Cardiac mitochondrial damage and inflammation responses in sepsis

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 x 10(6) 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-kappaB) 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-kappaB 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.

FIG. 1

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).

FIG. 2

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).

FIG. 3

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).

FIG. 4

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).

FIG. 5

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).

FIG. 6

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).