and serum was obtained by centrifugation. IL-1β, macrophage inflammatory protein-2, and TNF-α protein levels were determined with commercially available ELISA kits specific to mice (R&D Systems, Minneapolis, MN). The liver was rapidly resected for immediate use, snap frozen, or perfusion fixed for histology with 10% formalin.

Metabolism Studies

Steady-state oxygen consumption and carbon dioxide production rates (Vo2 and Vco2, respectively) were measured in resting, air-breathing mice under direct observation at a constant ambient temperature at the same time of day. The mice were placed individually in a miniature metabolic chamber, and after at least 10 minutes of acclimation, expired gas was collected at a calibrated flow rate for 5 minutes. O2 and CO2 gas concentrations were measured with a calibrated gas chromatograph (model 3800; Varian, Palo Alto, CA). Vo2 and Vco2 were computed with standard formulas corrected to stpd and btps, respectively. Resting energy expenditure (REE) was calculated with the modified Weir formula (26): REE (kcal/d) = [Vo2 (ml/min) × (3.941) + Vco2 (ml/min) × (1.11)] × 1.44.

Histopathology and Fluorescence Microscopy

Paraffin-embedded liver samples were sectioned at 5 μm and stained with hematoxylin and eosin for light microscopy. For transgenic C57BL/6J-TgN (cox8EGFP) mice (25), paraffin-embedded liver samples were sectioned at 5 μm and deparaffinized. The sections were treated with 0.1% saponin and washed with phosphate buffer. Slides were mounted with a SlowFade antifade kit (Invitrogen, Carlsbad, CA). Laser scanning confocal microscopy was performed with an LSM 410 microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY), and fluorescence images were collected with a 520-nm bandpass filter.

Semiquantitative Polymerase Chain Reaction

Total RNA was extracted from frozen liver with TRIzol reagent (Invitrogen). RNA samples then underwent reverse transcription and polymerase chain reaction (PCR) as described previously (6, 8).

mtDNA Copy Number by Real-Time PCR

Total cellular DNA was extracted from frozen liver with a GenElute mammalian genomic DNA kit (Sigma). The mtDNA copy number was obtained by real-time PCR as previously described (27).

DNA Fragmentation Gel Analysis

Total DNA was extracted with a GenElute mammalian genomic DNA kit (Sigma). Equal amounts of isolated DNA were electrophoresed on 1% agarose, using a series of standard molecular weight markers.

Western Blot Analysis

Liver protein extracts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were probed with fully characterized polyclonal rabbit anti–protein kinase C (PKC, diluted 1:750), protein kinase B (Akt, diluted 1:1,000), phosphorylated Akt (p-Akt, diluted 1:1,000), protein kinase A (PKA, diluted 1:500), phosphorylated PKA (p-PKA, diluted 1:500), or RIP140 (diluted 1:250) (Santa Cruz Biotechnology, Santa Cruz, CA) or caspase-3 (BD Biosciences, San Jose, CA). After the application of primary antibodies, membranes were washed and incubated with appropriate horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology and Jackson Laboratory). The membranes were developed by ECL (Santa Cruz Biotechnology) and protein was quantified on digitized images in the mid-dynamic range. Protein loading was confirmed by stripping the membranes and reprobing with antibody against tubulin (Sigma). At least four samples were used for densitometry measurements.

Statistical Analysis

Grouped data were expressed as means ± SD. Statistical analyses were performed by analysis of variance (ANOVA) and repeated measures ANOVA using StatView (SAS Institute, version 5.0.1; Chicago, IL). P [less-than-or-eq, slant] 0.05 was considered significant.

