Abstract
We have previously shown that mitochondria-targeted vitamin E (Mito-Vit-E), a mtROS specific antioxidant, improves cardiac performance and attenuates inflammation in a pneumonia-related sepsis model. In this study, we applied the same approaches to decipher the signaling pathway(s) of mtROS-dependent cardiac inflammation after sepsis. Sepsis was induced in Sprague Dawley rats by intratracheal injection of S. pneumoniae. Mito-Vit-E, vitamin E or vehicle was administered 30 minutes later. In myocardium 24 hours post-inoculation, Mito-Vit-E, but not vitamin E, significantly protected mtDNA integrity and decreased mtDNA damage. Mito-Vit-E alleviated sepsis-induced reduction in mitochondria-localized DNA repair enzymes including DNA polymerase γ, AP endonuclease, 8-oxoguanine glycosylase, and uracil-DNA glycosylase. Mito-Vit-E dramatically improved metabolism and membrane integrity in mitochondria, suppressed leakage of mtDNA into the cytoplasm, inhibited up-regulation of Toll-like receptor 9 (TLR9) pathway factors MYD88 and RAGE, and limited RAGE interaction with its ligand TFAM in septic hearts. Mito-Vit-E also deactivated NF-κB and caspase 1, reduced expression of the essential inflammasome component ASC, and decreased inflammatory cytokine IL–1β. In vitro, both Mito-Vit-E and TLR9 inhibitor OND-I suppressed LPS-induced up-regulation in MYD88, RAGE, ASC, active caspase 1, and IL–1β in cardiomyocytes. Since free mtDNA escaped from damaged mitochondria function as a type of DAMPs to stimulate inflammation through TLR9, these data together suggest that sepsis-induced cardiac inflammation is mediated, at least partially, through mtDNA-TLR9-RAGE. At last, Mito-Vit-E reduced the circulation of myocardial injury marker troponin-I, diminished apoptosis and amended morphology in septic hearts, suggesting that mitochondria-targeted antioxidants are a potential cardioprotective approach for sepsis.
Highlights
Severe sepsis, associated with staggering inflammatory responses and multi-organ failure, is a leading cause of death in intensive care units [1, 2]
Quantification of intact mitochondrial DNA (mtDNA) content out of total genomic DNA was achieved by a long distance PCR (LPCR) method, in which the ~14kb mitochondrial genome was amplified using mtDNA specific primers [50]
Mouse DNA was included in the long PCR (LPCR) reactions and utilized as an internal control, taking the advantage of the following two facts: (1) both rat and mouse mtDNA can be amplified by the same pair of primers due to sequence homology, and (2) their PCR products can be distinguished by restriction enzyme digestion
Summary
Severe sepsis, associated with staggering inflammatory responses and multi-organ failure, is a leading cause of death in intensive care units [1, 2]. In patients with severe sepsis, the degree of mitochondrial functional deficiency associates with clinical outcomes [5, 6]. A major source of mitochondrial damage comes from imbalanced production of reactive oxygen species (mtROS). During cell death and organ injuries, certain molecules released from impaired mitochondria function as danger-associated molecular patterns (DAMPs) [11,12,13,14]. The list of mitochondria-derived DAMPs includes mtROS [15], mtDNA fragments [11], N-formyl peptides [16,17,18], ATP [19, 20], cytochrome C [21, 22], cardiolipin [23] and carbamoyl phosphate synthetase I [24]
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