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.

Using a mitochondria-targeted vitamin E (Mito-Vit-E) in a rat pneumonia-related sepsis model, we examined the role of mitochondrial reactive oxygen species in sepsis-mediated myocardial inflammation and subsequent cardiac contractile dysfunction. Sepsis was produced in adult male Sprague-Dawley rats via intratracheal injection of S. pneumonia (4 × 10⁶colony formation units per rat). A single dose of Mito-Vit-E, vitamin E, or control vehicle, at 21.5 μmol/kg, was administered 30 min postinoculation. Blood was collected, and heart tissue was harvested at various time points. Mito-Vit-E in vivo distribution was confirmed by mass spectrometry. In cardiac mitochondria, Mito-Vit-E improved total antioxidant capacity and suppressed H₂O₂generation, whereas vitamin E offered little effect. In cytosol, both antioxidants decreased H₂O₂levels, but only vitamin E strengthened antioxidant capacity. Mito-Vit-E protected mitochondrial structure and function in the heart during sepsis, demonstrated by reduction in lipid and protein oxidation, preservation of mitochondrial membrane integrity, and recovery of respiratory function. While both Mito-Vit-E and vitamin E suppressed sepsis-induced peripheral and myocardial production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), Mito-Vit-E exhibited significantly higher efficacy ( P < 0.05). Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induced myeloperoxidase accumulation in myocardium, suggesting its effect on neutrophil infiltration. Echocardiography analysis indicated that Mito-Vit-E ameliorated cardiac contractility of sepsis animals, shown by improved fractional shortening and ejection fraction. Together, our data suggest that targeted scavenging of mitochondrial reactive oxygen species protects mitochondrial function, attenuates tissue-level inflammation, and improves whole organ activities in the heart during sepsis.

Sepsis is a leading cause of death in intensive care units, and cardiac dysfunction is an identified serious component of the multi-organ failure associated with this critical condition. This review summarized the current discoveries and hypotheses of how autophagy changes in the heart during sepsis and the underlying mechanisms. Recent investigations suggest that specific activation of autophagy initiation factor Beclin-1 has a potential to protect cardiac mitochondria, attenuate inflammation, and improve cardiac function in sepsis. Accordingly, pharmacological interventions targeting this pathway have a potential to become an effective approach to control sepsis outcomes. The role of autophagy during sepsis pathogenesis has been under intensive investigation in recent years. It is expected that developing therapeutic approaches with specificities targeting at autophagy regulatory factors may provide new opportunities to alleviate organ dysfunction caused by maladaptive autophagy during sepsis.

Rationale: Cilp1 (cartilage intermediate layer protein 1) is a secreted extracellular matrix protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive. Objective: To establish the function and mechanism of action of Cilp1 in postmyocardial infarction (MI) cardiac remodeling. Methods and Results: We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of patients with heart failure. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with NCilp1 (N-terminal half of the protein) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared with wild-type littermates. Conversely, NCilp1-transgenic mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 (mammalian Target of Rapamycin Complex 1) signaling were downregulated in KO hearts compared with wild-type hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and extracellular matrix gene expression analysis indicated that while MI caused significant stiffness in wild-type hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.

Mitochondria-derived danger-associated molecular patterns (DAMPs) play important roles in sterile inflammation after acute injuries. This study was designed to test the hypothesis that 17β-estradiol protects the heart via suppressing myocardial mitochondrial DAMPs after burn injury using an animal model. Sprague-Dawley rats were given a third-degree scald burn comprising 40% total body surface area (TBSA). 17β-Estradiol, 0.5 mg/kg, or control vehicle was administered subcutaneously 15 min following burn. The heart was harvested 24 h postburn. Estradiol showed significant inhibition on the productivity of H₂O₂and oxidation of lipid molecules in the mitochondria. Estradiol increased mitochondrial antioxidant defense via enhancing the activities and expression of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Estradiol also protected mitochondrial respiratory function and structural integrity. In parallel, estradiol remarkably decreased burn-induced release of mitochondrial cytochrome c and mitochondrial DNA (mtDNA) into cytoplasm. Further, estradiol inhibited myocardial apoptosis, shown by its suppression on DNA laddering and downregulation of caspase 1 and caspase 3. Estradiol's anti-inflammatory effect was demonstrated by reduction in systemic and cardiac cytokines (TNF-α, IL-1β, and IL-6), decrease in NF-κB activation, and attenuation of the expression of inflammasome component ASC in the heart of burned rats. Estradiol-provided cardiac protection was shown by reduction in myocardial injury marker troponin-I, amendment of heart morphology, and improvement of cardiac contractility after burn injury. Together, these data suggest that postburn administration of 17β-estradiol protects the heart via an effective control over the generation of mitochondrial DAMPs (mtROS, cytochrome c, and mtDNA) that incite cardiac apoptosis and inflammation.

Our previous research demonstrated that sepsis produces mitochondrial dysfunction with increased mitochondrial oxidative stress in the heart. The present study investigated the role of mitochondria-localized signaling molecules, tyrosine kinase Src and tyrosine phosphatase SHP2, in sepsis-induced cardiac mitochondrial dysfunction using a rat pneumonia-related sepsis model. SD rats were given an intratracheal injection of Streptococcus pneumoniae, 4×10⁶ CFU per rat, (or vehicle for shams); heart tissues were then harvested and subcellular fractions were prepared. By Western blot, we detected a gradual and significant decrease in Src and an increase in SHP2 in cardiac mitochondria within 24 hours post-inoculation. Furthermore, at 24 hours post-inoculation, sepsis caused a near 70% reduction in tyrosine phosphorylation of all cardiac mitochondrial proteins. Decreased tyrosine phosphorylation of certain mitochondrial structural proteins (porin, cyclophilin D and cytochrome C) and functional proteins (complex II subunit 30kD and complex I subunit NDUFB8) were evident in the hearts of septic rats. In vitro, pre-treatment of mitochondrial fractions with recombinant active Src kinase elevated OXPHOS complex I and II-III activity, whereas the effect of SHP2 phosphatase was opposite. Neither Src nor SHP2 affected complex IV and V activity under the same conditions. By immunoprecipitation, we showed that Src and SHP2 consistently interacted with complex I and III in the heart, suggesting that complex I and III contain putative substrates of Src and SHP2. In addition, in vitro treatment of mitochondrial fractions with active Src suppressed sepsis-associated mtROS production and protected aconitase activity, an indirect marker of mitochondrial oxidative stress. On the contrary, active SHP2 phosphatase overproduced mtROS and deactivated aconitase under the same in vitro conditions. In conclusion, our data suggest that changes in mitochondria-localized signaling molecules Src and SHP2 constitute a potential signaling pathway to affect mitochondrial dysfunction in the heart during sepsis.