RESULTS

Characterization of S. aureus Fibrin Clot Peritonitis in Mice

We produced a clinically relevant S. aureus sepsis model that would allow for the study of cellular injury and repair without significant mortality by adapting the rat fibrin clot peritonitis model of Ahrenholz and Simmons to mice infected with S. aureus (28). Prior work on direct intraperitoneal injection of S. aureus suggested a dose of 106 or 107 cfu as suitable for clot implantation (29). In a preliminary dose-ranging study with C57BL/6J mice, peritoneal implantation of an uninfected fibrin clot or one containing viable microorganisms (Figure 1A) produced dose-dependent lethality from S. aureus sepsis. A bacterial dose of 106 cfu yielded nearly 90% survival on Day 3 and approximately 80% survival on Day 7. Mice that received 107 cfu had an overall survival of approximately 60% after 7 days and the use of higher doses of S. aureus produced high mortality (data not shown). Mice that received uninfected clot (sham procedure) had no significant mortality.

Figure 1.
Survival, metabolism, and cytokine profiles during Staphylococcus aureus sepsis in mice. (A) Survival of C57BL/6J mice after peritoneal implantation of an uninfected (solid line) fibrin clot or one containing 106 (dashed line) or 107 (dotted line) cfu ...

Early cytokine release in sublethal S. aureus sepsis was characterized by serum ELISA in control mice (day 0) and in mice receiving 106 cfu of S. aureus on Days 1, 2, and 3 (Figure 1B). S. aureus peritonitis and sepsis induced significant production of macrophage inflammatory protein-2 on Day 1 and of IL-1β on Day 2, without a statistically significant rise in serum TNF-α.

The metabolic effects of S. aureus sepsis were measured by indirect calorimetry before and after implantation of uninfected clot and clot containing 106 or 107 cfu of S. aureus (Figures 1C–1F). All mice showed postoperative decreases in weight, Vo2, Vco2, and REE, reflecting the effects of anesthesia and the laparotomy. However, mice undergoing implantation of a sterile clot quickly returned to baseline weight and metabolic activity. Mice receiving 106 cfu of S. aureus experienced a prolonged postoperative decline in weight and metabolic activity. However, from Day 3 forward there was a steady increase in metabolic rate in these mice, with recovery of baseline values by the end of 1 week. Mice receiving 107 cfu of S. aureus experienced a more substantial decrease in weight and resting metabolism and did not recover baseline metabolic activity by the end of the experiment. Repeated measures ANOVA demonstrated a significant effect of group and time for weight loss, Vo2, Vco2, and REE in sepsis. Measured body temperature changes were small and inconsistent and no relationship with REE was observed (data not shown). Furthermore, some weight loss was inevitably due to reduced oral intake; however, this was clearly not significant enough to cause lethal shock.

S. aureus Peritonitis and Hepatic Cell Death

At an S. aureus dose of 106 cfu, there was no significant apoptosis or necrosis on liver histology by light microscopy through Day 7 of peritonitis. Compared with normal liver, the sections on Day 3 (Figures 2A and 2B) demonstrated a few more mitotic figures and foci of inflammatory cells. By Day 7, the sections contained only rare mitotic figures and occasional foci of inflammation (Figure 2C). Liver sections from mice receiving larger inocula of S. aureus exhibited prominent diffuse and focal inflammation and evidence of cellular injury.

Figure 2.
Evaluation of hepatic cell death during S. aureus sepsis. (AC) Representative liver histology from C57BL/6J mice during peritonitis at an S. aureus dose of 106 cfu. (A) Control liver section shows no significant structural abnormalities. (B) ...

To further evaluate the extent of hepatic cell death at a dose of 106 cfu, cellular DNA extracts were subjected to agarose electrophoresis (Figure 2D). The characteristic bands of apoptotic DNA fragmentation were absent, but a faint DNA degeneration smear, consistent with trace necrosis, was noted on Days 1 and 2. Consistent with this investigation, no increase in caspase-3 activity was demonstrated (Figure 2E), confirming a lack of caspase-dependent apoptosis in this model. Overall, fibrin clot peritonitis from S. aureus at a dose of 106 cfu caused minimal hepatocyte necrosis and apoptosis. On the basis of these findings, the limited mortality, and the reversible reduction in oxidative metabolism, the 106-cfu dose was chosen for detailed biochemical and molecular analyses.

Selective Mitochondrial Oxidative Stress during S. aureus Sepsis

To evaluate whether S. aureus sepsis at 106 cfu induces hepatic mitochondrial oxidative stress, mRNA levels for a key mitochondrial antioxidant enzyme, superoxide dismutase-2 (SOD2, Mn-SOD), were examined and compared with mRNA expression of the cytoplasmic SOD1 (Cu,Zn-SOD) (30). SOD expression by semiquantitative PCR (Figure 3) revealed a dramatic increase in SOD2 expression compared with SOD1 on Days 1 and 2, compatible with selective, early mitochondrial oxidative stress during S. aureus peritonitis.

Figure 3.
Hepatic superoxide dismutase expression during S. aureus sepsis. Mitochondrial (superoxide dismutase-2 [SOD2]) and cytosolic (SOD1) gene expression were measured by semiquantitative polymerase chain reaction (PCR) from total RNA of livers from controls ...

S. aureus Sepsis and Sequential Activation of Antiapoptotic, Prosurvival Protein Kinases

PKA, PKB/Akt, and PKC, key members of the ABC protein kinase family, have emerged as links between oxidative stress, energy metabolism, and cell survival. Therefore, we examined the activity of these three kinases as upstream regulators of cell survival. Western blot analysis of liver protein extract from controls and on Days 1, 2, and 3 of sepsis is shown in Figure 4. It is known that PKA activity depends on the intracellular cAMP concentration, and in our model PKA activity did not increase significantly until Day 3 (Figure 4A). Interestingly, PKB (Akt) activity was increased earlier in the course of sepsis and remained so throughout the study (Figure 4B). PKC-ε protein levels were increased on Days 2 and 3, completing the sequential activation of the ABC kinases (Figure 4C) and the mechanisms for cell survival.

Figure 4.
Hepatic ABC protein kinase expression during S. aureus sepsis. Hepatic protein was extracted from controls and mice on Sepsis Days 1, 2, and 3 of peritonitis; treated mice received an S. aureus dose of 106 cfu. Protein extracts were resolved by sodium ...

S. aureus Sepsis Induces Mitochondrial Biogenesis

We hypothesized that metabolic recovery from S. aureus sepsis would require mitochondrial biogenesis. To directly assess mitochondrial biogenesis, we determined the mRNA expression of three key regulators of biogenesis: NRF-1, NRF-2, and PGC-1α (Figure 5). We observed a marked increase in NRF-1 and NRF-2 on Day 2, followed by a return to basal values on Day 3. PGC-1α mRNA levels progressively increased during the study, reaching statistical significance on Day 3. These results demonstrate a significant increase in the nuclear-encoded transcription factors and an essential coactivator for mitochondrial biogenesis in S. aureus sepsis.

Figure 5.
Expression of nuclear transcriptional elements for mitochondrial biogenesis during S. aureus sepsis. Nuclear gene expression by semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) of total RNA prepared from livers of controls ...

Mitochondrial biogenesis depends on nuclear Tfam mRNA expression and synthesis and importation of Tfam protein into the mitochondria, where it directs mtDNA transcription and replication. Tfam mRNA expression in the liver during sepsis (Figure 6A) was markedly increased on Day 2 and returned to basal values on Day 3, thus paralleling the increased expression of NRF-1 and NRF-2, which regulate Tfam transcription. To evaluate Tfam protein function in the mitochondria we analyzed transcripts of cytochrome b, an OXPHOS gene encoded by the mitochondrial genome (Figure 6B). Cytochrome b mRNA levels increased significantly on Day 3 directly following the observed increase in Tfam gene expression on Day 2, consistent with enhancement of Tfam activity within hepatic mitochondria.

Figure 6.
Expression of mitochondrial transcription factor A, mitochondrial DNA (mtDNA) transcription, and mtDNA copy number during S. aureus sepsis. Expression of nuclear-encoded mitochondrial transcription factor A (Tfam) and mtDNA-encoded cytochrome b (Cyt b) ...

To determine whether mtDNA damage precedes biogenesis in S. aureus sepsis, mtDNA copy number was measured by real-time reverse transcriptase (RT)-PCR (Figure 6D). We observed a significant decrease in mtDNA copy number on Sepsis Days 1 and 2, characteristic of sepsis-induced oxidative damage. Complete restoration of copy number occurred on Day 3 through mtDNA replication, a critical component of mitochondrial biogenesis.

Because full expression of mitochondrial biogenesis in vivo may require relief of endogenous gene repressors, we evaluated RIP140, a nuclear transcriptional corepressor and a suppressor of mitochondrial biogenesis (31). The role of RIP140 in sepsis is unknown; therefore its expression was evaluated in our model by semiquantitative RT-PCR and Western blot analysis (Figures 7A and 7B). A significant loss of RIP140 mRNA expression was observed most prominently on Day 2, which corresponds with the evidence of activation of mitochondrial biogenesis noted earlier. Western blot for RIP140 protein levels (Figure 7B) revealed a steady downward trend in RIP140 protein levels through Day 3 (P = 0.09).

Figure 7.
Nuclear receptor interacting protein-140 (RIP140) suppression during S. aureus sepsis. Total RNA and protein were extracted from controls and mice on Days 1, 2, and 3 of peritonitis; treated mice received an S. aureus dose of 106 cfu. (A) RIP140 mRNA ...

To check the distribution of mitochondrial biogenesis in sepsis, we used transgenic mice (mt-GFP) with a construct linking the mitochondrial import sequence of cytochrome c oxidase subunit VIII to the GFP. Mitochondrial biogenesis leads to increased production and importation of mitochondrial proteins such as cytochrome c oxidase, which will be reflected in the mt-GFP mice by increased GFP within mitochondria (25). mt-GFP mice underwent peritoneal implantation of a fibrin clot containing 106 cfu of S. aureus and livers were harvested on Day 1 and Day 3 and compared with controls. In control liver (Figure 8A), background fluorescence was minimal and few hepatocytes showed brilliant mitochondrial fluorescence. Liver sections from Sepsis Day 1 (Figure 8B) demonstrated multiple but widely scattered hepatocytes with brilliant mitochondrial fluorescence. By Day 3 (Figure 8C) an intense, centrilobular green fluorescence was observed, indicating widespread activation of hepatic mitochondrial biogenesis.

Figure 8.
Confocal microscopy of liver sections from mitochondrial green fluorescent protein (mt-GFP) transgenic mice during S. aureus sepsis. Livers from control mt-GFP transgenic mice and on Sepsis Days 1 and 3 during S. aureus fibrin clot peritonitis were harvested ...

DISCUSSION

The major novel finding of this investigation is a mechanism for metabolic recovery during bacterial sepsis in a reproducible, clinically relevant S. aureus peritonitis model. We found, under dose-limited conditions, that S. aureus sepsis induces mitochondrial biogenesis in the liver and, most important, that biogenesis can restore mitochondrial number and oxidative metabolism after selective damage to these organelles. In addition, we demonstrated that full expression of biogenesis in sepsis involves relief of endogenous RIP140 suppression. We also implicate, for the first time, sequential activation of the ABC prosurvival protein kinases in the recovery from mitochondrial injury during S. aureus sepsis.

Clinical and experimental evidence associates sepsis with metabolic dysfunction, underscoring the complexity of the host response to sepsis. A major goal of this study was to link changes in oxidative metabolism to sepsis-induced mitochondrial injury and recovery. In resting animals, under direct observation, we carefully performed indirect calorimetry to obtain the REE. We also chose the liver as the sentinel organ; however, similar changes in other organs would be anticipated on the basis of prior research (7). Because respiration is responsible for approximately 90% of the body's oxygen consumption (Vo2), a decline in basal Vo2 corresponds to a change in mitochondrial respiration and vice versa (32). The significant sepsis-induced declines in Vo2, Vco2, and REE are all consistent with a generalized loss of mitochondrial function. Subsequent recovery of function coincides with molecular activation of mitochondrial biogenesis, thereby linking biogenesis to metabolic recovery. Moreover, the metabolic studies predict survival because mice receiving 106 cfu of S. aureus recover metabolic function and most survive, whereas those receiving 107 cfu of S. aureus fail to recover basal metabolism and have significant mortality. Systemic oxygen consumption may not specifically reflect hepatic mitochondrial oxygen use and both ETC loss and direct ETC inhibition will depress oxidative metabolism. In any event, the mitochondrial dysfunction and subsequent biogenesis would directly affect the observed metabolic changes.

The lower resting metabolic rate during S. aureus peritonitis was accompanied by decreased hepatic mtDNA copy number, which impairs OXPHOS (33). S. aureus sepsis also induced selective mitochondrial oxidative stress, noted by the dramatic increase in SOD2 expression relative to SOD1 (30). Mitochondria are particularly susceptible to oxidant injury from proximate reactive oxygen species production, and mtDNA is especially vulnerable to oxidative modifications (10, 33). Thus, the loss and recovery of mtDNA copy number is not simply a sign of transient oxidative stress but, rather, is indicative of the organized cellular program for mitochondrial reconstitution, that is, mitochondrial biogenesis.

Mitochondrial biogenesis requires bigenomic coordination between the mitochondria and the nucleus. After depletion of mtDNA on Day 1, we found an orderly appearance of mRNA for NRF-1, NRF-2, PGC-1α, and Tfam. By Day 3, mtDNA copy number was restored and transcription of the mitochondrial genome was increased (cytochrome b expression). Resting metabolism normalized as the nuclear regulators of biogenesis returned to baseline. Thus, activation of mitochondrial biogenesis appeared and subsided as a time-limited response to sepsis-induced mitochondrial dysfunction.

The full expression of mitochondrial biogenesis may require relief of an endogenous suppressor, such as RIP140, a ligand-dependent nuclear receptor corepressor that regulates mitochondrial biogenesis (31, 34). RIP140 protein is present in highly metabolic tissues such as muscle and liver, as well as in adipocytes, and depletion of RIP140 in 3T3-L1 adipocytes enhances the nuclear expression of essential mitochondrial genes for glucose metabolism, fatty acid β-oxidation, and oxidative phosphorylation (31). In this study, S. aureus sepsis in mice caused a decline in RIP140 mRNA levels and lower protein levels, thus corresponding to enhanced biogenesis in sepsis.

The timing and distribution of hepatic mitochondrial biogenesis in S. aureus sepsis were evaluated in mice (mt-GFP) with GFP tagged to the mitochondrial import sequence of cytochrome c oxidase subunit VIII. Control mt-GFP mice exhibit minimal background fluorescence due to basal mitochondrial maintenance. In S. aureus sepsis, however, a dramatic increase in brilliant mitochondrial fluorescence was found throughout the liver on Day 3, concomitant with molecular up-regulation of mitochondrial biogenesis and restoration of the resting metabolic rate. The minimal cell death and proliferation observed on histopathology indicate that biogenesis is promoting survival of existing hepatocytes and is not merely supporting cell replacement. However, the exact relationship between mitochondrial injury, mitochondrial biogenesis, and organ failure still remains unknown. Mitochondrial biogenesis is a cellular function, which in turn supports a large number of other functions that require energy. The time course of biogenesis suggests that the cell anticipates an impending crisis in energy availability and up-regulates biogenesis to avert cellular (organ) failure. Nonetheless, at the present time, it is difficult to relate mitochondrial injury and biogenesis to organ failure and this is a limitation of the study.

Mitochondrial biogenesis in sepsis acts in conjunction with sequential activation of the ABC kinases as a prosurvival, homeostatic mechanism. In S. aureus sepsis, Akt activity was increased on Day 1 and persisted through Day 3. Akt is activated by phosphatidylinositol-3-kinase during oxidative stress and promotes prosurvival cellular effects including inactivation of the proapoptotic protein Bad and regulation of forkhead transcription factors (35, 36). Thus, early Akt activation is consistent with the minimal apoptosis observed at a sublethal inoculum. In addition, phosphatidylinositol-3-kinase/Akt phosphorylation and activation of NRF-1 represent a critical early signal for mitochondrial biogenesis (27). Other links between Akt, glucose metabolism, and cell survival are also emerging: Akt promotes cell survival by stabilizing the interaction between hexokinases and the outer mitochondrial membrane and up-regulation of glucose transport (3739).

Among the PKC isozymes, PKC-ε is activated by inositol triphosphate–mediated calcium and diacylglycerol release, PDK-1 (3-phosphoinositide-dependent protein kinase 1) phosphorylation, and membrane translocation (40). Previously, in the cecal ligation and puncture model, significant early changes in PKC-ε levels were not detected (41); however, in our model, PKC-ε increased later, after 48 hours of sepsis. PKC-ε is cardioprotective after ischemia–reperfusion via stabilization of the permeability transition pore and inactivation of Bad and, given these findings, a regulatory role in mitochondrial biogenesis may emerge (42, 43).

PKA activity is conveyed by catalytic subunit release after cAMP binding, and active subunits are targeted to the mitochondrial membranes, where they bind to A-kinase anchoring proteins (44, 45). PKA at the inner membrane phosphorylates the 18-kD iron–protein subunit of complex I, enhancing respiratory activity (45). The G-protein Rab32, a regulator of mitochondrial fission, was described as a mitochondrially targeted A-kinase anchoring protein, providing another connection between PKA and mitochondrial biogenesis (46). In our model, PKA activity rose on Day 3, in conjunction with mitochondrial biogenesis and the restoration of resting metabolism. As with Akt and PKC, PKA localized to the mitochondria is also prosurvival through phosphoinhibition of Bad (35, 43, 47). Taken as a whole, these data illustrate a sequential activation (Akt → PKC → PKA) of the ABC protein kinases during S. aureus sepsis, consistent with their complementary roles in the regulation of energy metabolism.

The novel mechanisms defined by this investigation are summarized in the timeline of Figure 9. Early, low-lethality S. aureus sepsis, characterized by cytokine production, oxidative stress, and loss of mtDNA copy number in mice, transiently suppresses metabolism without appreciable hepatic cell death. As the host response evolves, prosurvival protein kinase activity increases in association with the activation of mitochondrial biogenesis. By Day 3, mtDNA copy number has been restored and by Day 7 resting metabolism has recovered. The firm establishment of mitochondrial injury on Day 1 suggests that this mechanism is an immediate, early hallmark of staphylococcal sepsis. Under rigorously defined injury conditions, the gradual recovery of normal metabolism, the high survival rate, and the minimal cell death and proliferation by histology indicate that mitochondrial biogenesis is an early and powerful prosurvival mechanism.

Figure 9.
Timeline of pathogenesis of mitochondrial damage and recovery in mice with S. aureus peritonitis and sepsis. Akt = protein kinase B; MIP = macrophage inflammatory protein; MtDNA = mitochondrial DNA; NRF = nuclear respiratory ...

Notes

Supported by NIH RO1 AI064789-01 (C.A.P.) and NIH T32 HL007538.

Originally Published in Press as DOI: 10.1164/rccm.200701-161OC on June 28, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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